Recent advances in food biopeptides: production, biological functionalities and therapeutic applications.

JBA-06873; No of Pages 37 Biotechnology Advances xxx (2014) xxx–xxx

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Article history: Received 19 April 2012 Received in revised form 5 February 2014 Accepted 5 December 2014 Available online xxxx

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Keywords: Functional peptides Enzymatic hydrolysis Protein hydrolysates Penetration routes Peptides bioavailability Multiple biological activities Antioxidative Antihypertensive In vitro and in vivo assessments

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The growing momentum of several common life-style diseases such as myocardial infarction, cardiovascular disorders, stroke, hypertension, diabetes, and atherosclerosis has become a serious global concern. Recent developments in the ﬁeld of proteomics offering promising solutions to solving such health problems stimulates the uses of biopeptides as one of the therapeutic agents to alleviate disease-related risk factors. Functional peptides are typically produced from protein via enzymatic hydrolysis under in vitro or in vivo conditions using different kinds of proteolytic enzymes. An array of biological activities, including antioxidative, antihypertensive, antidiabetic and immunomodulating has been ascribed to different types of biopeptides derived from various food sources. In fact, biopeptides are nutritionally and functionally important for regulating some physiological functions in the body; however, these are yet to be extensively addressed with regard to their production through advance strategies, mechanisms of action and multiple biological functionalities. This review mainly focuses on recent biotechnological advances that are being made in the ﬁeld of production in addition to covering the mode of action and biological activities, medicinal health functions and therapeutic applications of biopeptides. State-of-the-art strategies that can ameliorate the efﬁcacy, bioavailability, and functionality of biopeptides along with their future prospects are likewise discussed. © 2014 Published by Elsevier Inc.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Production of biopeptides . . . . . . . . . . . . . . . . . . . . The production of endogenous peptides . . . . . . . . . . . Production of biopeptides with enzymatic hydrolysis . . . . . Production of biopeptides with microbial fermentation . . . . Biopeptide generation during in vitro and in vivo proteolytic digestion Isolation and puriﬁcation of bioactive peptides . . . . . . . . . . Degradation kinetics and enzymes ﬂexibility . . . . . . . . . . . Enzymatic antagonism: case of competitive and feedback inhibitions Strategies needed to ameliorate enzyme catalytic rate . . . . . . . Biopeptide transportation modes and absorption routes . . . . . . Biopeptide resistance and bioavailability . . . . . . . . . . . . . General concerns related to in vitro biological activities . . . . . . General concerns related to in vivo biological activities . . . . . . . Role of biopeptides in scavenging oxidative stress . . . . . . . . . Role of biopeptides as antihypertensive agents . . . . . . . . . . Role of biopeptides in modulating the immune disorders . . . . . . Bi- and multi-function validations of biopeptides fragment . . . . . Biopeptides with antimicrobial and anti-fungal properties . . . . . Biopeptides with anticoagulant activity . . . . . . . . . . . . . .

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Department of Food Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia Department of Chemistry, Universiti of Sargodha, Sargodha 40100, Pakistan

Sami Saadi a, Nazamid Saari a,⁎, Farooq Anwar b,⁎, Azizah Abdul Hamid a, Hasanah Mohd Ghazali a

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Recent advances in food biopeptides: Production, biological functionalities and therapeutic applications

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⁎ Corresponding authors. E-mail addresses: [emailprotected], [emailprotected] (S. Saadi), [emailprotected] (N. Saari), [emailprotected] (F. Anwar), [emailprotected] (A. Abdul Hamid), [emailprotected] (H.M. Ghazali).

Please cite this article as: Saadi S, et al, Recent advances in food biopeptides: Production, biological functionalities and therapeutic applications, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biotechadv.2014.12.003

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Biopeptides with opiate-like activity . . . . . . . . . . . . . . . Biopeptides with anticancer and anti-tumor activities . . . . . . . Strategies needed for ameliorating biopeptides availability and potency Future prospects . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . Uncited references . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Parallel to the development of the area of functional foods, there is a threat of numerous diseases including oxidative stress, cardiovascular diseases, diabetes, and inﬂammation, which have a life-changing effect on the health of people around the world (Korkmaz et al., 2009; Martins et al., 2006; Siddiqui et al., 2004; Willcox et al., 2009). These types of physiological and morphological perturbations might have been associated with the excessive use of synthetic additives (Antignac et al., 2011), unbalanced diet (Barker, 2012), and augmentation in the infection rate (Grifﬁths et al., 2011). In addition, the spontaneous exposure to intensive irradiation has provoked many chronic diseases, which are expressed later as long-term maladies (Makri et al., 2004). The continuous propagation of these maladies the world over prompted scientists to probe the real causes of such health disorders through the in vitro or in vivo applications of natural ingredients as an alternative therapy. Within the last decade, many studies revealing the antioxidative, antihypertensive and immunomodulatory properties of functional foods have been documented. In fact, most of such studies present greater contribution of nutrients at the molecular level through establishing a ﬁeld of nutrigenomics rather than at the cell level, depending highly on the functional properties of the food materials (Afman and Müller, 2006). Recently, Bordbar et al. (2011) extensively reviewed the biological and medicinal properties of Sea cucumbers, showing that these natural materials possess versatile biological functions, thereby; they can be classiﬁed among the potential sources of functional foods, due to their high-value components and bioactives. In addition to several of the natural bioactive compounds (Marlida et al., 2000a, 2000b; Misnawi et al., 2002; Olusesan et al., 2011; Onsa et al., 2004; Saari et al., 1999), plant phenolics (Abdul-Hamid and Luan, 2000; Hassan et al., 2011; Hussin et al., 2007), as well as immunostimulating regulatory substances, particularly, antiinﬂamation drugs — based phytomedicine active compounds (Israf et al., 2007, 2010) have shown great potential for food preservation, microbial inhibition and medicinal applications. Peptides in turn are a kind of health protectant present in nature, which are made up of a series number of amino acid residues with variable chain lengths having low molecular weight in comparison to protein mass. Further, they have particular N-terminal and C-terminal amino acid residues. These peptides are inert in the nature within the parent molecule (protein). However, the subjection of the mother protein under enzymatic digestion initiates the production of more active fragments namely “biopeptides”. The attention that has been given to biopeptides among other active compounds such as phenolic and ﬂavonoids is due to their therapeutic and medicinal potential, and especially their potentiality in the regulation of food intake (Dougkas et al., 2011). Production, puriﬁcation and characterization of biopeptides from food sources are an immerging area of biochemical research. Much of the research in this regard is being focused on studying the bioavailability as well as on proving evidences on the medicinal potentiality of these active compounds as natural health protectants. However, signiﬁcant efforts have been undertaken recently by biochemists to investigate their biochemical transformation within the living organisms. For instance, specific studies on biopeptides or proteins as a type of polymer surface have used advanced quantitative and qualitative computational approaches

to identify the structure, kinetics, and mechanism of action of biopeptides (Vansco et al., 2005). While the majority of them paid attention on the biological functionalities of biopeptides including antioxidative (Bernardini et al., 2011; Torres-Fuentes et al., 2011; You et al., 2011; Tsopmo et al., 2011), antihypertensive (Contreras et al., 2011; Ryan et al., 2011; Ramchandran and Shah, 2011), antidiabetic (Anderson et al., 2002; Yuan et al., 2008; Lu et al., 2011; Shen et al., 2012) and immunomodulatory activities (Qian et al., 2011; Agyei and Danquah, 2011; Chu et al., 2011; Bonomi et al., 2011), by following either in vitro or in vivo assessments (Takai-Doi et al., 2009; Nakahara et al., 2010; Samaranayaka and Li-Chan, 2011). It is well known that food proteins are the main starting materials for the generation of biopeptides which mostly have 2–20 amino acids (Meisel and FitzGerald, 2003) and molecular masses of less than 6000 Da (Sun et al., 2004). Biopeptides can be incorporated, suspended and dispersed or encapsulated into different forms such as emulsions, liposomes, nutraceuticals, and other edible biopolymers to gaining their optimum functionality, bioavailability, stability and targeted effectiveness (Amar-Yuli et al., 2010; Livney, 2010; Patel and Velikov, 2011; Elzoghby et al., 2012). In fact, these strategies are given in order to protect the biological functionality of biopeptides during their transportation from the enterocyte domain (e.g. stomach and small intestine) into portal circulation (blood domain) without any denaturation or physical deformation. During the hydrolytic digestion by different proteases like pepsin, trypsin, chymotrypsin, and pancreatin, these active fragments are liberated from the degradable protein substrates. Being active compounds they start to interact with their preferred transporter agents based on afﬁnity parameters such as size, stereochemical structure, functional groups, and charges (Gardner, 1984). Under these interactions, the biopeptides are recognized and retained easily in their intact form in the portal circulation by the intervention of speciﬁc transporter agents. In addition, they can penetrate under particular modes of transmission such as paracellular routes (Gardner, 1988; Miguel et al., 2008), passive diffusion (Shimizu et al., 1997; Ziv and Bendayan, 2000; Satake et al., 2002), endocytosis (Gardner, 1988; Ziv and Bendayan, 2000), and lymphatic system (Deak and Csáky, 1984; Rubas and Grass, 1991). Owing to their structural characteristics and their amino acid residues positioned on the N-terminal and C-terminal, biopeptide fragments might exhibit natural bioresistance “shelf-life predictions”(Bell, 1997) under gastrointestinal tract conditions (e.g. acids and enzymes). This natural resistance is important for biopeptides to ensure maximum bioavailability when they reach to the blood domain. Therefore, it helps the biopeptides to contribute signiﬁcantly towards various biological functions, including opiate-like (Sienkiewicz-Szlapka et al., 2008), antihypertensive (Jia et al., 2010), antimicrobial (McCann et al., 2006), mineral binding (Cross et al., 2005), antithrombotic (Shimizu et al., 2008), immunomodulatory (Gauthier et al., 2006), hypocholesterolemic (Zhong et al., 2007) and antioxidative (Mendis et al., 2005a, 2005b) activities, while some of the peptide fragments could exhibit more than one biological function (Meisel and FitzGerald, 2003). Biopeptides, which are getting much attention among researchers, due to their high value compounds which exhibit an excellent nutritional platform has made them as an intermediate substances for different sectors like dairy industrial (Choi et al., 2012), nutraceuticals (AmarYuli et al., 2010) as well as therapeutic and vaccination (Zhang and

Introduction

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Production of biopeptides

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Production of biopeptides with enzymatic hydrolysis

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The enzymatic hydrolysis is the efﬁcient treatment destined for the production of biopeptides from proteins and it can induce the bioactivity of intact proteins as well. Milk proteins are an important source in 218 generating biopeptides with different biological activities including an219 tioxidative, antihypertensive, antimicrobial and immunomodulating 220 properties. Angiotensin converting enzyme's inhibitory activity is one 221 of the biological functions that may exert protective effects towards 222 human organisms by protecting them from several cardiovascular dis223 eases. The ﬁndings from the present study suggest that casein-derived 224 peptides may exert speciﬁc antioxidant and immunomodulatory effects 225 Q35 on cells in culture (Phelan et al., 2009). Bougatef et al. (2009) investigat226 ed the antioxidative activity of ﬁve hydrolysates from smooth hound 227 (Mustelus mustelus) meat obtained by various gastrointestinal prote228 ases: crude enzyme extract, low molecular weight (LMW) alkaline pro229 tease, and trypsin-like protease from M. mustelus intestine, pepsin from 230 M. mustelus stomach, and bovine trypsin. Results showed that the ﬁve 231 hydrolysates showed different degrees of hydrolysis and varying de232 grees of antioxidant activity. The incorporation of protein hydrolysates 233 into foods has been increasingly applied in the food industry. The for234 mulation of new food products by incorporating biopeptides as ingredi235 ents is of great interest for human organisms. The beneﬁt of this 236 technology is to enhance the daily diets of humans by supplementing 237 biological systems capable of regulating body functions. The physiolog238 ical effects of biopeptides in regulating blood pressure have prompted 239 the scientists to exploit different food materials such as ﬁsh, meat, 240 milk, eggs, beans, and plants. For instances, goby is a kind of ﬁsh that

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The production of biopeptides using microbial fermentation has drawn much of the dairy industry's attention due to their proteolytic activity as starter cultures. Milk products remain the captivating materials in generating potent biological peptides by the intervention of particular microbial proteases (Korhonen, 2009). Many examples are elaborated on the potentiality of microbial fermentation in the production of dairy products such as commercial probiotic bacteria, yogurt bacteria, and cheese starter bacteria, all these examples being the outcomes of the process of fermentation (Gómez-Ruiz et al., 2002). Milk based protein hydrolysates with an antihypertensive were produced by using 20 potential dairy yeast strains belonging to Kluyveromyces marxianus, Kluyveromyces lactis and Debaryomyces hansenii species (GarcíaTejedor et al., 2013). Another study has reported the use of a mixture of commercial starter culture of LAB strains during fermentation process and demonstrated that the angiotensin-converting enzyme-inhibitory activity is increased for the hydrolysates after subsequent hydrolysis using a microbial proteases (Chen et al., 2007). Interesting observations during secondary proteolysis of cheese ripening have been found to be dependent between the ripening stage of the cheese and the release of the biopeptides (Korhonen, 2009). The beneﬁcial roles of biopeptides that were obtained from milk products are considered as promising candidates in promoting various health functions such as bone functions, heart functions, controlling the stress, digestive systems, improving the immune defense and reducing the risk of obesity and development of type two diabetes (Zimecki and Kruzel, 2007).

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These types of biopeptides usually have an internal origin. They are secreted with particular routes either by neurons granules or from endocrine cells (Sasaki and Minamino, 2013). Much information has been elaborated on the generation of endogenous peptides that can occur under intrinsic proteolytic processes. Comprehensive peptidomics are required due to the limitation in the characterization of peptide hormones or neuropeptides. Biopeptides are considered as captivating biomarkers in the living systems (cells, tissue, and body ﬂuids) particularly in the recognition of certain maladies and diseases such as renal diseases, cancers, oral diseases and other types of diseases (Gruson and Bodovitz, 2010; Ling et al., 2010; Quintana et al., 2009; Pietrowska et al., 2012; Shen et al., 2010). The identiﬁcation and characterization of endogenous peptides have proposed many challenges and limitations, particularly in their domain efﬁcacies and their dynamics behaviors.

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The common approaches used in the production of biopeptides are 198 Q34 presented in the following sections. 199

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can be exploited for deriving biopeptide based hydrolysates. In this way, Nasri et al. (2013a, 2013b) examined goby proteins for the production of protein hydrolysates with potent angiotensin-converting enzyme inhibitory activities and antioxidative activities using gastro-intestinal proteases. They revealed that the biological function of goby protein hydrolysates are probably changes due to the hydrolysis time, peptide chain lengths, and types of amino acids. In another part of the research, which had been published recently Nasri et al. (2012) used same raw materials to target the anticoagulant activities. Results showed a signiﬁcant prolongation of both thrombin time (TT) and the activated partial thromboplastin time (APTT). Therefore, goby protein hydrolysates could be utilized to develop functional foods for preventing thrombosis.

Falla, 2006) sectors. Nowadays, food biopeptides as alternative natural ingredients and as a key regulatory of human organisms are becoming 183 the main target of researchers due to their capability to participate in 184 the same fashion as exhibited by synthetic drugs. Therefore, there is a 185 real need to focus and present a visible relationship between biopeptide 186 structure and their biological functionalities such as antioxidative, anti187 Q33 hypertensive and immunomodulatory functions, with particular atten188 tion on the beneﬁcial roles of proteolytic enzymes that facilitate the 189 production of these actives fragments (biopeptides). In fact, this review 190 was mainly framed to explore the recent biotechnological advances in 191 biopeptide production. Besides, the mode of action, biological and me192 dicinal health beneﬁts, and therapeutic applications of biopeptides are 193 covered. Efforts have also been made to discuss the modern strategies 194 that can improve and/or protect the efﬁcacy, bioavailability, and func195 tionality of these biomaterials.

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Biopeptide generation during in vitro and in vivo proteolytic 279 digestion 280 The emergence of a therapeutic and vaccination area helped the researcher to explore their ﬁndings in declining the risk effect of many diseases and maladies with least systemic effects. Several applications of bioactive peptides in the area of food, biotechnology, medicine, pharmacy have used robust protocols either at molecular or cellular levels to investigate many physiological and biological concerns related to human health and nutrition. Furthermore, the rapid screening and quantiﬁcation of the bioactive compounds as natural supplements and health protectants has drawn much of the researchers' attention due to their pharmacological interest. The resulting new bioactive compounds are highly dependent on selected starting material, mode of cultivation, techniques, and treatments used before the extraction, separation, and preparation of the ﬁnal samples. The optimization of the extraction process is an important step in the production of high yield of targeted compounds. In the current review more emphasis is given to the enzymatic hydrolysis due to its advantages including high yield, efﬁcient process and time saving treatment and it can be scaled easily in the laboratory. However, it is a recommended techniques and safe method during manipulation tests, because it does not leave residual organic solvents and excess of toxic substances resulting from the secondary metabolites during long fermentation process of foods (Brink & Huisin't Veld, 1999). Owing to their low molecular weight, amino acid sequences and less

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explains the degradation behavior of proteolytic enzymes before and after enzymatic hydrolysis. In addition, the enzymatic hydrolysis in the presence of a particular enzyme has shown a direct inﬂuence on the hydrophobicity content of amino acids generated in the reaction medium, which may give an additional advantage for this reaction mechanism (Moure et al., 2005). However, the continuous removing of the peptide bonds from the long chain of polypeptide under enzymatic hydrolysis has shown a beneﬁcial role in the induction rate of carboxyl and free amino groups, which in turn induced the solubility of active fragments. Depending on the molecular weight and type of protein precursor involved in the propagation of biopeptides, a hydrolysis mechanism can be used to predict their hydrophobicity rate (Calderón de la Barca et al., 2000). Several studies have revealed the important role of enzymatic hydrolysis in generating short biopeptides (lower molecular weight of less than 1000 Da (Kristinsson and Rasco, 2000; Moure et al., 2005)), with signiﬁcant biological potency (Cho et al., 2004). While, the study of Kristinsson and Rasco (2000) demonstrated that the extensive enzymatic hydrolysis might adversely inﬂuence the biological function of the isolated biopeptides. Table 1 lists the main enzyme groups that can be used to generate biopeptides. The efﬁcient role of these enzymes depends on their efﬁcacies, speciﬁcity, and other properties including molecular mass, polarity, and chargeability. Each enzyme is responsible for the generation of different fragments of peptides ranging between low, middle and high molecular weight. These enzymes can work either alone or in the presence of other enzymes, multiplying their mechanism of action under continuous breakdown of amino bonds, and therefore, increasing the chances of liberating further small active fragments (Smith, 1977). Emphasis should be given to highlight the most efﬁcient enzymes capable of attacking the protein complex, causing it to become a mixture of small

complex structure compared to that of protein, peptides (e.g. polypeptides) behaviors are predictable in unusual fashion in terms of absorption, 305 digestibility, and solubility afﬁnities (Yuan and Kitts, 1991; Vegarud et al., 306 2000; Kitts and Weiler, 2003). However, the modiﬁcation process of these 307 bio fragments by using enzymatic methods is preferred in order to im308 prove their resistance, and bioavailability. Basically, the enzymatic hydro309 lysis that occurs by several stimulated substances originated from 310 different sources like the gastrointestinal tract, plants, and microorgan311 isms as well as microbial fermentation in multiple pathways, has shown 312 signiﬁcant impact on the biological diversiﬁcation of many protein pre313 cursors, which in turn, helped to contribute signiﬁcantly to the occurrence 314 of these multifunctional peptide fragments (Korhonen and Pihlanto, 315 2006). For instance, fermentation technique, as one of the common 316 means of food preservation, especially in South East Asian countries 317 such as Malaysia, China, Japan and Korea, is considered to be the appropri318 ate option that might augment further the fragmentation mechanism of 319 protein into a small active peptide under favorable conditions of microbial 320 Q42 proteases (Rajapakse et al., 2005a, 2005b, 2005c). 321 Typically, to improve and modify the physiological functions of pro322 teins/peptides, the enzymatic hydrolysis is the best technique and in 323 most of the cases it needs to be controlled under speciﬁc operational con324 ditions (Calderón de la Barca et al., 2000; Yim and Lee, 2000; Moure et al., 325 2005). For example, enzymatic digestion of β-conglycinin and glycinin 326 using proteolytic enzymes has been found to successfully increase their 327 inhibition potent against oxidation supporting their use as potential 328 sources of antioxidants and functional food ingredients (Saito et al., 329 2003). This behavior might have been attributed to the fact that under 330 a spontaneous hydrolysis mechanism, the more active amino acids pres331 ent on the lateral chain sequence of biopeptides have better biological 332 activity (Matoba, 2002). The following schematic diagram (Fig. 1)

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Fig. 1. Schematizes the protein mass before (step 1: enzymes are in the active state) and after (step 2: deactivation of enzymes after complete hydrolysis) enzymatic hydrolysis; this ﬁgure permitted the understanding of enzyme behavior on protein biodegradation and their aggregation and assemblage on lateral surface of the attacked substrate. Once these enzymes are subjected to the optimal conditions of pH, temperature, and required doses, their reaction mechanism commence, helping them to penetrate after further incubation times, ensuring the highest enzyme activity, which in turn their endo-action begin to be more pronounced.

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Isolation and puriﬁcation of bioactive peptides

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The isolation and puriﬁcation techniques are the common means used in bioactive peptide production. For instance, the glutathione has been introduced in the research of functional foods since 1888, whereas its therapeutic application started to be effective after the 1950s. The emergence of robust technologies of puriﬁcation and separation science of bioactive systems starts to enforce the research area of bioactive peptides to be more pronounced and mechanistic (Sewald and Jakubke, 2002). Many of the applied procedures of protein puriﬁcation that can lead to appropriate separation for obtaining the exact bioactive peptide fragment that is destined to suppress, decline or modulate particular

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maladies and diseases have been introduced but not fully appreciated. Therefore, particular attention should be made and many of the consideration should be taken into account on the conditioning methods that is proposed for the puriﬁcation of bioactive peptides. Typically, the variability in molecular weights, charges, afﬁnity of bioactive peptides during separation and puriﬁcation caused many obstacles and because of these barriers and challenges related to the purity of peptides the scientists' proposed robust tools and advance instrumentation for solving these encountered issues. Among these tools we have as an example the size-exclusion chromatography (SEC) that is known with its limitation in terms of the resolving power (Sewald and Jakubke, 2002). Among the main different techniques that are used to extract the protein are solvent extraction and also by using different buffer saline systems. One of the simplest example on the mobile phase that is used to purify peptides from fermented protein hydrolysates is a mixture of the three elements including triﬂuoroacetic acid (TFA), water, and acetonitrile (Wang and de Mejia, 2005). Several steps were followed accordingly starting by centrifugation and then the ﬁltration of the supernatant and ﬁnally lyophilization of the sample for further liquid chromatography puriﬁcation and separation. For instance, HPLC is the common tool in the separation of bioactive compounds. However, the advance technology permitted the fast separation of these compounds by using RP-HPLC columns that facilitate the separation between the detected events particularly from a mixture of protein hydrolysates. In contrast the application of normal HPLC is preferred in case when the targeted bioactive peptides are hydrophilic peptides. Other chromatographic techniques including capillary electrophoresis (CE), capillary isoelectric focusing (CIEF), and ion-exchange chromatography (IEC) could reach to separate peptides based on their chargeability afﬁnities either positively or negatively charged bioactive peptides (Wang and de Mejia, 2005). In order to separate the peptides based on molecular weight in aqueous separation systems the size-exclusion chromatography (SEC) named as well as gel ﬁltration chromatography (GFC) is recommended, while for the non-aqueous separation systems the preferred one is the gel-permeation chromatography (Wang and de Mejia, 2005). The gelpermeation chromatography can be used to separate peptide fraction with different molecular sizes from buckwheat digestion (Li et al., 2002). Other techniques of separation and fractionation that can be employed in the puriﬁcation trajectory are ultraﬁltration, crystallization, and partition chromatography (Sewald and Jakubke, 2002). Various modes of separation and puriﬁcation processes have been done on different biomaterials. For instance, Gibbs et al. (2004) reported the generation of different peptides from various fermented food sources digested by a variety of enzymes including plasma proteases, pronase, Glu C protease, trypsin, and kidney membrane proteases. The peptides after that were subjected to further puriﬁcation and biochemical characterization. The generated bioactive peptides exhibited several levels of biological activities. As a result, the glycinin expressed high biological activity, followed by pronase, kidney membrane proteases and ﬁnally plasma protease digests of different fermented foods. They demonstrated that the speciﬁcity of the enzymes plays a key role in the presence of the targeted oligopeptides. They noticed that the proteases having lower speciﬁcity generate more oligopeptides and a higher yield of active fragments than the enzymes having vast speciﬁcity such as Glu C and trypsin. The pronase digest peptide of natto showed the highest ACE inhibitory activity followed by the kidney membrane hydrolysate. Further, an antithrombotic activity was observed by a peptide having same structure analogues of the hirutonin as described by DiMaio et al. (1992). More emphasis should be spent on the mechanism of action of bioactive peptides and its relationship to the biological activities within the living system. For example, Kuhn et al. (1993) investigated the guanylin one of the active peptide that is isolated from rat intestine and found to have a stimulating effect on the intestinal guanylate cyclase. They mention that this type of peptide was puriﬁed from the circulating guanylin of the human hemoﬁltrate. The identiﬁcation of the sequence concerning this peptide starting from the N-terminal showed 47 amino acid residues.

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fragments. These short peptides are preferred due to their stability and resistance under gastrointestinal tract conditions, and easy to be carried 365 by speciﬁc precursors, with low transmission energy (Windisch et al., 366 2005). Several studies indicated that peptides are not the only bioactive 367 compounds that can contribute signiﬁcantly to the decrease of the oxi368 dation risks, resulting from the free radicals; proteins themselves have 369 shown great impact in scavenging reactive oxygen species, minimizing 370 the hydroperoxides, cleaving speciﬁc oxidants by enzymatic interven371 tion, and chelating the preoxidation resulting from metal transition 372 (Elias et al., 2008, 2006; Diaz and Decker, 2005; Kong and Xiong, 373 2006). In addition to their special structure and high molecular mass 374 proteins are more fragile and unstable under gastrointestinal tract con375 ditions. Therefore, they cannot participate and act in the same fashion or 376 manner as are displayed by biopeptides. It is noticed from Table 1 that 377 pepsin, trypsin, chymotrypsin, thermolysin, and alcalase are the efﬁ378 cient enzymes in producing various active fragments. The degradation 379 action of these enzymes helped the generation of various biopeptides 380 having antioxidative, antihypertensive or immunomodulatory biologi381 cal functions (see Table 1). Several studies examined the hydrolysis of 382 protein mass by either using individual enzymes or combined enzymes 383 (Rokka et al., 1997; Mendis et al., 2005b; Saiga et al., 2006; Sauveur 384 et al., 2008; Jimsheena and Gowda, 2011). Typically, this strategy is use385 ful in enhancing the production of biopeptides according to the speciﬁc386 ity of enzymes used. It means the combination of more enzymes by 387 taking into account that the optimum conditions of each enzyme 388 might help to generate different biopeptide fragments having different 389 amino acids on the N-terminal and C-terminal positions. This type of 390 work has been remarked particularly in case of combined enzymes hav391 ing the same physiological working conditions like trypsin and chymo392 trypsin (Rokka et al., 1997; Saiga et al., 2006; Sauveur et al., 2008). 393 Normally, in any application mode of enzymatic hydrolysis certain 394 limitations can be raised (Xie et al., 2008). For instance, the biological 395 function of protein can be affected by proteases, having speciﬁc dose, 396 and molecular mass. Thus, they can facilitate its mode of action towards 397 peptides, provoking signiﬁcant variation in peptide prolongation and 398 chemical composition of amino acids situated on the N-terminal and C399 Q43 terminal of the sequence chain (H.M. Chen et al., 1995; J.R. Chen et al., 400 1995; Jeon et al., 1999; Wu et al., 2003). A speciﬁc study on enzymatic 401 hydrolysis, as reported by Peña-Ramos and Xiong (2002), indicated 402 that the utilization of different enzymes for producing hydrolysates 403 from native and heated soy protein yielded a mixture of peptides having 404 variable degrees of hydrolysis, which permitted ﬁnally the initiation of a 405 varying magnitude of antioxidant activity accordingly to different ranges 406 of the obtained hydrolysis level. It is remarkable that the efﬁcacy of 407 alcalase during enzymatic hydrolysis is better than hydrolysates digested 408 Q44 by other enzymes (Park et al., 2001; Qian et al., 2008a, 2008b). Further, it 409 is suggested that this enzyme can give more peptide yield and abun410 dance of short fragments in comparison to other proteases. Hence, the 411 resulting active fragments have been found to be more deﬁant to proteo412 Q45 lytic enzymes (Kim et al., 2001a, 2001b, 2001c; Park et al., 2001). Similar 413 behavior and same catalytic regime are exhibited by other proteolytic 414 enzymes, demonstrating a wide range of biological functions such as an415 tioxidative, antihypertensive and immunomodulating (see Table 1).

363 364

427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 Q46 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492

t1:3

Table 1 Main ﬁndings and observations noted during in vitro and in vivo generation of biopeptides. Enzyme used

t1:4 t1:5

Q1 Protease S “Amano”

t1:6

Q2 Proteolytic digest

t1:7 t1:8

(Bacillus subtilis)

Hydrolysates

Identiﬁed peptides

Techniques and biological properties

Observations/recommendation

Egg white albumin hydrolysates

P1: Ala-His-Lys, P2: Val-His-His, P3: Val-His-His-Ala-Asn-Glu-Asn, P1: Val-Lys-Leu P2: Val-Val-Lys-Leu P3: Val-Lys-Val P4: Pro-Lys-Ala-Val P5: Ile-Lys-Leu P6: Val-Pro-Ser-Gly-Lys/P7: Glu-Ala-Lys P8: Phe-Val-Ala-Gly-Lys/P9: Lys-Ala-Ile P10: Lys-Val-Ile P11: Lys-Asp P1: Val-Tyr P2: Ile-Tyr P3: Ala-Trp P4: Phe-Tyr P5: Val-Trp P6: Ile-Trp P7: Leu-Trp P1: Val-Asn-Pro-His-Asp-His-Gln-Asn P2: Leu-Val-Asn-Pro-His-Asp-His-Gln-Asn P3: Leu-Leu-Pro-His-His

The antioxidative property was conducted in vitro, where for P1 is the strongest, whereas for P2 and P3 is about ½ of the antioxidative displayed by P1. The antioxidative peptides from dried bonito protein hydrolysates were fractionated by ion-exchange chromatography and gel ﬁltration coupled by ODS HPLC for further separation.

This study has shown that the antioxidative property of Nobuaki et al. the hydrolysates was attributed to the chelating activity (1991) of their active peptide fragments. Insights in the structure–function relationship of the 11 Suetsuna (1999) peptides were exhibited based on their amino acid in the sequences, molecular weight, charges, and hydrophobicity properties.

The antihypertensive activity was conducted in vivo after a single oral administration of a dose of 1 mg/kg of body weight (BW) in spontaneously hypertensive rats (SHR).

Among these peptides P1, P2, P4, and P6, they showed signiﬁcant decrease in the blood pressure. These peptides exhibited a natural resistant to gastrointestinal proteases in vitro.

Dried bonito protein

Protease S “Amano” (from Bacillus stearothermophilus)

Hydrolysates of wakame (Undaria pinnatiﬁda)

t1:9 Q3 Protease S “Amano” t1:10 from Bacillus sp.

t1:11 t1:12 t1:13

Hydrolysates prepared from soybean protein, β conglycinin (7S protein) Alcalase 2.4L (EC.3.4.21.62, Insoluble soy protein (InSP) hydrolysates from Bacillus licheniformis 2.4 AU/g).

t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21

Orientase 90 N an endopeptidase prepared from B. subtilis. Protease XXIII an endopeptidase prepared from A. melleus Protease XXIII, from Aspergillus oryzae

Protein hydrolysates

Tuna dark muscle by-product hydrolysates

P1: Leu-Pro-Thr-Ser-Glu-Ala-Ala-Lys-Tyr (Orientase 90 N). P2: Pro-Met-Asp-Tyr-Met-Val-Thr (Protease XXIII).

Tuna cooking juice hydrolysates

The peptide sequences comprised four to eight amino acid residues, including Val, Ser, Pro, His, Ala, Asp, Lys, Glu, Gly, or Tyr.

t1:22 Q4 Crude enzyme extract t1:23 from sardine t1:24 (Sardina pilchardus)

Hydrolysates of sardinelle (Sardinella aurita) protein by-products

t1:25

Sea bream scales hydrolysates

P1: Leu-His-Tyr* P2: Leu-Ala-Arg-Leu P3: Gly-Gly-Glu P4: Gly-Ala-His P5: Gly-Ala-Trp-Ala P6: Pro-His-Tyr-Leu P7: Gly-Ala-Leu-Ala-Ala-His P1: Gly-Tyr P2: Val-Tyr P3: Gly-Phe P4: Val-Ile-Tyr

Alkaline protease

Peptides of P1, P2, and P3 in this study are expected to be All of the antioxidative peptides isolated from β-conglycinin contained a proline residue in the sewith high antioxidative properties in the unpuriﬁed quences. It is suggested that the variation in the antioxihydrolysates in spite of their low yields. dative may be due to puriﬁcation steps.

Immunomodulating properties were examined by following the phagocytic activity using peritoneal macrophages and proliferation of murine spleen lymphocytes. The antioxidative property was evaluated in vitro, and then the hydrolysates were subjected to further puriﬁcation and fractionation using different chromatographic techniques such as GFC and HPLC.

References

Sato et al. (2002)

H.M. Chen et al. (1995), J.R. Chen et al. (1995)

Lower molecular weight and positively charged peptides Kong et al. (2008) were effective in stimulating the immunomodulating activity. This study has shown that both hydrolysates obtained by Hsu (2010) orientase 90 N and protease XXIII from tuna dark muscle by-product could be used to produce natural antioxidative agents. The use of OR and PR would shorten the hydrolysis time as well.

R O

After the solid content of the hydrolysates was concentrated from one to 5 times the antioxidative activity becomes strong for 2 fractions (B and C) out of 6 fractions (A, B, C, D, E, and F). Seven anti-oxidative peptides (mixture of B and C) were isolated from the hydrolyzates by reversed-phase HPLC. The hydrolysates was fractionated by size exclusion chromatography, then subjected to further fractionation and puriﬁcation using reverse-phase high performance liquid chromatography (RP-HPLC).

The crude hydrolysates showed the highest 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging effect. This study has shown that tuna cooking juice hydrolysates is a promising source for deriving antioxidative ingredients.

Jao and Ko (2002)

The ﬁrst peptide showed high antioxidative activity (e.g. DPPH radical-scavenging activity) among other peptides. Authors suggest that sardinelle hydrolysates are good source of natural antioxidants.

Bougatef et al. (2010)

The anti-hypertensive effect of the peptides isolated from protein hydrolysates by chromatographic methods and was assessed by following ACE inhibitory activity in vitro and blood pressure in vivo in spontaneously hypertensive rats (SHR). A single oral administration of 300 mg/kg of BW has shown to be able in lowering the blood pressure signiﬁcantly (p b 0.05).

Results indicated that the presence of amino acid Tyr at the C-terminal position of the peptides contributed signiﬁcantly to ACE-inhibitory activity. This study showed that sea bream scales hydrolysates is a potential source for generating potent biomaterials (biopeptides) able to impair particular diseases such as hypertensions.

Fahmi et al. (2004)

S. Saadi et al. / Biotechnology Advances xxx (2014) xxx–xxx

t1:1 t1:2

Protease from Bacillus sp. SM98011

Marine shrimp (Acetes chinensis)

P1: FCVLRP P2: IFVPAF P3: KPPETV

t1:27 t1:28 t1:29

Bromelain (from pineapple) and alcalase (from Bacillus licheniformis)

Hydrolysates of body wall protein of sea cucumber (Acaudina molpadioidea)

P1: MEGAQEAQGD

The hydrolysates displayed both of antioxidative and antihypertensive activities. Three out of ﬁve peptides were puriﬁed from the hydrolysates by following ultraﬁltration, gel permeation chromatography, and RP-HPLC. The hydrolysates were fractionated using gel ﬁltration, ion-exchange chromatography, and RP-HPLC. The peptide was veriﬁed in vitro by assessing the ACE-inhibitory activity. In addition to its stability under gastrointestinal proteases and then conﬁrmed in vivo via an oral administration of a dose of 3 μg/kg in SHR.

t1:30 t1:31 t1:32 t1:33 t1:34

Proteinase K from Tritirachium album and thermolysin from Bacillus thermoproteolyticus rokko

Haruan (C. striatus) myoﬁbrillar protein

P1: VPAAPPK P2: NGTWFEPP (Both from thermolysin)

t1:35 t1:36 t1:37 t1:38

Thermolysin + proteinase A, trypsin, proteinase K, tyrosinase, pepsin, papain, and protease

Beef sarcoplasmic protein hydrolysates

P1: GFHI (proteinase K) P2: DFHING (alcalase) P3: FHG (thermolysin + proteinase A) P4: GLSDGEWQ (Thermolysin + proteinase A)

t1:39

t1:40 t1:41

Alkaline protease

Sardine muscle

P1: Lys-Trp P2: Arg-Val-Tyr P3: Met-Tyr

Lun et al. (2006)

This study revealed that novel peptides are identiﬁed from marine shrimp (Acetes chinensis). These types of hydrolysates are possibly capable to induce an antihypertensive effect in vivo after intact digestion.

The hydrolysates and puriﬁed peptides showed a good antihypertensive activity both in vitro and in vivo. It is suggested the ACE-inhibitory activities was varied based on the MW distribution, whereas the fractions with low MW displayed potent ACE-I activity. Promalein and alcalase contributed to high degree of hydrolysis. Ultraﬁltration was efﬁcient in enhancing the ACE inhibitory activity of sea cucumber. It was observed that the presence of proline residue at The hydrolysates were fractionated by using the lateral side of the peptide (C-terminal position) is ultraﬁltration and size exclusion chromatography. The responsible for the ACE inhibitory activity. This study fractions that showed high ACE inhibitory activity was subjected to RP-HPLC and then the best fraction in terms suggested that this type of protein material has the ability to generate a potent biopeptides to decrease of ACE activity was sent to electrospray blood pressure. ionization-time-of-ﬂight mass spectrometry (ESI-TOF MS/MS). P4 at a dose of 100 ppm showed to inhibit E. coli, Bacillus The authors expected that the ACE inhibitory peptides derived from cereus, Salmonella, and Listeria monocytogenes but did beef sarcoplasmic protein not show any effects on Pseudomonas aeruginosa, and hydrolysates have displayed Staphylococcus aureus. both of antimicrobial and cancer P1 showed a slight reduction in breast cancer (MCF-7) cell viability and signiﬁcant decreasing (75%) in stomach cell cytotoxic effect. The key parameters for peptides cancer (AGS) cell at applied dose of 400 μg/ml. It structure and the mechanism effect of stimulates the macrophage for the production of nitric immunomodulating have not oxide (NO). P2 played a role as nutrient to enhance SGS cell viability. yet deﬁned in this study. The cytotoxic effect was negative for all types of peptides on lung cancer (A549) cells. This study revealed that all the isolated Out of eleven peptides screened for ACE-inhibitory peptides showed a competitive inhibition peptides only P1 displayed the maximum inhibitory activity. Furthermore, the sequence hom*ology indicated towards ACE. In contrast, P3 showed non-competitive inhibition towards ACE. that P2 was found in the primary structure of Thus sardine muscle may use as potential angiotensin I, II and III. protein source for deriving a potent ACE-inhibitory peptides. The active hydrolysates were subjected to ultra-ﬁltration The results suggested that the peptides derived from bovine casein hydrolysate are promising and the resulting permeates (3 kDa) revealed high ACE candidates in treating cardiovascular diseases by inhibitory activity in vitro. incorporating them into food and pharmaceutical The hydrolysates were examined in products as vivo and displayed an antihypertensive effect with a antihypertensive agents. signiﬁcant decreasing of the systolic blood pressure in SHR after repeated oral administration of the hydrolysate. The hydrolysates showed high ACE Antihypertensive peptides were isolated from this inhibitory activity. All the peptides material by following ultra-ﬁltration, have hydrophobic amino acid residue gel ﬁltration, and RP-HPLC. (aromatic or branched side chains) F or Y at the C-terminal position which they are contributed signiﬁcantly to ACE inhibitory activity. This study suggested that shark meat hydrolysates might have potential utilization both in food application and clinical nutrition associated to human health.

Zhao et al. (2009)

Ghassem et al. (2011).

Jang et al. (2008)

Matsufuji et al. (1994)

AS1.398 neutral protease (from Bacillus subtilis)

t1:42 Q5 Protease SM98011 was t1:43 extracted from the t1:44 culture of Bacillus sp.

Bovine casein hydrolysate (bovine milk)

Shark meat hydrolysate (marine protein)

P1: Arg-Tyr-Pro-Ser-Tyr-Gly (κ-casein; f25–30) P2: Asp-Glu-Arg-Phe (κ-casein; f15–18)

P1: Cys-Phe P2: Glu-Tyr P3: Met-Phe P4: Phe-Glu

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S. Saadi et al. / Biotechnology Advances xxx (2014) xxx–xxx

t1:26

Jiang et al. (2010)

H. Wu et al. (2008), J. Wu et al. (2008)

(continued on next page)

Enzyme used

Hydrolysates

Identiﬁed peptides

t1:45 t1:46 t1:47 t1:48

Aspergillus protease followed by trypsin/chymotrypsin and small intestinal juice

Chicken breast muscle hydrolysates

t1:49 t1:50 t1:51 t1:52

Aspergillus protease followed by trypsin/chymotrypsin and small intestinal juice

Chicken breast muscle hydrolysates

t1:53

Peptic digest

Sardine muscle hydrolysates

Saiga et al. (2003) The relationship between the N-terminal of peptide structure and ACE inhibitory peptides is still going on and remain ambiguous. It has been noticed that by replacing Pro for Hyp of P1 there is a diminution in the activity by 10-fold, meaning that Hyp with its hydroxyl group may exert a pivotal role towards the catalytic site of ACE, resulting in strong binding capacity of peptides to enzyme. Saiga et al. (2006) P1 was synthesized to be designed as P4: After conducting the in vivo study using SHR and getting The author suggested that the presence of Phe as an Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp- high ACE inhibitory activity (previous study) the author aromatic residue at the C-terminal position of the peptide fragment was able to enhance the ACE inhibitory in this 2nd part of study were tried to elucidate the Gly-Phe mechanism of action in between active peptide and ACE activity, meaning that Phe is considered as the key After an in vivo decomposition of P4 a enzyme in vivo. They noticed that after decomposition of parameter in peptide structure–function relationship. novel peptide the original fragment P4 the novel fragment P5 displayed was generated strong activity. P5: Hyp-Gly-Leu-Hyp-Gly-Phe This study has shown that sardine muscle can be used to Suetsuna and Ukeda P1: Leu-Gln-Pro-Gly-Gln-Gly-Gln-Gln. The hydrolysates were evaluated for an antioxidative (1999) generate bioactive peptides having an antioxidative property in vitro by following superoxide scavenging property, so this biomaterial can be applied to slow activity and hydroxyl radical scavenging assays. down certain diseases such oxidative stress. The peptidic fraction was puriﬁed using ion-exchange chromatography, gel ﬁltration and further isolation was conducted using HPLC. Suetsuna et al. The hydrolysates exhibited highest superoxide anion P1: Tyr-Phe-Tyr-Pro-Glu-Leu Antioxidative peptides have been (2000) scavenging activity (SOSA), DPPH radical scavenging successfully separated from the activity. This study suggested that dipeptides could be hydrolysates of casein protein. The hydrolysates were puriﬁed by using a series of chromatographic techniques easily penetrated into portal circulation via speciﬁc including ion exchange, gel ﬁltration and octadecylsilano dipeptides-transporter. (ODS)-HPLC. Tsopmo et al. Results indicated that moderate antioxidative activity P1: HNPI The milk hydrolysates were ultra ﬁltrated, separated, (2009) was displayed by P1, whereas P2 did not show any P2: PLAPQA and analyzed for antioxidative peptides in vitro. The scavenging effect. The addition of Try into infant formula following three samples were examined for milk increased the antioxidative property. Trp is a potent antioxidative activity including non-digest milk, completely digested milk, and derived ultra-ﬁltrates. An free radical scavenger that has the ability to reduce HPLC was used to obtain the active peptides (P1 and P2) oxidative stress. from the derived ultra-ﬁltrate (best sample). P1: Asp-Val-Cys-Gly-Arg-Asp-Val-AsnThe hydrolysates digested by pepsin enzyme showed the This study has shown that P1 able to protect DNA against S.-H. Lee et al. H2O2 induced DNA damage. This peptide showed Gly-Tyr highest antioxidative activity among other types of (2010), S.-J. Lee hydrolysates. HPLC was used to purify the selected compatible antioxidative activity of vitamin C, thus P1 is et al. (2010) peptide P1 and electron spin resonance (ESR) a potent fragment possessing an antioxidative activity. spectrometer was applied to determine the hydroxyl radical scavenging activity. Je et al. (2004) This study revealed that the selected peptide P1 was P1: Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala Angiotensin converting enzyme inhibitory activity was investigated in vitro. The hydrolysates were fractionated found to act non-competitively towards the active site of ACE enzyme. into 5 fractions using an ultra-ﬁltration membrane. The This study suggested that peptides structure activity best hydrolysate showing high activity was further relationships still not fully established because a large puriﬁed using chromatographic number of peptides having different C-terminal were techniques. observed. J. Wang et al. P1: Oyster protein hydrolysate was examined in Results showed good antihypertensive activities both Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe vitro using ACE inhibitory activities and in vitro and in vivo. The peptide showed a good stability (2008), J.P. Wang et al. (2008) further separated using chromatographic techniques. against heat, pH, and gastrointestinal proteases. Kinetic The stability for ACE inhibitory activity was assessed study revealed that the mechanism of action of the under controlled conditions of heat, pH, and resistance peptides was in the non-competitive manner towards against gastrointestinal proteases. The the active site of ACE enzyme. hydrolysate (FII) was veriﬁed in vivo via an oral administration of 20 mg/kg hydrolysate dose in SHR.

Hydrolysate of casein derived milk protein

t1:56

Pepsin P-7000, pancreatin P1750

Human Milk

t1:62 Q7 Pepsin

Hydrolysates of duck processing by-products

α-chymotrypsin Pepsin

References

Pepsin (from porcine gastric mucosa, EC 3.4.23.1)

t1:61

Observations/recommendation

The ACE inhibitory activity of the extract after hydrolysis treatment became strong. An in vivo study was carried out using SHR after an oral administration of the extract. The results indicated that the extract was able to lower the blood pressure. The peptides of the hydrolysates were isolated using RP-HPLC. The isolated peptides displayed same structure hom*ologous to that of collagen.

P1: Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-LeuHyp-Gly-Phe P2: Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-LeuHyp-Gly-X, P3: Gly-Val-Asn-Gly-Glu-Glu-Gly-Val-ProGly

t1:54 t1:55

t1:57 Q6 Flavourzyme, neutrase, t1:58 protamex, alcalase, t1:59 proteases, papain, t1:60 pepsin, trypsin and

Techniques and biological properties

Hydrolysates of Alaska pollack frame protein

Oyster (Crassostrea talienwhanensis Crosse) proteins hydrolysates

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S. Saadi et al. / Biotechnology Advances xxx (2014) xxx–xxx

Table 1 (continued)

Pepsin, trypsin, papain, α-chymotrypsin, alcalase and neutras

P1: Hoki frame protein hydrolysate (APHPH) Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr-HisPro-Ala-Cys-Pro-Asp-Phe-Asn produced by pepsin

t1:66 t1:67

Trypsin, α-chymotrypsin, and pepsin

Hoki (Jonius belengerii) skin gelatin

t1:68 t1:69

P1: His-Gly-Pro-Leu-Gly-Pro-Leu

Trypsin, α-chymotrypsin and pepsin

Hydrolysate of jumbo P1: Phe-Asp-Ser-Gly-Pro-Ala-Gly-Val-Leu squid (Dosidicus P2: gigas) skin gelatin Asn-Gly-Pro-Leu-Gln-Ala-Gly-Gln-ProGly-Glu-Arg

t1:70 t1:71 t1:72 t1:73 t1:74

Alcalase, α-chymotrypsin, mackerel intestine crude enzyme (MICE), Neutrase, Papain, pepsin, pronase E, trypsin

P1: Arg-Pro-Asp-Phe-Asp-Leu-Glu-ProYellowﬁn sole Pro-Tyr (Limanda aspera) frame protein hydrolysates (YFPHs) obtained by pepsin followed by MICE

t1:75

Pepsin

Hydrolysate of algae protein waste byproduct obtained from microalgae, Chlorella vulgaris

P1: VECYGPNRPQF

Tuna frame proteins hydrolysates

P1: Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-ValSer-Asn-Trp-Ser-Pro-ProLys-Try-Lys-Asp-Thr-Pro

t1:76 Q8 Alcalase, Neutrase, pepsin, t1:77 papain, α-chymotrypsin t1:78 and trypsin

t1:79 t1:80 t1:81

Pepsin, α-chymotrypsin, trypsin, alcalase, neutrase and papain

Tuna dark muscle hydrolysates

The antioxidative activity was conducted in vitro by following free radical scavenging assay-using ESR, the best hydrolysate obtained by pepsin was further fractionated into four groups using ultraﬁltration and puriﬁed by consecutive chromatographic techniques. Cell culture was conducted to assess the cytotoxicity effect of peptide and its protective effect against DNA damage. The best hydrolysate was extracted by using trypsin and different antioxidant assays were assessed including superoxide radical scavenging activity, carbon-centered scavenging activity, and DPPH radical scavenging activity. The isolated peptide was examined for lipid peroxidation and for different enzymatic reaction using cell culture such as superoxide dismutase assay, glutathione peroxidase assay and catalase assay. The hydrolysate was subjected to ultraﬁltration and tested for lipid peroxidation inhibition. The resulting fraction was then puriﬁed using chromatographic methods. Peptides were then tested for antioxidative properties in vitro by following hydroxyl radical scavenging assay, carbon centered radical scavenging assay, metal chelating activity assay, culture cells for assessing oxidation induced cell viability. The hydrolysate was separated into ﬁve parts using ultraﬁltration membrane. The antioxidative activity of YFPHs was examined in a linoleic model system and compared to that of α-tocopherol. The fraction showing strong antioxidative activity was isolated with different chromatographic techniques. The antioxidative activity of the isolated peptide was examined by following DPPH radical, ABTS radicals, superoxide radical, peroxy radical, and hydroxyl radical assays. In addition to cytotoxicity and oxidation induced DNA damage assays.

P1: Trp-Pro-Glu-Ala-Ala-Glu-Leu-MetMet-Glu-Val-Asp-Pro

Results indicated that the hydrolysate obtained by pepsin showed the highest antioxidative effects. The hydrolysates were efﬁciently quenched propagated free radicals and displayed a potent inhibition against lipid peroxidation higher than alpha-tocopherol as positive control. The peptide reduced radical-mediated cytotoxicity and protected DNA.

Kim et al. (2007)

The hydrolysate digested by trypsin displayed a potent antioxidative activity in terms of radical scavenging activity, followed by carbone-centered, and lastly by DPPH radicals. The hydrolysate showed signiﬁcant inhibition of the peroxidation in a linoleic acid model system. It is suggested that gelatin-derived radical-scavenging peptide (GRSP) can be implicated to maintain the redox balance due to its antioxidant activity.

Mendis et al. (2005b)

This study revealed that trypsin was a potent enzyme in developing antioxidative peptides. Tryptic digest showed consistent short peptide with high degree of hydrolysis. It showed that hydrophobic amino acids contributed signiﬁcantly to antioxidative peptides. The ability of hydrophobic peptides can be considered as the key structural parameters of peptide, particularly in protecting cellular damage. The amino acid residues may increase the interaction between peptide and lipid. Authors suggested that the antioxidative activity probably associated to MW and type of amino acid residues.

Mendis et al. (2005a)

Results showed that P1 has signiﬁcant protective effects on DNA and decline cellular damage caused by propagated free radicals. This peptide showed also a good resistance under gastrointestinal enzyme digestion. Authors suggested that inexpensive algae protein waste could be a new source for generating an antioxidant agent. The hydrolysate digested by pepsin displayed an antihypertensive activity better than other enzymes. The kinetic study demonstrated that peptide was found to participate in non-competitive manner towards the active site of the ACE enzyme. The isolated peptides showed to decrease signiﬁcantly the systolic blood pressure of SHR. MTT assay revealed that this peptide with no cytotoxicity effects. Results demonstrated that the hydrolysate produced by pepsin showed the highest activity. From the kinetic study, the peptide was found to exert a non-competitive action towards ACE enzyme. It is suggested that the presence of Val (hydrophobic) and Pro at the C-terminal position of the peptide may contribute signiﬁcantly to ACE inhibitory activity.

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Antihypertensive activity was evaluated in vitro by assessing ACE-inhibitory activity. The hydrolysates were subjected to ultraﬁltration and the best fraction was further puriﬁed using chromatographic techniques. The kinetic study was assessed to establish the relationship between peptides structure and their function. On the other hand, an in vivo study was conducted by measuring the systolic blood pressure of SHR after an oral administration of the bioactive peptides. Antihypertensive activity of the hydrolysates and peptide after isolation was measured in vitro by following ACE inhibitory peptide activity. The kinetic study of the peptide was determined and the effects of peptide on systolic blood pressure was conducted in vivo by oral administration of a peptide dose of 10 mg/kg of BW in SHR.

Jun et al. (2004)

Sheih et al. (2009)

S. Saadi et al. / Biotechnology Advances xxx (2014) xxx–xxx

t1:63 t1:64 t1:65

S.-H. Lee et al. (2010), S.-J. Lee et al. (2010)

Qian et al. (2007)

(continued on next page)

t1:85

Enzyme used

Identiﬁed peptides

Techniques and biological properties

Observations/recommendation

P1: VKAGFAWTANQQLS

The antioxidative activity was conducted in vitro by evaluating lipid peroxidation inhibition assay, direct free radical scavenging activity by ESR spectrometer. The best hydrolysate showing good antioxidative was further puriﬁed using consecutive chromatographic techniques. The cytotoxicity of the peptide was carried out using MRC-5 and ECV304 cell lines. The hydrolysate was tested for ACE inhibitory activity in vitro and then puriﬁed using ODS-5-HPLC in order to get the following peptides (P1, P2, P3, and P4). A systolic blood pressure was determined after an oral administration of a synthetic dose of the peptide of 50

Results showed that the hydrolysate produced by pepsin Je et al. (2007) was the best from antioxidative level. The isolated peptide showed to scavenge the peroxidation of linoleic acid based emulsion system. The peptide exhibited a potent antioxidative action by quenching free radical in a dose dependent manner.

U Pepsin

Wakame powder hydrolysate

P1: Ala-Ile-Tyr-Lys P2: Tyr-Lys-Tyr-Tyr P3: Lys-Phe-Tyr-Gly P4: Tyr-Asn-Lys-Leu

α-chymotrypsin

Giant squid (Dosidicus gigas) muscle protein hydrolysates

P1: Asn-Ala-Asp-Phe-Gly-Leu-Asn-GlyLeu-Glu-Gly-Leu-Ala P2: Asn-Gly-Leu-Glu-Gly-Leu-Lys

This study demonstrated that there is no correlation found in between the ACE inhibitory peptide activity in vitro and in vivo. The four types of peptides showed compatible values in reducing blood pressure compared to Captopril. They suggested that these peptides might degrade under gastrointestinal conditions into short mg/kg BW in SHR for up 6h. A drug (Captopril) with a peptide (dipeptides) which may facilitate their dose of 10 mg/kg was used during in vivo evaluation. absorption into portal circulation. The antioxidative property was evaluated by measuring Results demonstrated that the best hydrolysate was the inhibition against lipid peroxidation. The hydrolysate obtained by trypsin enzyme. The isolated peptides play a role as chain-breaking antioxidant by scavenging was fractionated using ultraﬁltration and the best fraction was puriﬁed using consecutive chromatographic propagated free radicals and inhibiting the peroxidation in the designed model system. The addition of low methods. The isolated peptides were veriﬁed from peptide dose of 50 μg/ml enhanced the cell viability. cytotoxicity effects in vitro using Human embryonic Electron spin trapping revealed that the peptide lung ﬁbroblasts, MRC-5. While the antioxidative property was evaluated using electron spin resonance in scavenge free radical in the following order carbon-centered N hydroxyl N superoxide radicals. It is order to determine free radicals by following hydroxyl suggest that the relative content of hydrophobic amino radical scavenging assay, carbon-centered radical acid (80%) may the result of antioxidative activity. scavenging assay, superoxide radical scavenging assay. Results demonstrated that P1 signiﬁcantly decreased The antihypertensive activity was evaluated in vitro blood pressure at a dose of 6 mg/kg BW. This study using ACE inhibitory peptide activity. The hydrolysate showing high activity was subjected to puriﬁcation such suggested that P1 is well absorbed in the gastrointestinal tract and transported to the tissue of the body. Further GFC and RP-HPLC in order to obtain P1. Systolic blood researches on structure–function relationship, pressure measurement was conducted using SHR after an oral administration of synthetic peptide doses ranging absorption mechanism, and metabolism are strategized. from 2 to 6 mg/kg BW. By using pepsin, the obtained hydrolysate exhibited high The egg white protein hydrolysate was hydrolyzed for up 3 h and conﬁrmed in vitro for both antioxidative and value of angiotensin converting enzyme inhibitory activity and displayed high value of radical scavenging antihypertensive biological functions. ACE inhibitory activity in vitro. activity and RSA were assessed. They noticed that there was no correlation found in between antioxidant and ACE inhibitory activity. The isolated peptide was found to be able in delaying the low density lipoprotein lipid oxidation induced by Ca+2 Results showed that the best hydrolysate was obtained A number of proteolytic enzymes were used to release the bioactive peptide from Arachin as the major storage by using pepsin rather than other enzymes. Out of three peptides, the P1 was the most potent peptide in protein of peanut. The degree of hydrolysis and their exhibiting the highest value of ACE inhibitory activity. corresponding ACE inhibitory activity of the Based on the molecular docking simulation the result hydrolysates were measured. Three types of peptides were successfully released from the selected hydrolysate showed that the presence of Pro at the C-terminal of the and then synthesized. Molecular docking simulation was peptide sequence and the length of peptide advocate the ACE activity potency. conducted to raise some insights concerning peptide structure and ACE inhibitory activity relationship. Immunomodulatory functions of the hydrolysates were Results showed that the hydrolysates could play both conducted using cell lines (THP-1 and Caco-2) as models roles of growth promoting and growth inhibiting by following cell proliferation and in vitro bioavailability peptides mixture. It is suggested that the hydrolysates produced by pepsin and pancreatin can regulate cell assays. proliferation that is highly depending on cell type, culture conditions and degree of hydrolysis.

References

Suetsuna and Nakano (2000).

Rajapakse et al. (2005a, 2005b, 2005c)

Pepsin

Fresh marine shrimp (Acetes chinensis) hydrolysates

P1: Leu-His-Pro

Crude egg white protein hydrolysates

P1: Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu

Cao et al. (2010)

R O

t1:89 t1:90

Pepsin, trypsin, chymotrypsin, and pancreatin

Arachin protein of Peanut (Arachis hypogaea) hydrolysates

P1: NAQRP P2: NLAG P3: IETWNPNNQ

t1:91

Pepsin followed by pancreatin

Hydrolysate of chickpea (Cicer arietinum) of protein isolate

Hydrolysates

Davalos et al. (2004)

Jimsheena and Gowda (2011)

Girón-Calle et al. (2010)

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t1:88

Hydrolysates

Alcalase, α-chymotrypsin, Tuna backbone neutrase, papain, pepsin, hydrolysates and trypsin

t1:86 Q9 Pepsin, trypsin and

t1:87

t1:82 t1:83 t1:84

Table 1 (continued)

Trypsin, clostripain, and subtilisin

Sodium caseinate and β-Casein hydrolysates

t1:93

Trypsin

Sodium caseinate and β-casein hydrolysates

t1:94 t1:95 t1:96

P1: VKEAMAPK (98–105) P2: AVPYPQR (177–183) P3: VLPVPQK (170–176)

Trypsin, alcalase, α-chymotrypsin, and pepsin.

Conger eel (Conger myriaster) muscle protein hydrolysates

P1: Leu-Gly-Leu-Asn-Gly-Asp-Asp-Val-Asn

Lipoxygenase inhibitory peptides were derived from the Results showed that all peptides inhibited LOX with Tryptic digest after fractionation using FPLC or HPLC. The different extent. It is suggested that the C-terminal of the sequence chain of peptides is the dominating element in active fraction was analyzed by ESI-MS (Part 1). promoting LOX inhibitory activity. They suggested as well as that the effect of casein derived peptides on the activity of lipoxygenase is a true inhibition that could result from iron complexation and/or mechanism of antioxidation. The antioxidative of the following synthesized peptides Results revealed that peptides of tryptic hydrolysates were able to inhibit enzymatic and non-enzymatic (Part 2) was examined in vitro by following peroxidation. They suggested that the biological activity their effects against enzymatically induced was remained effective and did not inﬂuence by linoleic acid oxidation: (LA/LOX) oxidation system; proteolysis and dephosphorylation of the proteins. An chemically induced oxidation of linoleic acid: interesting conclusion was pointed out suggesting that (LA/AAPH) oxidation system; Hemoglobin catalyzed the trapping of free radical by the hydrolysates/peptides oxidation of linoleic acid hydroperoxide: was associated by peptide sequence encrypted in the (Hpode/Hb) oxidation system; DPPH radical oxidation mechanism. scavenging activity and ferric chelating ability. The hydrolysate was fractionated based on the molecular This study suggested that low molecular weight fraction of less than 1 kDa showed high level of antioxidative weight using ultraﬁltration and the best fraction was puriﬁed using consecutive chromatographic techniques. even was signiﬁcantly higher than α-tocopherol. Peptide of 928 Da derived from the hydrolysates and relative The antioxidative tests were conducted by measuring the effects against lipid peroxidation in a model system, content of hydrophobic amino acids are the key structural parameters of the hydroxyl radical scavenging activity, and peptides that contributed to antioxidative carbon-centered radical scavenging activity. activity. The presence of two acidic Asp amino acids and two Gly residues may contribute signiﬁcantly to antioxidative activity. Results demonstrated that both of the The hydrolysates obtained from bovine milk αS1-casein were examined in vitro using cell culture and the effect hydrolysates digested by trypsin and pancreatin signiﬁcantly inhibited the proliferation of of the hydrolysates on cell proliferation as one of the lymphocytes and rabbit Peyer's patch cells. Pepsin and immunomodulatory property was conﬁrmed. chymotrypsin digests were found to be inactive towards mitogen-stimulated cells as conﬁrmed in vitro. Results showed the ability of trypsin and The WPIHs were fractionated using an isoelectric chymotrypsin in producing a biological system focusing technique that permitted to categorize the capable to modulate immune disorders in the fractions based on their pH value. Then the fractions of low, medium, and high pH were puriﬁed using HPLC. The cells. The obtained fractions were found to stimulate immunomodulatory properties were assessed by in vitro cells proliferation except at high doses in the proliferation assay of murine splenocyte in the presence presence of ConA. Peptide fractions signiﬁcantly stimulated the secretion of IL-2 and absence of concanavalin A (ConA) and their and IFN-γ. subpopulations were analyzed by ﬂow cytometry. The collected fractions of neutral and acidic pH ranges Cytokine analyses were conducted by measuring the showed to enhance the secretion of cytokines and level of IL-4 and IL-10 using commercial ELISA kits. stimulation of cell proliferation. This study showed that tryptic hydrolysates contributed The antihypertensive activity was examined in vitro by signiﬁcantly to ACE inhibitory activity. Authors assessing the ACE inhibitory peptide derived whey suggested that the molecular docking of LL on ACE active protein hydrolysates. Ultraﬁltration was employed to fractionate the hydrolysates into three fractions then the site revealed that LL makes contact with hydrophobic interaction of amino acid residues Ala354, Ala356, active fraction was subjected to consecutive chromatographic techniques. The molecular mechanism Phe391, Phe512, and Val518, and via hydrophilic and the interaction between P1 and ACE were studied as interactions with residues His353, 383, 387, 410, 513, Glu384, 411, and Arg522. The binding well. energy of P1 when it was in the free state was much lower to that of P1 + ACE. Tyr523 residue (aromatic portion) showed to maintain the complex stabilization during the interaction.

Rival et al. (2001a)

Rival et al. (2001b)

Ranathunga et al. (2006)

t1:97 Q10 Pancreatin, trypsin and

pepsin/chymotrypsin

t1:98 Q11 Trypsin/chymotrypsin

t1:99 Q12 Trypsin

Bovine milk αS1-casein and β-casein hydrolysates

Peptide-rich hydrolysates

Hydrolysates of whey Peptide fractions protein isolate (WPIHs)

Whey protein hydrolysates

P1: Leu-Leu (LL)

Otani & Hata (1995) and reviewed by Gill et al. (2000)

Saint-Sauveur et al. (2008)

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Pan et al. (2012)

(continued on next page)

t1:92

t1:101

t1:103

Enzyme used

Hydrolysates

Identiﬁed peptides

Techniques and biological properties

Observations/recommendation

References

α-Chymotrypsin

Yellowﬁn sole (Limanda aspera) frame protein hydrolysates

P1: Met-Ile-Phe-Pro-Gly-Ala-Gly-Gly-ProGlu-Leu

The hydrolysate was fractionated based on molecular weight using ultra-ﬁltration and the best fraction of less than 5 kDa was further puriﬁed using consecutive chromatographic techniques. The kinetic study of P1 with ACE and was studied by Line weaver–Burk plots. A systolic blood pressure was measured after an oral administration of peptide dose of 10 mg/kg BW on SHR and the effects was compared with captopril following same procedure. The antioxidative property of the hydrolysate was compared with α-tocopherol after fractionation using ultraﬁltration and divided into low, medium, and high molecular weight fractions. The peptides were isolated from the hydrolysate by using consecutive chromatographic techniques. The antioxidative activity was measured by following the effects of peptide on lipid peroxidation in cultured human liver cell.

Results indicated that the best ACE inhibitory activity was obtained with low molecular weight fraction using ultraﬁltration. The isolated peptide P1 showed to exert a non-competitive inhibition towards the active site of ACE enzyme. A signiﬁcant reduction in the systolic blood pressure was observed after 3 h to 9 h after injection. It is suggested that the C-terminal tripeptide that contains hydrophobic amino acids at this position may exert a potent inhibitory activity. Results demonstrated that lecithin-free egg yolk digested by alcalase is a promising protein source to produce antioxidant bioactive peptides. The presence of His residue with imidazol group was attributed to the chelating and lipid radical trapping ability. On the other hand, Tyr as a potent hydrogen donor residue may contribute signiﬁcantly to antioxidative activity. Results indicated that the hepatocytes cultured in the presence of P2 revealed low amount of lipid peroxidation compared with non-treated sample. This study showed that low molecular fraction of less than 1 kDa that was found to be rich with hydrophobic amino acids exhibited high value of antioxidative activity concerning both DPPH•+ and O2•−assays. On the other hand, the fractions with intermediate hydrophobicity revealed maximum ABTS•+ activity. Authors suggested that the antioxidative activity was attributed to molecular weight and hydrophobicity of the peptides. As results, fraction number 7 showed the highest activity among other fractions, resulting in signiﬁcant inhibition for lipid peroxidation better than α-tocopherol. They suggested that F7 is a promising function ingredient for scavenging free radical and chelating metal ion transition. The acidic and basic amino acid residues may contribute signiﬁcantly to metal ion chelating activity. Results indicated that low molecular fraction showed the highest ACE inhibitory activity. They suggested that the presence of Leu at C-terminal amino acid plays an important role in the expression of ACE inhibitory activity. Results showed that alcalase digest was selected to be the best from ACE inhibitory activity point of view. The kinetic study based on Lineweaver–Burk plots revealed that P1 acts as competitive inhibitor towards ACE. They suggest that peptides containing hydrophobic residues are potent inhibitors for ACE, and they revealed that the tripeptides sequence at the C-terminal position of the substrate is one of the keys parameter for understanding peptide structure–function relationships. Results showed that P2 had a potent antioxidative activity due to its ability for scavenging the peroxidation of lipid (linoleic acid) in an emulsion as model system. The cell viability of cultured liver cells was enhanced by the addition of the peptide. Results demonstrated that the antioxidant activity might attribute to the repeating motif Gly-Pro-Hyp at the C-terminal position.

Jung et al. (2006)

Alcalase

Lecithin-free egg yolk P1: Leu-Met-Ser-Tyr-Met-Trp-Serhydrolysates Thr-Ser-Met. P2: Leu-Glu-Leu-His-Lys-Leu-Arg-SerSer-HisTrp-Phe-Ser-Ser-Arg

Alcalase from Bacillus licheniformis

Alcalase

Zein 92% (a byproduct obtained from corn starch processing) protein hydrolysates

P1: Tyr-Ala P2: Leu-Met-Cys-His

Chickpea protein hydrolysates (CPH)

P1: Asn-Arg-Tyr-His-Glu

Alcalase

Egg white protein hydrolysates

P1: Arg-Val-Pro-Ser-Leu

t1:105 t1:106

Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin

Marine rotifer (Brachionus rotundiformis) hydrolysates

P1: Asp-Asp-Thr-Gly-His-Asp-Phe-GluAsp-Thr-Gly-Glu-Ala-Met

Alaska pollack skin gelatin hydrolysates

P1: Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-ProHyp-Gly-Pro-Hyp-GlyPro-Hyp-Gly. P2: Gly-Pro-Hyp-Gly-Pro-Hyp-GlyPro-Hyp-Gly-Pro-Hyp-Gly

Alcalase

Antioxidant peptide was puriﬁed from the hydrolysate using chromatographic separation techniques and the best fraction with high antioxidative activity was assessed for DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, metal ion chelating activity and against autooxidation caused by linoleic acid. The hydrolysate was fractionated and puriﬁed and the resulting peptide (P1) was synthesized and then evaluated in vitro for ACE inhibitory activity.

t1:104

t1:107

The hydrolysate was fractionated using ultraﬁltration and puriﬁed by following consecutive chromatographic methods. The antioxidative assays used were ABTS•+, DPPH•, and superoxide anion (O2•−).

R O

Angiotensin converting enzyme inhibitory peptide was separated from the hydrolysate using different chromatographic techniques. The kinetic study of peptide was studied as well.

The hydrolysate was fractionated using ultraﬁltration and the peptides were separation using a series of chromatographic techniques. The antioxidative property was measured using the thiobarbituric acid (TBA) method, whereas the cell viability was measured with MTT assay.

Park et al. (2001)

Tang et al. (2010)

Zhang et al. (2011)

Liu et al. (2010)

Lee et al. (2009)

Kim et al. (2001b)

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t1:102

t1:100

Table 1 (continued)

Alcalase, pronase E, and collagenase

t1:109Q13 Alcalase, α-chymotrypsin, t1:110 Neutrase, pronase E and

t1:111 t1:112 t1:113

t1:114

t1:115 t1:116

t1:117 t1:118

t1:119

trypsin

Papain, bovine pancreatin 6.0, bromelain, neutrase 1.5MG and alcalase 2.4L

Gelatin hydrolysate of bovine skin

Gelatin hydrolysate from Alaska Pollack (Theragra chalcogramma) skin

Grass carp muscles hydrolysate

The digested hydrolysate using pronase E was fractionated and the antioxidant activity was determined by TBA method. Three peptides were successfully puriﬁed using consecutive chromatographic methods. The antioxidants of puriﬁed peptides were measured using TBA, cell viability of cultured liver cells assay and peroxidation of linoleic acid in a model system. An antihypertensive activity was conﬁrmed from the hydrolysates after puriﬁcation steps including gel ﬁltration chromatography ion-exchange chromatography and RP-HPLC.

P1: Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp P2: Gly-Pro-Hyp-Gly-Pro-Hyp-GlyPro-Hyp-Gly P3: Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp

P1: Gly-Pro-Met P2: Gly-Pro-Leu

P1: Pro-Ser-Lys-Tyr-Glu-Pro-Phe-Val

Bromelain and alcalase

Sea cucumber (Acaudina molpadioidea) hydrolysates

P1: MEGAQEAQGD

Results showed that the second hydrolysate using pronase E exhibited high antioxidative activity against peroxidation of linoleic acid in a model system. They revealed that the viability of cultured liver cells was enhanced signiﬁcantly by the addition of the peptide. A conclusion gelatin derived bovine skin can be used as natural antioxidant.

Results indicated that small peptides (di-or-tri-peptides) are useful as an oral drug for treating hypertension. They suggested that a continuous enzymatic hydrolysis with different digestive enzymes and fractionation using different ultraﬁltration membranes produced small peptides that contributed signiﬁcantly to ACE inhibitory activity. Results demonstrated that basic peptides showed the The antioxidative functions of the hydrolysates were assessed by using lipid peroxidation assay and hydroxyl highest activity better than acidic and neutral peptides. Another point is that the hydrophobic peptides radical scavenging activity assay. The best hydrolysate contributed match better to antioxidative than was then puriﬁed using consecutive chromatographic hydrophilic peptides. They suggested that techniques including ion-exchange chromatography, the peptide sequence would have signiﬁcant high-speed counter-current chromatography, gel contribution to antioxidant ﬁltration chromatography, and RP-HPLC. activity. The hydrolysate was fractionated into 2 range of low and Results demonstrated that low molecular fraction showed high ACE inhibitory activity. The ACE inhibitory high molecular weight using ultraﬁltration technique. activity was 3.5 times intensiﬁed after incubation using The 2nd fraction was puriﬁed using gel ﬁltration, pepsin, trypsin, and chymotrypsin. The peptide ion-exchange chromatography, and RP-HPLC. The stability under gastrointestinal enzymes was veriﬁed as decreased signiﬁcantly the systolic blood pressure after 3 well. The effects of peptide were conﬁrmed h and the SBP was maintained after 5 h. The peptide in vivo using SHR after an oral administration of the showed low ACE inhibitory activity in vitro compared to peptide at a dosage of the synthetic compound captopril. 3 μM/kg BW. Results showed that the hydrolysate digested by The antioxidative functions of the hydrolysate were neutrase exhibited high level of antioxidative activity. veriﬁed in vitro using DPPH, hydroxyl scavenging DPPH and hydroxyl radical have been found to be higher activity, superoxide radical scavenging activity, and than superoxide radical scavenging activity. The inhibition of lipid peroxidation in a model system. The synthetic peptide displayed a potent antioxidative viability test and radical-induced cell culture assay was activity by inhibiting the lipid peroxidation conducted by using MRC-5 and RAW264.7 cells. better than α-tocopherol. In addition, this peptide enhanced cell viability t-BHP induced cytotoxicity. Results revealed that some of the peptides from chicken Both hydrolysates were fractionated and puriﬁed using muscle hydrolysates and ovalbumin were failed to consecutive chromatographic techniques. The ACE inhibitory activity was conﬁrmed in vitro and in vivo by exhibit an antihypertensive effects. Three groups of systems were tested to conﬁrm the activity. These following intravenous administration in SHR. groups were (1) the inhibitor (peptide that did not show the effect with ACE; (2) peptide that hydrolyzed with ACE but with least effect and (3) pro-drug inhibitor (the peptide that lead to real inhibition after digestion using ACE/gastrointestinal proteases). Systolic blood pressure was found to be signiﬁcant for group 1 and 3. The P1 was hydrolyzed by ACE and produced P2. Systolic Results showed that P2 was 8 fold higher in ACE inhibitory activity than P1. Authors suggested that P1 blood pressure was determined using SHR after an oral could be regarded as pro-drug type ACE inhibitory administration of P1, P2 and captopril. peptide. Both P1 and captopril showed maximum decrease in systolic blood pressure after 4 h, whereas P2 was found to decrease the systolic blood pressure sharply after 2 h.

Kim et al. (2001a)

Byun and Kim (2001)

Ren et al. (2008)

Zhao et al. (2009)

Alcalase, chymotrypsin, neutrase, papain and ﬂavorase

Defatted rice endosperm protein hydrolysates

P1: Phe-Arg-Asp-Glu-His-Lys-Lys P2: Lys-His-Asp-Arg-Gly-Asp-Glu-Phe

Zhang et al. (2010b)

Thermolysin, pepsin, trypsin and chymotrypsin

Chicken muscle hydrolysates Ovalbumin

P1 (Myosin) FQKPKR P2 (Ovalbumin) NIFYCP

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Fujita et al. (2000)

Thermolysin

Dried bonito hydrolysates

P1: LKPNM P2: LKP

Fujita and Yoshikawa (1999)

(continued on next page)

t1:108

Enzyme used

Hydrolysates

Identiﬁed peptides

Thermolysin

Dried bonito hydrolysates

P1: Ile-Lys-Pro-Leu-Asn-Tyr →

Techniques and biological properties

Ile-Lys-Pro P2: Ile-Trp-His-His-Thr → Ile-Trp P3: Leu-Lys-Pro-Asn-Met → Leu-Lys-Pro P4: Asp-Tyr-Gly-Leu-Tyr-Pro → Leu-Tyr-Pro

Thermolysin

Chum salmon muscle P1: Phe-Leu hydrolysates P2: Leu-Phe

Observations/recommendation

Results showed that out of eight peptides only four peptides displayed signiﬁcant ACE inhibitory activity. After digestion four novel peptides were initiated from their original fragments. These active fragments were found to exhibit potent ACE inhibitory activity, which were possibly attributed to the C-terminal of di-or-tripeptides of their sequence chain. Results demonstrated that P1 exhibited better activity, The antihypertensive activity (ACE) of the isolated whereas P2, which is the reverse peptide, showed a peptides was assessed in vitro by following an angiotensin converting enzyme inhibitory peptides. The potent antihypertensive activity more than P1. The ﬁrst inhibition of P1 was found to be non-competitive, while kinetic of ACE-inhibitory peptide was studied in vitro the second one was found to be competitive. In addition using six-Try containing dipeptides. to P1, and P2 another six peptides possessing Try at the C-terminal were found to act as non-competitive inhibitors towards ACE. On the other hand, reverse sequence where Try at the N-terminal showed competitive inhibition. As conclusion, the sequence dipeptides may affect the ACE inhibitory activity signiﬁcantly. The results demonstrated that by using Corolase PP, a Whey protein derived from bovine α-lactalbumin and potent antioxidative peptide could be produced from β-lactoglobulin was investigated from antioxidative this functional food material. Out of 42 peptide point of view using ﬁve different proteolytic enzymes. fragments identiﬁed by HPLC-MS/MS, P1 exhibited the The radical scavenging activity was conducted by meahighest value of radical scavenging activity better than suring ORAC-ﬂuorescein (ORAC-FL) assay. The best hythat displayed by butylated hydroxyanisol (BHA). drolysate was fractionated using RP-HPLC and further Authors suggested that the conformation of peptides and separation was conducted using HPLC-MS/MS. peptidic bonds could lead to synergistic and antagonistic effects compared to the activity of their constitutive amino acids. The results showed that the highest levels of ACE The antihypertensive activity of the hydrolysate obtained from hard clam meat hot water-soluble extract inhibitory activity were obtained from the ﬁve fractions obtained from the hydrolysate using size exclusion and digested by Protamex enzyme was evaluated by chromatography and Sephadex-G-25 column. The using ACE-inhibitory assay in the presence of ACE hydrolysate showed mix-type of inhibition kinetics, enzyme, and HHL as substrate. Peptide content was measured based on OPA method and the ACE-inhibitory while the mode of action exhibited by Captopril was found to be in competitive inhibition. The results activity assay was performed using RP-HPLC. The indicated that the polypeptides were further breakdown inhibitory hydrolysate was further tested for stability under the action of pepsin and pancreatin enzymes and a by the proteolytic enzyme to small peptides that might signiﬁcantly contribute to ACE-inhibitory activity. Lineweaver-Burk plot was used to evaluate the inhibition mode. The hydrolysate was fractionated and puriﬁed using HPLC. Eight peptides were obtained from the thermolysin digest. These peptides were digested again and the resulting novel peptides were assessed for angiotensin converting enzyme inhibitory peptide.

References Yokoyama et al. (1992)

Ono et al. (2005)

t1:122Q14 Pepsin, trypsin, t1:123 chymotrypsin, t1:124 thermolysin, and t1:125 corolase PP

t1:126

Table 1 (continued)

Protamex (PX)

Whey proteins bovine (α-lactalbumin and β-lactoglobulin) hydrolysates

P1: Trp-Tyr-Ser-Leu-Ala-Met-Ala-AlaSer-Asp-Ile.

Hydrolysates of hard Clam (Meretrix lusoria) meat

P1: Tyr-Asn

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Hernandez-Ledesma et al. (2005)

Tsai et al. (2008).

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t1:121

t1:120

α-chymotrypsin)

t1:128Q16 Mackerel intestine crude t1:129 enzyme (MICE)

t1:130

t1:131 t1:132

Alaska pollack frame protein hydrolysates

P1: Leu-Pro-His-Ser-Gly-Tyr

The results demonstrated potent inhibitions of the antioxidative peptides against lipid peroxidation compared to results obtained by using ascorbic acid and α-tocopherol. The peptide showed a good activity and compatible scavenging effect of ascorbic acid for hydroxyl radical formation. The peptide exhibited also signiﬁcant effects in quenching superoxide and carbon-centered radicals but lower than ascorbic acid. The amino acid residues Ala, Val, Leu, and Pro with non-polar aliphatic groups exhibited high reactivity to the hydrophobic groups of PUFAs, whereas other aromatic amino acid residues such as Tyr, His, Phe, and Trp had the ability to stabilize the ROS via direct electron transfer. Gly, Asp, Glu, and Tyr showed to support protons through their ability in quenching unpaired electrons. Results showed that the low molecular fraction The antioxidative activity of the hydrolysates was displayed a potent antioxidative activity. This fraction assessed in vitro against lipid peroxidation of a linoleic acid in an emulsion model system. The hydrolysate was was used to produce P1, which in turn exhibited adequate inhibition (35%) against hydroxyl radicals fractionated into ﬁve fractions using ultra-ﬁltration using electron spin resonance (ESR) spectroscopy. The technique. The puriﬁed peptide after consecutive inhibition of lipid peroxidation may attribute to the chromatographic separation was tested for hydroxyl presence of imidazol ring that has the ability to trap the radical scavenging activity. radicals propagated from lipids during peroxidation. It is suggested that the antioxidative activity was associated to the amino acid residues and molecular weight. The material used was examined for immunomodulating The results showed a dose dependent manner in stimulating the proliferation of lymphocytes. The activity after fractionation using consecutive isoelectric point of the peptide was found to be 3.82 with chromatographic techniques to obtain active fraction, molecular mass of 2133.52 Da. The isolated peptide and the identiﬁed peptide was assayed for lymphocyte showed no signiﬁcant hom*ology with other peptides and proliferation, and other biophysical characterization such as capillary isoelectric focusing electrophoresis and displayed an immune potency of 60% for a period of 30 thermal stability using Far-ultra-violet circular min under heat temperature of 80 °C. The analysis of the dichroism (far-UV CD) spectrometry. spectra of P1 revealed that the secondary structure of the

This study was conducted to evaluate the antioxidative properties of fresh muscle based hydrolysates and its generated bioactive peptides. The antioxidative activity was assayed by following lipid peroxidation of linoleic acid in an emulsion model system. After puriﬁcation by LC-(ESI-Q-TOF-MS/MS). The sequenced peptide was checked for hydroxyl radical scavenging assay, superoxide radical scavenging assay, and carbon centered radical scavenging assay.

P1: LeuVal-Gly-Asp-Glu-Gln-Ala-Val-Pro-AlaVal-Cys-Val-Pro

Mytilus coruscus muscle Protein hydrolysate

Jung et al. (2007a, 2007b)

Je et al. (2005a, 2005b)

Without using enzymes

Bovine placenta water soluble extract

P1: Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr

Fang et al. (2007)

Lactobacillus delbrueckii ssp. bulgaricus LB340

Fermented skim milk hydrolysates

Peptide fraction (F2)

The supernatant was fractionated using ultraﬁltration technique and the resulting low molecular weight fraction (b1 kDa) was puriﬁed using chromatographic techniques. Out of six fractions obtained, the F2 was veriﬁed by following DPPH radical scavenging activity, stimulation effect of murine spleen lymphocyte proliferation and ACE inhibitory activity.

peptide gradually decreased as the temperature increased. This study suggested that the low molecular weight fraction of less than 1 kDa could be used as multifunctional candidate in lowering the effects of oxidative stress, hypertension, and immune disorders. The size of bioactive peptide may range from 2 to 20 amino acids, but the most support ones are di-and-tripeptides due to their intact absorptions compared to the long peptide chain.

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Qian et al. (2011)

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t1:127Q15 Pepsin + (trypsin +

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fragments shall exhibit high activity with desired biological functions. It is well known that the ultraﬁltration membrane reactors could generate high yield of uniform biomaterials with preferred molecular weight (Korhonen and Pihlanto, 2006). In part, the broad usages of milk proteins made them as abundant sources for the production of bioactive peptides and because of these milks become more captivating and dynamic biological ﬂuids. Thus, many of the national and international health centers used these materials to enrich and supplement certain products in order for them to maintain and serve many of the biological activities including antioxidative stress, antihypertensive and immunomodulating functions. For instance, antithrombotic peptides were recovered and derived from caseinomacropeptide (CMP) that is subjected to enzymatic digestion (Bouhallab and Touzé, 1995). On the other hand, the antihypertensive peptides were obtained by using different proteases and were puriﬁed by reverse-phase HPLC on a Delta Pak C18 column (Haileselassie et al., 1999). In addition, the best fractions were puriﬁed again using the same column coupled by a binary gradient. Je et al. (2007) by using different proteases successfully puriﬁed antioxidative peptides using consecutive chromatographic methods. Finally, the peptides were discriminated by Q-TOF ESI mass spectroscopy and the resulting sequence was exhibited on the following form VKAGFAWTANQQLS (1519 Da). Kim et al. (2007) reached to extract bioactive peptides (Glu-Ser-Thr-Val-Pro-GluArg-Thr-His-Pro-Ala-Cys-Pro-Asp-Phe-Asn) having an antioxidant activity from hoki frame protein hydrolysate (APHPH). After ultraﬁltration membranes and consecutive chromatographic methods the generated system showed to possess a molecular mass of less than 1801 Da. Megías et al. (2004) reported an ACE inhibitory peptide (Phe-Val-AsnPro-Gln-Ala-Gly-Ser) generated under the action of pepsin and pancreatin using G-50 GFC and C18 RP-HPLC from sunﬂower protein. Jang and Lee (2005) in turn implicated a simultaneous puriﬁcation technique (e.g. Ultraﬁltration, gel-ﬁltration, and RP-HPLC) in order to generate angiotensin converting enzyme (ACE) inhibitory peptides as an antihypertensive peptide from sarcoplasmic protein extracts derived beef rump. Liepke et al. (2001) managed to separate biopeptide fragments by means of reversed-phase chromatography after intensive veriﬁcation of the antimicrobial activity of the obtained fractions via a sensitive radial diffusion assay and by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The results reﬂect that the generated bioactive peptides from human milk proteins during proteolytic hydrolysis might have a signiﬁcant contribution in the host defense system of the newborn. Finally, many of the preparation conditions concerning different chromatographic separation systems should be taken into account for the success of the end products. For example, the kind of initial extraction, nature and chemical behavior of the targeted biomaterials, selection of the right column and appropriate chromatographic conditions in addition to the organic solvents involved in the formation of mobile phase as well as the steps of puriﬁcation used. As an example, silica and Sephadex LH-20 columns, as well as C18 HPLC are preferred to be implicated in the ﬁnal puriﬁcation (Aneiros and Garateix, 2004). The variability of the active compounds and their chemical nature within a food matrix has made it difﬁcult for scientists to follow a speciﬁc technique in order to separate the targeted constituents from the mixture systems (crude extract or protein hydrolysates). It has been stated that the presence of active compounds having similar chemical structure analogues makes their separation very difﬁcult due to their complexity during the isolation procedures (Duarte et al., 2012). These cases can occur particularly when the targeted active compounds are found in the form of hom*ogenous and heterogeneous complex systems. In addition to extraction and isolation techniques a series of separation methods can be used in order to achieve the pure compounds. The fractionation of biopeptides by chromatographic techniques is effective, especially when the hydrolysates are enriched using ultra-ﬁltration techniques, and therefore the desired activity may accumulate in a particular fraction. The fractionation in most cases requires long periods to concentrate the required amount of bioactive system. In addition to some encountered problems like large numbers of solvent-consuming steps, which can lead to the lashing of the

This particular fragment is compatible to the positions of amino acids 22 to 68 of the pro-hormone of human proguanylin derived from the cDNA 529 sequence. Further characterization revealed that the mass spectral results 530 of the cyclic peptide exhibited less molecular mass of 10,336 Da. The ob531 tained result was conﬁrmed with the molecular mass of the peptide pre532 dicted from the cDNA sequence, which corresponds to the molecular 533 weight expressed by the C-terminus at position 22. Meanwhile, signiﬁ534 cant inducement (increasing) in the T84 cells having cyclic GMP content 535 was noticed via the circulating guanylin markedly, revealing that cyclic 536 peptide-like hormone of guanylin is circulating as a 10.3-kDa peptide in 537 human blood. Speciﬁc study on bioactive peptides derived from for 538 human plasma they used hemoﬁltrate. The examination was done by 539 collecting the blood of voluntary patients having chronic renal failure. 540 The utilization of human plasma is (1) abundant and has many advan541 tages due the large quantities and potential sources for deriving biological 542 peptides and (2) hemoﬁltration is considered the ﬁrst stage of plasma pu543 riﬁcation for potential biopeptides. The narrow pore size of the hemoﬁlter 544 of less than 20,000 Da permitted to diminish the amount of the ﬁltered 545 fraction signiﬁcantly. In fact the procedure was commenced by the isola546 tion as a ﬁrst step according to a modiﬁcation protocol of the isolation 547 that is used by Mutt (1978) to isolate the polypeptide. The sample after 548 collection was transferred immediately into the acidiﬁed solution of 549 pure hydrochloric acid (HCl; pH 3.5). A series of biochemical screenings 550 were done for each fraction in T84 cells and then the active fractions 551 were collected and injected into RP-HPLC for further puriﬁcation. Finally, 552 the fraction was submitted for capillary zone electrophoresis and the re553 sults showed by this fraction revealed one single peak; the highly puriﬁed 554 Q47 fraction was loaded into the mass spectrometer for the analysis of amino 555 Q48 acid sequence of the puriﬁed peptides. Rajapakse et al. (2005a, 2005b, 556 2005c) studied the antioxidative effects of giant squid (Dosidicus gigas) 557 muscle protein used for the production of lower molecular weight pep558 tides with ultraﬁltration (UF) and conﬁrmed from an antioxidative 559 point of view in vitro. Results revealed that the viability of cytotoxic em560 bryonic lung ﬁbroblasts of these peptides was induced signiﬁcantly 561 (p b 0.05) at low applied doses. Although the molecular interaction re562 sponsible for scavenging the propagated free radicals is not fully appreci563 ated, the relative content of hydrophobic amino acid (more than 75%) of 564 the starting material used proposed a hypothesis that these types of 565 amino acids might have a signiﬁcant contribution to the obtained activi566 ties. Harwig et al. (1993) developed a preparative method to purify rabbit 567 and human defensins named as continuous acid-urea-polyacrylamide gel 568 electrophoresis (CAU-PAGE). Their ﬁnding revealed that defensins were 569 stable from the N-terminus. Further, the biological effect of this puriﬁed 570 biomaterial veriﬁed in vitro towards antimicrobial activity has been 571 found to be similar with that exhibited by defensins that is puriﬁed by 572 chromatographic methods. Recently the utilization of membrane separa573 tion techniques facilitated and permitted to enrich the hydrolysate mix574 tures according to a particular range of peptide molecular weights 575 (Korhonen and Pihlanto, 2003). However, the continuous hydrolysis 576 using ultraﬁltration membranes via enzymatic hydrolysis are widely ap577 plied through conventional batch hydrolysis (Korhonen and Pihlanto, 578 2006). These techniques have drawn much of the researchers' attention 579 Q49 in the area of functional foods, speciﬁcally the enzymologists. Thus, they 580 are convinced to be implicated for total generation of various bioactive 581 peptides from protein via proteolytic enzymes. In fact these strategies 582 will help to ameliorate the functionality and other physicochemical and 583 nutritional features of the end products (Martin-Orue et al., 1999; Perea 584 and Ugalde, 1996). Fitzgerald and Murray (2006) investigated the antihy585 pertensive of peptide fractions derived from different cheese varieties in 586 spontaneously hypertensive rat (SHR) models. The active fractions have 587 been successfully puriﬁed by hydrophobic interaction chromatography. 588 As results signiﬁcant decrease in the systolic blood pressure (SBP) was 589 noticed from the range of 7.1 to 29.3 mm mercury (Hg). Therefore, and 590 under the action of serum proteinases/peptidases the protein complex 591 start to degrade into further peptide fragments via gastrointestinal condi592 tions. Under this mechanism of fragmentation the generated active

527 528

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659 660

compounds and they become not effective in order to cible the biological effects within the living system.

661

Degradation kinetics and enzymes ﬂexibility

N C O k1

704 705 706 707 708 709 710 711 712 713 714 715 716 717 718

They reported that the formulated end product (e.g. biopeptides) is considered the loss part of the protein involved in the reaction mechanism. They expressed as well as the generation of the end product in terms of the dissociation rate (k2), that is commonly known as kcat and deﬁned as “the maximum end product generated under controlled conditions of saturated substrate, expressed per unit time per unit enzyme”. Currently, most of the biochemists are mainly focusing on comprehensive understanding of the key parameters of these catalytic systems by providing new insights on their catalytic reaction. For this reason, Estevez et al. (2002) reported that the enzyme efﬁciency or catalytic efﬁciency is a measure of substrate speciﬁcity and the constant parameter Km (mol·l− 1), which gives an indication on the adhesion force generated between the enzyme and its speciﬁc targeted material. Moreover, the maximum velocity that can be achieved by the enzyme can give an indication on vmax. Finally, to rejoinder enzyme kinetic properly it is important to understand their physiological circ*mstances.

703

Enzymatic antagonism: case of competitive and feedback inhibitions

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Enzymatic antagonism is the mechanism of action, which can exhibit the substance that exerts a reverse action to its enzyme, by wasting the enzyme's time to occupy the catalytic site in the same time, decreases the capability of the enzyme to adhere with its preferred substrate, and therefore, blocks its catalytic mechanism. The reaction scheme below shows the basic mechanism of antagonism in the presence of an inhibitor, which is expressed by the Ki and determined via competitive inhibition. Where, k1 and k−1 represent the association and redissociation rates of the enzymatic reactions of enzyme–substrate complex, respectively, whereas, k2 and k−2 represent the association and redissociation rates of the enzymatic reactions between enzyme and its inhibitor (I).

727

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721 722 723 724

728 729 730 731 732 733 734 735 736 737 738

↑↓k ½Enzyme—Inhibitorcomplex: 2

þ½Peptide þ ½Inhibitork2

Always referring to the Michaelis–Menten equation (Michaelis and Menton, 1913), which is improved further by Briggs and Haldane (1925), this fundamentally kinetic equation in the presence of competitive inhibitor, is expressed as follows: ν¼

740 Q55 Q56 741 742 743

υmax ½Protein : ½Inhibitor þ ½Protein Km 1 þ Ki 745

Moreover, Jr et al. (2004) stated that the feedback inhibition is the result of the continuous release of peptides by enzymatic hydrolysis, which might raise the competition between the active site of the peptides and its original substrate, and therefore, induced the reduction in the reaction rates of hydrolysis. Further, it could be the result of the continuous releasing of the peptide under hydrolysis process, where in this case some amount of the peptides and its original substrate would participate towards the active site of the enzyme. Thus, the beginning of the reaction rates can be measured based on the consumption rate of both peptide and substrate during an onset time (Adler-Nissen, 1986).

746 747 748 749 750 751 752 753 754

½Enzymes þ ½Protein ⇄½Enzyme—Proteincomplex →½Enzyme þ½Peptide

719 720

½Enzymes þ ½Protein ⇄½Enzyme—Proteincomplex→½Enzyme

Two general catalytic pathways are employed in order to estimate 663 the reaction rate, particularly when the substrates are subjected to an 664 enzymatic degradation by using proteolytic enzymes. Thus, the ﬁrst cat665 alytic pathway is by exploring the Michaelis–Menten equation and the 666 second one is by following the catalytic degradation using ﬁrst-order ki667 netics. Clear evidence was given to the ﬁrst-order kinetics, where the 668 acceptability of this hypothesis is attributed to that during mechanism 669 of degradation, the controlled rate is the result of the ﬁrst degradation 670 of the tertiary structure of the protein by catalytic enzyme (Vorob'ev 671 et al., 1996). However, the rates of the end products of the respective 672 substrate and product concentrations in this case are generally articulat673 ed either in terms of the degree of hydrolysis or degradable peptide 674 bonds. Ruan et al. (2010) studied the degradation of egg white protein 675 (EWP) and characterized its kinetics under the action of proteolytic en676 zyme of pepsin, showing the importance of enzyme inactivation and 677 substrate inhibition in sustaining the bioreaction mechanism. They re678 ported that a mathematical model could be established on form loga679 rithmic equation: h = (1/b) ln (1 + abt), displaying a correlation 680 between hydrolysis degree as a function of time. As a result, signiﬁcant 681 variation in the structural properties can occur once the substrate is sub682 jected to continuous catalytic enzyme cycles under favorable conditions 683 of pH, concentration and reaction temperature, particularly in case of 684 stimulating the reactive conformations. This action may allow the hy685 drolysis to take place at speciﬁc stages, which in most of cases are ﬁnal686 ized either by the binding of the substrate or by releasing of the active 687 compounds. McGeagh et al. (2011) demonstrated that the endo action 688 of the catalytic enzymes and their rapidity in traversing distinct confor689 mations is an extra advantage to their particular catalytic activity. For 690 this reason, the enzyme ﬂexibility is important also, especially in case 691 when the ﬂuctuations can occur at speciﬁc reactive conformations 692 (see Fig. 2). 693 Meanwhile, to enhancing the production of biopeptides, it is impor694 tant to understand the behavior of the enzymes towards the substrate 695 and vice versa. This strategy can help to understand the fundamental 696 of enzymatic reaction and the main factors associated with the mode 697 of actions in order attain more reproducible results. However, the build698 ing up of more information about enzymes kinetic should be based on 699 Q54 the ﬁrst fundamentals proposed by Michaelis and Menton (1913), 700 which is improved later by Briggs and Haldane (1925). They demon701 strated that the enzymatic reaction is expressed as follows:

It means to study kinetic properties of such substrate under mechanism of enzymes action, frequently necessitates an in vitro test on the puriﬁed enzyme, with more emphasis on its pharmokinetics trend. Further, a huge information on enzyme modeling, particularly metabolic network will help to gather information on the catalytic properties and its relation to the genotypes growth conditions of the living systems.

662

Strategies needed to ameliorate enzyme catalytic rate

755

Cowan and Lafuente (2011) reported that many efforts are being focused by the chemists to support the protein research area using different enzymatic and chemical modiﬁcations and sometimes by combining both chemical and physical with genetic engineering approaches to explore the protein functionalities on a larger scale. However, the recent technological progress permitted the development of several robustness techniques, allowing screening of the proteins and peptides either from a simple matrix system, or from a complex mixture of compounds to enhance their functionalities such as thermal stability, bioavailability, and biological stimulating activities. Lee and Dordick (2002) extensively elaborated the fundamental for a comprehensive understanding the main techniques employed in generating different kinds of enzymes that have shown an important role on large scale of biopeptides production and their commercialization as well. Basically, the necessity of these fundamentals are more supported to clarify the mechanism effect of tailored enzymes, more considering their parameters including regioselective, enantioselective and stability in order for

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Fig. 2. Illustrates the biodegradation pathways of polypeptides under the mechanism of action of speciﬁc and non-speciﬁc enzymes, demonstrating different steps of the degradation and catalyzing domains exerted by enzymes. For instance, in case of speciﬁc enzymes, which can react only with one site, four essential steps are distinguished, where the ﬁrst step is characterized by the reconnaissance between polypeptide receptors and activated enzyme pole. Second step is the rotation in geometrical structure of enzymes under speciﬁc attractive forces. The third step reﬂecting the compatible adhesion between enzymes site and polypeptide receptors, thus promoting the breakdown of chain sequence of polypeptides. Final step (4) indicated the dissociation mechanism between the enzyme and polypeptides receptors, meaning the end of the fragmentation action. Same behaviors and same catalytic regimes that have been exhibited by speciﬁc enzyme are displayed by non-speciﬁc enzymes, where the only difference here is that in case of speciﬁc enzymes there is a monotectic interaction between polypeptide receptors and activated enzyme site, whereas for non-speciﬁc there is a polytectic interaction, meaning that the non-speciﬁc enzymes are more ﬂexible than that of speciﬁc enzymes, permitting them to be switched on or switched off depending on certain factors such as afﬁnity, avidity (overall strength of binding) and attractive and repulsive forces including electrostatic, columbic, van der wall, hydrophobic and hydrogen forces.

773 774 775 776 777 778 779 780 781 782 783 784

them to be more rigorous during any relevant method of enzyme transformations, particularly in the case of the synthetic ones. Furthermore, Carrea and Riva (2000) stated that the employment of enzymes via media rather that aqueous media, showed a lot of positive points. For instance, the application of these enzymes revealed noticeably higher values in the solubility of substrates, easy to adapt with synthetically enzymes rather than other degradable ones. The sensitivity can be controlled easily by just a simple tailoring of the reaction medium, without modifying the enzyme parameters. It has been remarked that there is an induction in the catalytic rate of the enzymatic reaction more than 109 fold as compared to their nonenzymatic counterparts (Radzicka and Wolfenden, 1995). It is well acknowledged that reaction mechanisms

take place in the presence of catalytic agent, which in turn adhere on the active site of the substrate by creating a link with certain amino acid, thus, contributing signiﬁcantly to the catalytic transformation process (Peracchi, 2001). Further, the position of the enzyme on these residues (amino acids) is the best way for maintaining and facilitating several biochemical modiﬁcations such as proton transfer reactions and electrostatic environment modulation. It is believed that other manipulation techniques related to the extension in the chain of polypeptides coupled by chimeric and rational design of proteins have major effect on stability gains (Ó'Fágáin, 2003). In addition, other chemical processes beyond the system have attracted much attention and remained useful, particularly on the new insights in the cross-linked enzyme crystals

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2008). To increase the reproducibility of enzymes during industrial application certain parameters like stability of enzymes and their adaptation under newly physiological environmental conditions should be well controlled for the success of the end products (Sheldon, 2005; Cantone et al., 2007; Durand et al., 2007). Moreover, the emergence of metagenomic gene, metabolimic and proteomic discoveries enforced the research on enzymes and because of these new designs and protocols concerning the enzyme immobilization and bioreactors are developed (Zhang et al., 2005; Avnir et al., 2006; Rusmini et al., 2007; Betancor and Luckarift, 2008; Sheelu et al., 2008; Cabana et al., 2009; Siqueira et al., 2010; Hernandez and Fernandez-Lafuente, 2011). Several ﬁndings on proteins reveal that these complex systems are dynamics and captivating (Olsson et al., 2006a, 2006b; Claeyssens et al., 2006; Roca et al., 2006; Ruiz-Pernia et al., 2008; Castillo et al., 2008; Kanaan et al., 2010; McGeagh et al., 2011). Therefore, their biomolecular simulations can make it easy to assess the dynamic behavior of the molecules by going more in detail on enzyme reactions often in combination to atomistic modeling for better understanding of the mechanism of action and their mode of transmission, biotransformation and how they involve in different metabolomics processes (e.g. anabolism and/or catabolism) within the living organism.

837 838

Biopeptide transportation modes and absorption routes

857

Basically peptides are active substances composing of more than two amino acids linking to each other by an amide bond, facilitating their categorization into dipeptide (one amine bond jointed with two lateral amine acids), tripeptides (two amine bonds forming a short sequence between three amino acids), tetrapeptides (three amine bonds attaching successively to four amino acids) and ﬁnally the polypeptides (more than three amine bonds, possessing high molecular mass compared to that of dipeptide, tripeptides and tetrapeptides) (Perczel et al., 2000). Their molecular weight is very low in comparison to the molecular mass of protein. After enzymatic hydrolysis using different kinds of protease, the short peptides comprising one or two amine bonds that have been released under gastro intestinal tract condition are penetrated through the brush border membrane by either physical transmission phenomenon or by the intervention of speciﬁc proteins transporter agents (see Fig. 3). The short peptides have been found to be more active and their rapidity of absorption is favored, thus, in certain cases their penetration routes are expected to be much vital than amino acids (Webb, 1990). For

858

R O

N C O

(CLECs) mechanism and changes occurred due to the attachments action of the covalent resulting from the active surfaces and polymers. 799 Nowadays, the enhancement of the catalytic rate of such enzyme is 800 being one of the targets of the biochemists via substrate channeling for 801 protecting the instable substances within a complex mixture system, 802 toxic metabolite inhibition and transmission, kinetics forced, antagonism, 803 absorption routes using different modes, controlling the metabolic ﬂuxes 804 and their regulation within the living organisms (Vriezema et al., 2007; 805 Zhang, 2009; Dueber et al., 2009; Wilner et al., 2009; Zhang, 2010a, 806 2010b; Agapakis et al., 2010; Wang et al., 2011; Zhang, 2011). Many of 807 the modern techniques including freeze drying coupled by lyoprotectants 808 helped to preserve this catalytic activity with desired biologically func809 tions (Dabulis and Klibanov, 1993; Triantafyllou et al., 1995) or often in 810 combination to protein engineering to develop other kinds of mutant en811 zymes (Arnold and Volkov, 1999). However, the recent biotechnological 812 advances have shown a synergistic effect towards many strategies includ813 Q57 ing genetically modiﬁed enzymes (Rodrigues et al., 2011), which is later 814 subjected to additional amelioration through chemical adaptation (Gron 815 et al., 1990; Davis et al., 1999; Matsumoto et al., 2002) or immobilization Q59816 Q58 (Chiang et al., 2008; Ortiz-Soto et al., 2009; Van-Loo et al., 2009; Vallin et al., 2010). In addition, the channel substrates that are designed to inQ61 Q60 817 crease the network interaction with other biopolymers and also the pre818 caution rewarded to avert any losses in the efﬁcacies relating to the 819 cofactors or intermediates all of them contributed signiﬁcantly to the en820 zyme evolution (McGeagh et al., 2011). This is the reason why many of 821 the biochemists have focused much attention to the enzymes production, 822 puriﬁcation and modiﬁcation, starting from the basic framework going 823 deeply into mathematical modeling for a better understanding the mech824 anism of action of these catalytic systems (García-Viloca et al., 2004; 825 Q62 Claeyssens et al., 2006; Olsson et al., 2006a, 2006b). 826 Nevertheless, at this moment and due to the huge available informa827 tion on enzymes, these catalytic systems perform various activities and 828 function in the deﬁciency of immensity water. Therefore, they can be eas829 ily modulated as well as their stabilities and selectivities could be altered 830 and tailored (Klibanov, 2001). Owing to their own activity characteristics, 831 selectivity and speciﬁcity, enzymes have been widely utilized for multi832 purpose uses such as engineering laboratories for reason of investigation 833 and development of modern enzymes like Xenozymes (genetically mod834 iﬁed enzymes), industrial application relating to fermentation and bio835 chemical transformation, especially during food manufacturing (Schmid 836 et al., 2001; Straathof et al., 2002; Pollard and Woodley, 2007; Woodley,

797 798

Fig. 3. Illustrates different routes of penetration relating to several mechanism of transportation in the presence of speciﬁc carriers, facilitating the absorption of active peptides. Once these substances handled by speciﬁc agents under number of reaction mechanisms, their transmission modes recognized depends on the living system roles, and therefore the active peptides transported either by: endocytosis route, intervention of speciﬁc paracellular carrier, on form passive diffusion route, or through a lymphatic system route. However, the importance of peptide permeability, which is controlled by the capillarity absorption phenomena plays an important role in inducing the entrance of these active fragments into the intestinal lymphatic system.

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With regard to the other biological functions such as antihypertensive and immunomodulating properties, the assessment of the antioxidative activity of biopeptide by using an in vitro test is getting an agreement by the scientists, due to its validation data exhibited during practical usages. However, the hypothesis concerning the relationship between the antioxidative properties that is revealed in vitro and antioxidative capacity that is veriﬁed in vivo is still getting acceptance and it needs more practical evidences to be considered. This is because the majority of active substances are expected to be catalyzed and modiﬁed further in the intestine domain. Low molecular mass and other properties like hydrophobicity are expected to be one of the dominating factors responsible for the biological activities of these active fragments. Basically this characteristic may become more pronounced if these generated systems are in the short forms, which permit their intact passage into blood domain, thus, causing many biological effects in the tissue level (Gardner, 1988a, 1988b). Several researches demonstrated that the peptide chain comprising two to six amino acids have been found to be transmitted into portal circulation readily than those having long amino acid residues (Grimble, 1994). Further, the afﬁnity to the absorption is possibly dominated by structural and function parameters like hydrophobicity and molecular size, which could affect signiﬁcantly the transportation and integrity of peptides (Shimizu et al., 1997). Under appropriate circ*mstances, this physiological process commences to accomplish the absorption of peptides on their intact form through ordinary transmission pathways. Another study on the drug delivery system indicated that the hepatic metabolism can be escaped when the transmission of this active systems occur via the gastrointestinal lymphatic route (Wasan, 2002). On the other hand, multiple conclusions on biopeptide researches have shown the efﬁcacies of short peptides and their ability to be retained into portal circulation by different mode of transportation compared to that exhibited by protein and free amino acids with regard to their molecular mass variation. However, the low molecular mass (short sequence chain) of the peptide increases their prospect of transportations via different system of transmissions, whereas their opportunities of penetration or crossing the portal circulation will decrease once their chain amino acids sequence elongate. This suggestion is in agreement with the ﬁnding of Roberts et al. (1999). Speciﬁc study on biopeptide indicated the important presence of proline or double proline on the lateral chain of peptides, because they contribute to maintain good resistance and rigidity for peptide against enzyme attachments, thereby exerting better biological functions (FitzGerald and Meisel, 2000). Another study indicated that the excess amount of biopeptides might lead to subsaturation of peptide transporters, which in turn delayed their entrance into blood domain (Matsui et al., 2002). Therefore, the insufﬁcient studies on bioavailability of peptides in vivo compared to the pronounced results that have been shown during an in vitro test is attributed to the oral administration of these active fragments and also due to the long way of transmission, starting from mouth mastication, stomach digestion and ﬁnally intestinal absorption (Erdmann et al., 2008). Once these active substances remain for a long time within the stomach and under the action of gastric enzyme secretion and later by pancreatic enzymes, the biopeptides in this case will become more fragile and their transportation will be retarded. For this reason, the microencapsulation is needed

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(1) The paracellular route, which is based on the diffusion mechanism via stretched connection constructed by the surrounding cells; this mode of transport in fact is highly depending on water-soluble peptides, but independent on energy sources (Gardner, 1988; Miguel et al., 2008). (2) The passive diffusion which is an auto-mechanism independent on energy source and usually it takes place on transcellular level; supporting too much the hydrophobic peptides (Shimizu et al., 1997; Ziv and Bendayan, 2000; Satake et al., 2002). (3) By the intervention of speciﬁc transporter, which allow the exit of certain active fragments from the enterocyte domain into the portal circulation, this mode of transport usually acts more in the presence of short peptides having hydrolysis-resistance (Gardner, 1984). (4) Via an endocytosis, where this phenomenon based principally on the binding afﬁnity of molecules recognized by the receptors of the cells and their penetration trade into cell maintained by vesiculization, which is usually adaptable with the large polar peptides (Gardner, 1988; Ziv and Bendayan, 2000). (5) Via lymphatic system, where the absorption of peptides in this case occurred between two barrier systems, starting from the exo-system (intestinal tract) going into the endo-system (intestinal lymphatic), this mode of transmission, in fact more suitable to the highly lipophilic peptides, because they can be retained easily into the portal circulation (Deak and Csáky, 1984; Rubas and Grass, 1991).

949

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Biopeptide resistance and bioavailability

R O

887

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detection in the diminution of the electrical resistance of trans epithelial, which is strong evidence that the administration of some food compounds might increase the fragility of peptides spontaneously (Shimizu, 1999). An additional beneﬁt to functional properties of protein compared to the pure peptides is that in rat intestine certain solvent drugs locating between the stretched junctions of surrounded cellular walls initiated to activate in the presence of other organic compounds such as carbohydrates and amino acids, which in turn enhance the absorption of long peptide fragments (Pappenheimer and Volpp, 1992).

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instance, some speciﬁc carrier of peptide named as PepT1 can be functioned depending on its broad substrate speciﬁcity and due to its mode of activity, which highly necessitates the driving force resulting from the electrochemical proton gradient generated during the transmembrane reaction (Yang et al., 1999). Moreover, various intracellular peptidases could be involved as well as in the hydrolysis mechanism of protein present in the enterocyte domain, thereby, permitting the liberation of the maximum active fragments inside the cytoplasm. Hence, other amino acids residues will be oriented to the portal circulation via speciﬁc transporter agents, allowing them to crossing the basolateral membrane as one of their preferred routes of penetration (Ganapathy and Leibach, 1999). Basically, there are ﬁve principal routes for peptides:

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On the other hand, Gardner (1988), and Grimble (2000) expected that few doses of these active fragments and under complete enzymatic 917 digestion can break out, allowing them to cross into the portal circula918 tion without any physical or chemical changes. Consequently, it is ap919 parent that many factors relating to the peptide structural such as 920 hydrophobicity exhibited an important role in enhancing the accelera921 tion of peptides absorption in addition to their low molecular weight, 922 thus sustaining their major pathways of transportation (Shimizu et al., 923 1997). Moreover, the existence of peptidases on the membrane surface 924 might induce the retardation of penetration rate of peptide fragments 925 by hydrolyzing them before their intact absorption. As a result, the pres926 ence of valine-tyrosine into marginal blood domain might explain by 927 the total occupation of the speciﬁc transporters responsible for trans928 mitting this peptide into portal circulation (Matsui et al., 2002). It is 929 outlined that any active substance may has some limitation, for this rea930 son, an additional work needed to be carried out by conducting an 931 in vivo tests, verifying all models of transportation in order to be more 932 pronounced than that exhibited during an in vivo examination of 933 Caco-2 cell monolayer (Vermeirssen et al., 2004). Further, the presence 934 of other foreign compounds might inﬂuence the rate of degradation and 935 transportation of these vital molecules due to the compaction, resulting 936 from the assemblage of certain molecules to each other, which may delay 937 the time for speciﬁc transporter to recognize the target molecule that is 938 Q66 designed to be carried (Charman et al., 1997). This suggestion is given be939 cause during an in vivo examination using Caco-2 cell monolayer, there is

942 943 944 945 946 947 948

952 953 954 955 956 957 958 959 960 961 962 963 964 965 Q67 966 967 968 969 970 971 972 973 974 Q68 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003

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Several in vitro tests have been employed to verify the biological function of many isolated active compounds, for instance in vitro rapid system named cell model culture is considered the ﬁrst choice for fast screening of the biological activity of such vital substances, especially on the assessment of bioavailability and other biochemical transformation mechanism of biopeptides during metabolism. In fact, the manipulation of this model is easier and more reproducible than animal studies and clinical trials related to the human organism investigation. By using cell culture as one of the in vitro tests (see Fig. 4), the results generated for antioxidative activity are found to be more pronounced, and this is an additional advantage for biopeptides as natural health protectants towards human organisms, particularly in declining the propagated free radicals (Liu and Finley, 2005). Further, the role of these chemical elements have involved in maintaining and sustaining many of the vital

Basically, once the biological activities such as antioxidative, antihypertensive and immunomodulatory are more pronounced during in vitro manipulations screening, in this case, the in vivo study should be carried out to ensure the bioavailability of these active systems and their stability under upper GI digestion conditions. Typically this stage

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General concerns related to in vivo biological activities

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General concerns related to in vitro biological activities

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mechanisms such as enzyme modulation, antibacterial activity, anticancer activity (Vermeirssen et al., 2002; Liu and Finley, 2005; Vermeirssen et al., 2005; van Breemen and Li, 2005), immunostimmulation, regulation of perturbed hormone secretion and gene expression, particularly in case of cell differentiation or proliferation (Chu et al., 2002; Sun et al., 2002; Waladkhani and Clemens, 1998). Moreover, they play an important role especially in diminishing platelet aggregation; they also have anticholesterol and antihypertensive modulating activities (Libby et al., 2002). Several studies have used many manipulation tests to verify whether or not these active fragments and their applied dosages have a cytotoxic effect towards the cell culture models and also, their ability to diminish the intracellular oxidation and further inﬂammatory responses (Mosmann, 1983; Elisia and Kitts, 2008; Wolfe and Liu, 2007). For example, the scavenging of the generation of intracellular ROS was carried out by peptides like PR (Wang et al., 2009), and cell-penetrating peptide (SS-31) (Zhao et al., 2005).

1010

to maintain this stability. In certain cases, it is suggested that even under the degradation action of proteolytic enzymes in vivo, this behavior in fact may help to generate the appropriate active fragment compared to the mother fragments, which in turn can exhibit signiﬁcant biological functions towards the living organism, this suggestion is supported particularly in case when the biopeptides are present in the form of long chain amino acids like polypeptides (Li et al., 2004).

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Fig. 4. Illustrates different assays, veriﬁcation modes, and processes related to biopeptides, starting from production until practical application. All these steps are explained as follows: (1) and (2): Indicate the trade of biopeptides, which is produced to ensure certain biological requirements such as the antioxidative and antihypertensive properties in our living systems. (3) Shows the microencapsulation technique of these active fragments to ensure their bioavailability from the enterocyte domain into portal circulation as long as possible. (4): It reﬂects the incorporation of these biopeptides into fermented foods such as cheese, yogurt or lyophilized infant milk products. (5): Integrating biopeptides into spontaneously hypertensive rat (SHR) via oral administration for further in vivo tests. (6): Indicates that the cells are discarded from SHR after speciﬁed feeding regime that is adequately expected to fulﬁll the biological target, where the animals here are sacriﬁced using CO2, then their isolated cells are survived under optimum condition of the culture medium. (7): This step shows an in vitro test on cell level by examining the proliferation assay of bioactive peptides. (8) and (9): Represent an in vitro test on molecular level (e.g. blood and its fractions are used as starting material for manipulation test) in order to verify the immunostimulating activity of bioactive fragment. (10) and (11): In vitro veriﬁcation of the antihypertensive and antioxidative activities of biopeptides, respectively.

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Owing to their efﬁcacies in declining the propagation of free radicals, biopeptides show a great impact on the protection of the whole living systems from rigorous risks including both short and long-term maladies (Klompong et al., 2007, 2008a, 2008b; Ngo et al., 2011). For instance, Thiansilakul et al. (2007) found that scad muscle hydrolysates using ﬂavourzyme is better than alcalase from an antioxidative point of view, because the generated peptides using ﬂavourzyme showed to have a robust scavenging activity towards DPPH radicals and reducing power as well, whereas in the presence of alcalase as the catalytic element, permitted only to obtain a slight Fe2 + chelating ability. Je et al. (2007) revealed the efﬁcacies of papain, neutrase, alcalase, trypsin, and ﬂavourzyme in exhibiting the highest inhibition potency on lipid peroxidation instead of using other proteases. Peng et al. (2009) demonstrated that after an induction time of hydrolysis to 5-h using alcalase, whey protein isolate (WPI) displayed signiﬁcant reducing capacity towards free radicals. In the same way, Park et al. (2004) introduced Electron spin resonance (ESR) spectroscopy to valorize edible seaweed (Sargassum horneri) after an enzymatic extraction process; they stated that the majority of free radicals have been scavenged. Without negligent herbs and plants and due to their high levels of polysaccharides, these materials in fact are considered also potential sources for deriving active compounds including biopeptide, phenolic, and ﬂavonoid substances. However, speciﬁc attention was given to Alfalfa leaf protein by Chen and Qiu (2003), Xie et al. (2008) in order for it to be used as novel source of antioxidative ingredient. After further fractionation and ultraﬁltration, Xie et al. (2008) stated that the most puriﬁed peptides have shown to eliminate free radicals and also, bio-macromolecules peroxidation (Hook et al., 2001). Particular attention has been given by Sheih et al. (2009), to investigate algae protein waste in order to generate an antioxidative peptide by using pepsin; the obtained peptide is sequenced in the form VECYGPNRPQF, after further chromatographic separation and isolation processes. The examination of this active fragment resulted in

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Role of biopeptides in scavenging oxidative stress

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signiﬁcant DNA protection, prevention of hydroxyl radical and scavenging of cellular damage. Furthermore, no cytotoxicity has been detected for this peptide during their bioavailability assessment under gastrointestinal conditions. The incorporation of biopeptides into food products is one of the strategies that have been widely used by the dairy industrial technology, for instance, Farvin et al. (2010a, 2010b) veriﬁed the antioxidative properties of milk-enriched with ﬁsh oil, and during the assessment they noted that the lower molecular fractions (3–10 kDa and b3 kDa) are found to have the most effect on iron-chelating activity rather than other fractions having high molecular weight. Zhong et al. (2007), stated that the silver carp protein, particularly, SCPH-V that is hydrolyzed by pepsin exhibited the maximum free radical-scavenging capacity and antioxidative stress in human intestinal epithelial Caco-2 cells. As a conclusion, they mention that peptide fractions are dependent on their molecular weight and type of amino acid incorporated in the sequence chain, whereas their antioxidant activities are correlated positively with the total number of hydrophobic amino acids. Kudo et al. (2009), have successfully puriﬁed three short antioxidative peptides namely 5A, 5C and 6C by using pancreatin and amano protease enzymes from potato protein hydrolysates compared to the long peptides chain obtained by While, Hsu et al. (2009) from tuna cooking juice hydrolysates using orientase. It has been noticed that pancreatin and amano protease are good in terms of generating short biopeptides compared to orientase digest. They concluded that the presence of Pro, Tyr, and His among AAs of the sequence chains perhaps is responsible on the antioxidative activity. In part, Ma et al. (2006) on the digestion of buckwheat protein by using pepsin–pancreatin enzymes and puriﬁed by LC–MS/ MS. They reported that the most identiﬁed peptides are Pro-Trp, ValPro-Trp, Trp-Pro-Leu, and Val-Phe-Pro-Trp, all of which exhibit maximum ABTS+• scavenging capacity. Cheison et al. (2010) stated that trypsin can contribute to determine the appropriate amino acid at the speciﬁc termini of the designed peptides that is oriented to react with speciﬁc biological target. Zhang et al. (2010), on rice endosperm protein (REP) which is hydrolyzed using various proteases indicated that the amino acids located on the N-terminal of the antioxidative peptide fragments are Phe and Lys, whereas on their C-terminal sides there are Lys and Phe, respectively. Li et al. (2007) reported that the most effective antioxidative peptide after hydrolysis of porcine skin collagen using many proteases is Gln-Gly-Ala-Arg, and due to their amino acid residues incorporated in the sequence chain its proton-donating activity has been found to be strong. Bernardini et al. (2011) exploited thermolysin to degrade sarcoplasmic proteins derived from bovine livers and demonstrate their potentiality in liberating antioxidative peptides. The amino bond linking the tow amino acid residues (tryptophan and arginine), according to Huang et al. (2010) are the result of the strong oxygen radicalscavenging effect exhibited in a sample of egg white protein. Zhang et al. (2011) successfully identiﬁed an antioxidative peptide named as Asn-Arg-Tyr-His-Glu by using consecutive chromatographic techniques in combination to ABI 4700 proteomics analyzer for a sample of chickpea protein hydrolysates (CPH). Guo et al. (2009) reported that the three identiﬁed dipeptides (Lys-Tyr, Arg-Tyr, and Tyr-Tyr) comprising the Tyr amino acid, which is situated at the C-terminal of the three sequence chains are characterized by phenolic hydroxyl poles, which show to inhibit the free radicals signiﬁcantly through the donating hydrogen atoms mechanism positioned in their hydroxyl group. Sarmadi and Ismail (2010) suggested that the structure of amino acids and their sequence ﬂow in peptide could change the antioxidative capacity signiﬁcantly. Torres-Fuentes et al. (2011) investigated that the best chelating activities could be obtained by combining the high His levels ranging from 20 to 30% and short peptide fragment. Cheng et al. (2010) identiﬁed 2 antioxidative peptides having the following amino acid residues on Nterminal (Ser/Ile) and C-terminal (Tyr/Gln) of both lateral sides of active fragments from a sample of potato protein hydrolysate (PPH) fractionated using ammonium sulfate precipitation. Lu et al. (2010) successfully puriﬁed two peptides (NHAV and HVRETALV) by MALDI-TOF/TOF MS/ MS and they revealed that the application of small dose of 10 μg/ml

1077 1078

required human clinical trials and animal studies (Fig. 4) to record strong evidences about the working mechanism of biopeptides and their natural protective effect against several maladies and cardiovascular diseases (Samaranayaka and Li-Chan, 2011) as well as oxidation damages associated to DNA, lipids and proteins (Collins, 2005; Grifﬁths et al., 2002). On the other hand, an accepted animal model, which is called “Spontaneously Hypertensive Rats”, is one of the animal strains that is getting much acceptance by all researchers, due to its similar biological behaviors to the human organisms, particularly in case of an essential hypertensive human (Fitzgerald et al., 2004). However, many of the researches on antihypertensive activity of biopeptides used SHR during their investigation either on long-term administration of these vital substance or on short-term administration (Wang et al., 2010; Nakano et al., 2006; Hernández-Ledesma et al., 2007; Miguel et al., 2007; Takai-Doi et al., 2009). A speciﬁc in vivo study has been reported recently by Nakahara et al. (2010) on Dahl salt-sensitive rat model to examine the antihypertensive function of active peptide enriched soy sauce-like seasoning and they demonstrated that the in vivo action of ACE inhibitory activity is independent of its in vitro action, thus there is no relation found between them. Nowadays, biopeptides can be consumed in different forms like nutraceutical supplements or as dietary ingredients. However, many precautions about these active substances should be taken into account as safety concerns such as their mechanism of actions, mode of delivery, least systemic effects on the living system, toxicity, and required dosage, particularly in case of the synthetic peptides. For this reason, the majority of the in vivo studies concerning the immunomodulatory activity of peptides are not pronounced compared to those done during in vitro tests, this is because of changes in the experimental protocols, starting materials, mode of puriﬁcation, and also variation in the animal models used to verify the mechanism effect in vivo (Gauthier et al., 2006).

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1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243

Role of biopeptides as antihypertensive agents

1278

Biopeptides can be deﬁned as authentic components with mono-biand multi-functional biological activities exhibited from their amino acid residues, which made them capable to serve different mechanism of action like intonation, stimulation, depression, and inhibition once they exist with adequate dosages in the living organism. The angiotensin-1-converting enzyme (ACE) is well deﬁned that has a direct link with the rennin–angiotensin system that can maintain and regulate the human blood tension and it reported to cleave the dipeptides at the C-terminal of HHL. Therefore, the mechanism of ACE is that when the blood pressure increase there is notiﬁcations that ACE is present with abundant amount, which in turn ACE stimulates the blood perturbation by converting angiotensin I released from angiotensinogen by the intervention of rennin to angiotensin II as vasoconstrictor potent agent (Pihlanto-Leppälä, 2001a, 2001b). ACE has been reported also to be able in degrading the bradykinin and stimulates the secretion of aldosterone in the adrenal cortex (Petrillo and Ondetti, 1982; Ondetti and Cushman, 1984). The antihypertensive activity of bovine milkderived biopeptides and other food proteins such as legumes/beans, gelatin, and yeast has been reviewed extensively by Ariyoshi (1993a, 1993b) and Yamamoto (1997). The ACE inhibitory has been reviewed by Meisel (2001) showing the main outlook of biopeptides derived from milk protein that are destined to consumers and procedures. It was also studied by Ryan et al. (2011) in a sample of muscle protein derived meat products (domestic animals and ﬁsh) and also by Wilson et al. (2011) who elaborated the nutritional value of biopeptides, more focusing on the ACE and prolyl endopeptidase (PEP) as an antinutritional agent isolated from marine and their wastes bioproducts. In the same way, Kim and Wijesekara (2010) reviewed the marine biopeptides and their nutraceutical, pharmaceutical, and

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brane and reverse HPLC reached to purify tow peptides (YGYTGA and ISELGW) as the most prominent active fragments from human milk under the action of proteolytic enzymes (pepsin and pancreatin) digests. An interesting ﬁnding on multifunctional (antioxidative and antifatigue activities) of loach peptide (LP) has been investigated recently by You et al. (2011) following both in vitro (for antioxidant activity) and in vivo (for anti-fatigue activity) tests. This active fragment (LP) promoted several health beneﬁts such as chelating the cupric ion, prolonging swimming time for mice exhaustion compared to the control, lowered lactic acid doses and blood urea nitrogen, ameliorated the endogenous cellular, increased superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and scavenges the peroxidation of lipid in a linoleic acid-based emulsion system, respectively. Table 2 shows the selected antioxidative biopeptide fragments according to the different types of proteolytic enzymes used, including pepsin, trypsin, α-chymotrypsin, alcalase, papain, protease XXIII, colorase PP, and neutrase. In fact, the catalytic action of these enzymes appears to be different among enzymes. As results, wide varieties of active fragments are generated in the reaction medium. These active substances have exhibited variable molecular weight, which is due to the type of fractionation and puriﬁcation used to isolate these vital compounds. For instance, the molecular weights of preponderance antioxidative peptides are in the range of 500 to 1800 Da (Je et al., 2005a, 2005b; Jun et al., 2004; Ranathunga et al., 2006; Wu et al., 2003). Basically, fractionation using membrane cut offs with narrow pores size could enhance the permeability of short peptides with low molecular mass. Further, the chromatographic separation technique HIC plays an important role in the fractionation of biopeptides. This type of technique is more efﬁcient than fractionation using ultraﬁltration and more reproducible under controlled conditions. The selection of the right column for separation taking into account the afﬁnity of biopeptides to the following criteria: lipophilicity, hydrophilicity and chargeability, mobile phase and type of solvent used, ﬂow rate and working temperature all of these parameters and conditions should be veriﬁed and ﬁxed properly. In addition, the right control of enzyme parameters, fractionation, and puriﬁcation using ultraﬁltration, dialysis, and chromatographic techniques coupled by robust standard protocols might contribute signiﬁcantly to reach the desired active fragments. It is apparent from Table 2, that the six-peptide fragments can be stratiﬁed according to their molecular weight on the following order: The leading antioxidative peptides are AOBP2 and AOBP3 with low molecular weight, followed by AOBP1 and AOBP4 with middle molecular weight and ﬁnally AOBP4 and AOBP6 with high molecular weight compared to those having low and middle molecular mass. However, the short peptides have the chance to display more resistance, high activity and fast penetration into portal circulation. Owing to these characteristics such as hydrophobicity, hydrophilicity and chargeability, the short peptides have the ability to be absorbed completely without any physical or chemical changes. Moreover, Val or Leu at the N-terminus of the peptides, and Pro, His, Tyr, Trp, Met, and Cys in their sequences (H.M. Chen et al., 1995; Elias et al., 2008; Uchida and Kawakishi, 1992) are hydrophobic amino acid residues. They can express similar behavior to Val or Leu, which in turn can induce the abundance of short active fragments at the water–lipid interface. Furthermore, they can facilitate the intact penetration of the biopeptides through the lymphatic system, and therefore, causing signiﬁcant inhibition on the propagated free radicals (Ranathunga et al., 2006). It is accepted that the small active fragments have more resistance than those having high molecular weight. The long chain of peptides possesses many biomarkers on its contact surface. Therefore, the endo and exo actions of the enzyme, particularly the non-speciﬁc enzymes might cause signiﬁcant changes on its long chain sequence, particularly on the nature of the amino acids exist on C-terminal and N-terminal. This behavior might delay the recognition time by the transporter agent to hold the appropriate peptides. It can

1180 1181

also attribute to the incompatibility of the peptide biomarkers and activated site of transporter agent occurred due to the physical or chemical deformation or perhaps due to the compatibility in between activated biomarkers of enzymes and transporter agent. In this case, the peptide will be reinforced under the action of speciﬁc reaction phenomenon to be absorbed by other routes such as endocytosis or by taking other ways like the lymphatic system, which is usually supporting large polarity and high lipophilicity peptides. It is well accepted that the lateral Nterminal and C-terminal amino acid of the sequence chain play an important role in the orientation of the absorption mode of these active fragments. As it was remarked from Table 2 that, the leading amino acid of the N-terminal of the six selected antioxidative peptides are his, Asn, Pro, and Asp. While, the prominent amino acids situated on the C-terminal of the lateral side of the sequence chain of active peptide are Leu, Glu, Val, Thr, Ile, and Tyr. It has been reported that the reactivity of peptides depends on the structural form of amino acids present in the sequence chain like aromatic amino (Trp, Tyr, and Phe), imidazole (His), and nucleophilic sulfur (Cys and Met) (Elias et al., 2008). It is hypothesized that the C-terminal amino acid is considered as a ﬁxed side, whereas the N-terminal amino acid is a variable side, thus, the dominant amino acids of the six active peptide fragments are the amino acids exist on the C-terminal of the lateral side of the sequence chain. The N-terminal amino acids are important, especially when the peptide fragment equilibrated by the same amino acid on both of the C-terminal and N-terminal sides, and therefore, the antioxidative property in this case becomes more signiﬁcant. On the other hand, the presence of two different amino acids on the N-terminal and C-terminal sides of the sequence chain of biopeptides made them as versatile fragments having polytechtique poles. Thus, might exhibit various biological functions such antihypertensive and immunomodulatory properties. For this reason, the recent technological advances such as nanotechnology coupled by structure elucidation may help to protect this variable side from further enzymatic degradation and the binding of other active substances like lipids and oligosaccharides.

1178 from these peptides showed to cause protective effect against cell death 1179 Q95 and oxidative apoptosis. Tsopmo et al. (2011) by using molecular mem-

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AOBP2: [Asn-X2-Glu]

AOBP1: [His-X1-Leu]

AOBP4: [Pro-X4-Thr]

AOBP5: [Trp-X5-Ile]

AOBP6: [Asp-X6-Tyr]

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

✓ ✓ ✓

✓

✓ ✓

✓

R O

✓

✓ ✓

✓

✓ ✓

✓

Protein source: Hoki (Johnius belengerii) skin gelatin Chickpea protein hydrolysate (CPH) Loach (Misgurnus anguillicaudatus) Tuna (Thunnus obesus) dark muscle Bovine α-lactalbumin and β-lactoglobulin Duck by products Enzyme applied: Pepsin Trypsin α-Chymotrypsin Alcalase Papain Protease XXIII Corolase PP Neutrase Puriﬁed technique used: Ultra-ﬁltration Chromatographic separation Active peptide fragment: Low molecular weight Middle molecular weight High molecular weight N-terminal amino acid: Non-polar amino acid (hydrophobic) Polar amino acid (hydrophilic) Basic amino acid (positively charged) Acidic amino acid (negatively charged) Aromatic amino acid Aliphatic amino acid C-terminal amino acid: Non-polar amino acid (hydrophobic) Polar amino acid (hydrophilic) Basic amino acid (positively charged) acidic amino acid (negatively charged) Aromatic amino acid Aliphatic amino acid Reference name

AOBP3: [Pro-X3-Val]

✓

✓ Mendis et al. (2005b)

✓ Zhang et al. (2011)

✓

✓ ✓ ✓

✓

t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29 t2:30 t2:31 t2:32 t2:33 t2:34 t2:35 t2:36 t2:37 t2:38 t2:39 t2:40 Q17t2:41 Q18

Evaluation parameters

t2:3

Table 2 Relationship between biopeptides structure and antioxidative biological function.

✓ ✓

✓

✓ ✓ You et al. (2010)

✓ Hsu et al. (2010)

✓ Ledesma et al. (2005)

S.-H. Lee et al. (2010), S.-J. Lee et al. (2010)

t2:1 t2:2

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Abbreviations: AOBP: antioxidative biopeptides; X1: Gly-Pro-Leu-Gly-Pro; X2: Arg-Tyr-His; X3: Ser-Tyr; X4: Met-Asp-Tyr-Met-Val; X5: Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp; X6: Val-CysGly-Arg-Asp-Val-Asn-Gly.

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therapeutic potentials in scavenging the most frequent cardiovascular diseases. Extensive research works have been done to screen this substance, for instance, Cao et al. (2010) examined the ACE of Acetes chinensis as one of the marine shrimps by using pepsin enzyme digestion. They showed that Leu-His-Pro which is in the range of molecular weight of 1320 Da–311 Da exhibited the highest ACE inhibition activity in spontaneously hypertensive rat (SHR) models. Jamdar et al. (2010) treated the peanut protein hydrolysate (PPH) by Alcalase, spending particular attention on how the degrees of hydrolysis (DH) could inﬂuence the biological functions. They revealed that once the DH increased there is a notiﬁcation in the increasing all of the ACE inhibitory activity, DPPH radical-scavenging activity, and ferrous ion chelating activity of PPH samples, in the same time they observed that the reducing powers are dropped signiﬁcantly. Pan et al. (2011) conﬁrmed Leu-Leu (LL) as novel ACE obtained from whey protein that is digested by trypsin and puriﬁed by size exclusion chromatography on Sephadex G-25, and G10 columns and RP-HPLC. Ferreira et al. (2007) stated that hydrophobic amino acids situated on the lateral side of biopeptides fragments; exactly at the C-terminal position is considered the preferred substrates for activating the ACE. Ko et al. (2006) indicated that the enhancing of absorption might ameliorate the bioavailability of oligopeptides to exhibit higher antihypertensive capacity, which is highly depending on the mechanism behavior of cell membrane permeation. Jimsheena and Gowda (2011) examined NAQRP peptide released under different proteases from arachin protein of peanut. They demonstrated that Pro residue advocates ACE inhibitor potency. Jung et al. (2006) successfully

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1312 1313

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t2:42 t2:43

identiﬁed an ACE from yellowﬁn sole (Limanda aspera) by using αchymotrypsin. Zhang et al. (2006) suggested that the presence of the following amino acid residues phenylalanine, isoleucine and glycine at ratio of 1:2:5 may contribute signiﬁcantly to the highest ACE activity. Lo et al. (2006) reported that at 90 min, subsequent digestion by pancreatin of unheated and blanched sterilized isolated soy protein (ISP) the ACEinhibitory activity was decreased compared with peptic digestion alone. H. Wu et al. (2008), J. Wu et al. (2008) studied the ACE of two puriﬁed peptides namely Val-Ser-Val (IC50 = 0.15 μM) and Phe-Leu (IC50 = 1.33 μM) from defatted canola meal samples using Alcalase. Speciﬁc attention has been given to ACE-inhibitory activity to improve the functional properties of extracted proteins, for instance, Campos et al. (2010) investigated the ACE-inhibitory activity using Alcalase®, Flavourzyme® and pepsin–pancreatin to generate hydrolyzed Vigna unguiculata protein extract, whereas, Huang et al. (2011) studied its potency in corn peptides (CP). Ramchandran and Shah (2011) successfully conﬁrmed the ACE effect of yogurt- and probiotic yogurt-based diets on the weight gain, serum lipid proﬁle, and blood pressure (BP) in spontaneously hypertensive rats (14 week old). Pan et al. (2005) reported that Val-Pro-Pro and Ile-Pro-Pro peptides possessing strong ACE inhibitor and antihypertensive activities are obtained by three steps using RP-HPLC. Jang et al. (2008) identiﬁed GFHI, DFHING, FHG, and GLSDGEWQ peptides that shown both antimicrobial and anticancer. Yust et al. (2003) demonstrated that short peptides contain the amino acid “Met” in combination to other hydrophobic amino acids is a good source for ACE. Tiengo et al. (2009) showed that the ACE-inhibitory

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N C O

Asn (PPEIN)(κ-CN; f156–160) with IC50 = 0.29 ± 0.01 mg/ml and Pro-Leu-Pro-Leu-Leu (PLPLL) (β-CN; f136–140) with IC50 = 0.25 ± 0.01 mg/ml. On the other hand, Jiang et al. (2010) isolated two novel ACE-inhibiting peptides Arg-Tyr-Pro-Ser-Tyr-Gly (κ-casein; f 25–30) with an IC50 = 54 ± 1.2 μg/mL and Asp-Glu-Arg-Phe (κ-casein; f15–18) with IC50 = 21 ± 0.8 μg/mL. Jia et al. (2010) in turn accelerated the release of hydrophobic amino acids from defatted wheat germ protein during hydrolytic digestion by using an ultrasonic pretreatment. Another study, which was reported by Pripp and Ardo (2007), established the relationships between ACE and the bitter taste of peptides based on the hydrophobicity properties of the amino acid residues. Table 3 shows the selected antihypertensive biopeptide fragments according to the different types of proteolytic enzymes used, including pepsin, trypsin, chymotrypsin, alcalase, pancreatin, proteinase K, bromelain, thermolysin, protease, Aspergillus protease, gastric proteases and protamex. As it was remarked from Table 3 that the amino acids on the N-terminal of the antihypertensive peptides fragment are Asn, Arg, Gly, Met, and Tyr. Further, the prominent amino acids positioned on the C-terminal of the lateral side of the sequence chain of active peptide are Leu, Pro, Asp, Phe, and Asn. It is apparent from Table 3, that the six-peptide fragments can be stratiﬁed according to their molecular weight on the following order: The leading antihypertensive peptides are AHBP1, AHBP2 and AHBP6 with low molecular weight, followed by AHBP3 with middle molecular weight and ﬁnally AHBP4 and AHBP5 with high molecular weight compared to those possessing low or middle molecular mass. It has been reported that the presence of Pro at the C-terminal of the sequence chain protect the peptide from further degradation (Matsufuji et al., 1994; Vermeirssen et al., 2004). Furthermore, it has been found that the absorbance capacity of short peptides having two or three amino acids is much faster than free amino acids themselves (Webb, 1990). Meanwhile, the larger peptides (10–51 amino acids) can be enter the blood circulation on intact form by crossing the intestine seeking different biological routes and mechanism of transmission. Therefore, the efﬁciency of the peptides has been found to be diminished once the chain length of them is extended (Roberts et al., 1999). However, the appropriate biopeptides having positive results on ACE are possibly associated and dominated by the amino acid residues including Pro, Lys, and Arg located on the Cterminal sequence of the peptides, these amino acids have been found the most detectible on the C-terminal residue having signiﬁcant ACEI-inhibitory potency (Meisel, 1997). Furthermore, the C-terminal containing Tyr residue progressively diminished the systolic blood pressure in SHR models in comparison to the rapid reduction in the systolic blood pressure detected by dipeptides possessing the Phe at the C-terminal as well (Suetsuna, 1998). Thus, short peptides could be accommodated by crystallography compared to large peptide fragments during the conformational test of the active site of ACE (Natesh et al., 2003). It is hypothesized that during hydrolytic digestion, enzymes exhibit high afﬁnity to attack the substrate or inhibitors comprising an aromatic or branched side chain of amino acid residues at the C-terminal amino acid of the active fragment. It is concluded that the prominent amino acid residues contributing signiﬁcantly to ACE-inhibitory peptides activity are situated at the C-terminal of the sequence chain containing Tyr, Phe, Trp, and Pro. Gómez-Ruiz et al. (2004a, 2004b) reported that the L-conﬁguration for the amino acid at the C-terminal position and the variation in cis– trans conformations of Pro might use to control the behavior of ACEinhibitory peptide with its interacted enzyme.

1426 1427

Role of biopeptides in modulating the immune disorders

1483

It is well acknowledged that many of active fragments have been proved to have many biological functions such as cell-mediated immune properties, antimicrobial effect and humoral roles (Agyei and Danquah, 2011). For these reasons, all efforts are reworded to ensure their evidences in the practical usages (e.g. in vivo veriﬁcation) to ensure a good reproducibility of these active substances from pharmaco*kinetic,

1484

R O

activity of peptides digested by alcalase from concentrated amaranth protein give an indication on the enhancement in the resistance of pep1362 tides under gastrointestinal hydrolysis. Ghassem et al. (2011) studied 1363 two abundant fragment of peptides VPAAPPK (IC50 = 0.45 μM) at 1364 791.155 m/z and NGTWFEPP (IC50 = 0.63 μM) at 1085.841 m/z generat1365 ed from Haruan myoﬁbrillar protein under proteinase K and thermolysin 1366 actions. Results showed robust ACE-inhibitory activity for both peptide 1367 fractions, concluding that the existence of two Pro residues at the C1368 terminal sequence is responsible for the high ACE inhibitory activity of 1369 these peptides. Fujita et al. (2000) clariﬁed the ACE-inhibitory activity 1370 of peptides by using three models of inhibitors named as (1) inhibitor 1371 type, (2) substrate type and (3) pro-drug-type inhibitor. This technique 1372 in fact is conducted after getting negative results concerning the ACE-I 1373 activity in order to conﬁrm the effect of peptides towards ACE-I potency 1374 whether it exist in a sample of chicken muscle and the peptic digest of 1375 ovalbumin hydrolyzed by thermolysin enzyme or not. Alemán et al. 1376 (2011) reported that Leu or Hyp residues play an important role in the 1377 ACE-inhibitory activity when they present together on the sequence 1378 chain of active fragment. Rivas et al. (2007) studied the effects of pulsed 1379 electric ﬁelds (PEF) technology (15–40 kV/cm; 0–700 μs) and thermal 1380 processing (84 °C and 95 °C, 15–120 s) on an ACE-I peptides, showing 1381 that, there are no differences in the ACE-I capacity in terms of method 1382 treatments. Contreras et al. (2011) produced the ACE-I peptides αs1-CN 1383 f(90–94), with sequence RYLGY, and αs1-CN f(143–149), with sequence 1384 AYFYPEL from casein hydrolysis process. Liu et al. (2010) reported that 1385 among the 19 peptides that were discovered in the fractions. Only 1386 Arg-Val-Pro-Ser-Leu (RVPSL) shows great ACE-inhibitory activity by 1387 using HPLC assay. Hatanaka et al. (2009) stated that a hydrolyzed Ant1388 arctic krill (Euphausia superba) peptide powder (AKPP) signiﬁcantly 1389 lowered the systolic blood pressure in SHRs by administrating single 1390 Q102 oral doses of 1, 10, or 100 mg. J. Wang et al. (2008), J.P. Wang et al. 1391 (2008) studied heat and pH stability under gastrointestinal conditions 1392 for a sample of Oyster (Crassostrea talienwhanensis Crosse) proteins 1393 that is subsequently hydrolyzed with pepsin. Results indicated that 1394 Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe had the good and strong enzyme 1395 resistant properties against gastrointestinal proteases. According to 1396 Kodera and Nio (2006) the most potent inhibitor was identiﬁed to be 1397 NWGPLV (IC50 = 21 μM), where systolic blood pressure was signiﬁ1398 cantly reduced after the oral administration of doses exceeding 100 1399 mg/kg. Lee et al. (2006) showed that the water-soluble extract from 1400 broccoli had 76.9% ACE inhibitory activity, whereas low values of ACE 1401 inhibitory activities were obtained in the presence of other organic sol1402 vent extracts. Ma et al. (2006) isolated and identiﬁed the following ACE 1403 inhibitory peptide Gly-Pro-Pro; having IC50 value of 6.25 μg protein/ml 1404 from buckwheat (fa*gopyrum esculentum Moench) by using an 1405 electrospray-LC–MS. Motoi and Kodama (2003) investigated the hypo1406 tensive activity of Ile-Ala-Pro on spontaneously hypertensive rats. Re1407 sult was signiﬁcant, yielding an ACE inhibitory activity with IC50 value 1408 of 2.7 μM. Foltz et al. (2009) stated that the N-terminal amino acid res1409 idues including Asp, Gly, and Pro as well as the C-terminal amino acid 1410 residues involving all of Pro, Ser, Thr, and Asp contributed signiﬁcantly 1411 to the stabilization of the active fragments once they subjected to the lu1412 minal enzymatic hydrolysis. However, the retardation in the absorption 1413 of ACE inhibitory activities of these active fragments in vivo is probably 1414 linked to their low intestinal permeability. Lin et al. (2011) they devel1415 oped a pilot-scale production to enhance the antihypertensive effect, 1416 where they found that Ala-Tyr is one of the major peptides of protein 1417 derived corn gluten meal (CGM) having strong ACE. Qu et al. (2010) 1418 suggested that under optimum condition of hydrolysis alcalase might 1419 achieve the highest antihypertensive activity of peptide. Majumder 1420 and Wu (2011) compared the ACE activity of the three novel peptides 1421 IQW, IRW and LKP with the activities those exhibited by the synthetic 1422 ones. They showed that puriﬁed peptide activities have been found to 1423 be compatible with the synthetic ones. Mao et al. (2007) subjected 1424 the Yak (Bos grunniens) milk casein under the action of alcalase and 1425 the results yielded two novel ACE-inhibiting peptides Pro-Pro-Glu-Ile-

1360 1361

1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 Q103 1480 1481 1482

1485 1486 1487 1488 1489

Table 3 Relationship between biopeptides structure and antihypertensive biological function. AHBP4: [Met-X4-Asp]

AHBP5: [Gly-X5-Phe]

AHBP6: [Tyr-X6-Asn]

✓ ✓ ✓ ✓ ✓ ✓ ✓

✓ ✓ ✓

✓

R O

✓

✓ ✓

✓

✓ ✓

✓

✓ Liu et al. (2010)

✓

✓ ✓

✓ ✓ ✓

✓ ✓ ✓ Ghassem et al. (2011)

✓ Zhao et al. (2009)

Saiga et al. (2006)

✓ Tsai et al. (2008)

✓ Jimsheena and Gowda (2011)

✓ ✓

✓

✓ ✓

Protein source: Arachin from peanut protean Egg white protein Fish myoﬁbrillar protein (Haruan. L) Sea cucumber Chicken breast protein (muscle) Hard clam meat Enzyme applied: Pepsin Trypsin Chymotrypsin Pancreatin Alcalase Proteinase K Thermolysin Bromelain Protease Aspergillus protease Gastric proteases Protamex Puriﬁed technique used: Ultra-ﬁltration Chromatographic separation Active peptide fragment: Low molecular weight Middle molecular weight High molecular weight N-terminal amino acid: Non-polar amino acid (hydrophobic) Polar amino acid (hydrophilic) Basic amino acid (positively charged) Acidic amino acid (negatively charged) Aromatic amino acid Aliphatic amino acid C-terminal amino acid: Non-polar amino acid (hydrophobic) Polar amino acid (hydrophilic) Basic amino acid (positively charged) Acidic amino acid (negatively charged) Aromatic amino acid Aliphatic amino acid Reference name

AHBP3: [Asn-X3-Pro]

t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23 t3:24 t3:25 t3:26 t3:27 t3:28 t3:29 t3:30 t3:31 t3:32 t3:33 t3:34 t3:35 t3:36 t3:37 t3:38 t3:39 t3:40 t3:41 t3:42 t3:43 t3:44 t3:45

AHBP2: [Arg-X2-Leu]

AHBP1: [Asn-X1-Pro]

Evaluation parameters

t3:3

t3:1 t3:2

S. Saadi et al. / Biotechnology Advances xxx (2014) xxx–xxx

Abbreviations: AHBP: antihypertensive biopeptides; X1: Ala-Gln-Arg; X2: Val-Pro-Ser; X3: Gly-Thr-Trp-Phe-Glu-Pro; X4: Glu-Gly-Ala-Gln-Glu-Ala-Gln-Gly; X5: Phe-X-Gly-Thr-X-Gly-LeuX-Gly; X6: none.

1490 1491

therapeutic potency and immunomodulatory efﬁcacy point of views. However, the immunomodulatory properties of biopeptides are seemed to be not appreciated yet compared to those exhibited for antioxidative and antihypertensive functions. This is perhaps due to the many challenges occurred during the practical application, especially during the in vivo study, which mainly based on cell culture assessment and also, due to the complexity of chemical reaction and mechanism effect resulting by the end of primary and secondary metabolites within the living system. Limited insights have been elaborated on immunomodulatory properties of biopeptides. For instance, Qian et al. (2011) indicated that the fraction F6 that is obtained from the fermented skim milk displayed a potent immunostimulating function on the proliferation of murine spleen lymphocytes. Eriksen et al. (2008) screened different kinds of milk protein samples (e.g. cow and goat) from immunomodulatory function point of view. They suggested that during hydrolysis of milk protein samples certain components released and showed to produce activation signals that are contributed later to inhibit the proliferation of the lymphocytes. In part, Girón-Calle et al. (2010) suggested that chickpea-derived peptides possess an immunomodulatory activity, which in turn scavenged the propagation of tumors in the colon. Kong et al. (2008) indicated that the immunomodulating activity and positively charged peptides ratio have been found to be correlated positively. They suggested as well as that lower molecular weight of these active fragments are efﬁcient in provoking the activity of immunomodulatory

1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513

1496 1497

1494 1495

1492 1493

t3:46 t3:47

peptides. Sheu et al. (2004) puriﬁed a new immunomodulatory protein (APP) from mushroom and revealed that the resulted peptide has the ability to enhance signiﬁcantly the production of NO and TNF-α by LPS-induced RAW 264.7 macrophages at dosages ranging from 5 to 20 μg/ml. Particular study on partially puriﬁed peptides (PFP) motif with D-Pro from bovine β-casein was done by Bonomi et al. (2011). They revealed that the changing of the last proline (P206) caused a negative impact on the immunosuppressory activity, whereas its substitution with amino acid residue having same structural analogues of proline did not show any effect. Clement et al. (2010) reported that the three isolated immunomodulatory proteins from raw garlic exhibited mitogenic activity towards murine splenocytes, thymocytes, and human peripheral blood lymphocytes. Sauveur et al. (2008) revealed that peptide fractions derived from a whey protein isolate (WPI) and degraded by combined trypsin: chymotrypsin enzymes on form of enzymatic digest (ED) fractions successfully stimulated the proliferation of splenocytes with and without ConA. Haddani et al. (2006) supplemented CHO cell cultures by various peptide mixtures. They revealed that the addition of 4 g/l of small peptides with molecular mass of less than 500 Da signiﬁcantly stimulates cell growth. Fang et al. (2007) investigated the immunomodulatory of Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr peptide function from bovine placenta water-soluble extract. However, the incorporation of this active system at different doses showed to stimulate the proliferation of lymphocyte and non-signiﬁcant effect of hom*ology was remarked

1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537

S. Saadi et al. / Biotechnology Advances xxx (2014) xxx–xxx

N C O

1592

Bi- and multi-function validations of biopeptides fragment

1593 1594

Table 5 generates the global idea on how the biopeptides could exhibit more than one biological function as it stated by Meisel and FitzGerald (2003) in the introduction. It has been pointed out from Table 5 that there is a cross-link biological network between the Nterminal and C-terminal amino acids responsible for the antioxidative, antihypertensive and immunomodulatory biological functions. Based on this observation, the margining of the N-terminal amino acids of the three biological functions (antioxidative, antihypertensive and immunomodulating) with their C-terminal amino acids revealed a bi-

1595 1596 1597 1598 1599 1600 1601

function validity among certain bioactive peptide fragments. From Nterminal position the bi-function validity can be detected between AOBP2 and AHBP1, these type of fragments have been veriﬁed previously by Jimsheena and Gowda (2011); Zhang et al. (2011), for only one biological function, but in fact they possess both of the antioxidative and antihypertensive activities. Same observation has been noted between the following peptide fragments: AOBP2–AHBP3, AOBP2–IMBP1, AOBP5– IMBP6, AHBP5–IMBP2, and AHBP6–IMBP5 where these types of fragment exhibited antioxidative and antihypertensive activity, antioxidative and immunomodulatory activity and the last one is between antihypertensive and immunomodulating biological activities. Moreover, from the Cterminal position the AOBP1–AHBP2, AOBP4–IMBP5, AOBP6–IMBP6, AOBP6–IMBP4, AHBP1–IMBP1, AHBP1–IMBP3, AHBP3–IMBP1, and AHBP3–IMBP3 displayed antioxidative with antihypertensive functions and antioxidative with immumodulatory functions, and ﬁnally antihypertensive with immunomodulatory functions. Finally, from the N-terminal position the multi-function has been detected between AOBP2–AHBP1/ AHBP3–IMBP1, demonstrating the wide contribution of biopeptides towards the human organisms through their capability in regulating food intake, scavenging free radicals and modulating the immune disorders (See Table 5). Nevertheless, Tsopmo et al. (2009) claims that under circ*mstances of hydrolytic enzymes a potent radical scavenging activity was detected for amino acid “Trp” when it was liberated within human milk. Many of the mechanisms of action could be used to express the activity and/or capacity of antioxidative peptides such as aldehyde adduction, metal ion chelation and free radical scavenging (Chen et al., 1996; Zhou and Decker, 1999). They reported that the presence of Trp or Tyr residues at the C-terminal position of the sequence chain showed to cause robust scavenging activities towards the free radicals, but inadequate scavenging capacity against peroxy nitrite. Chan and Decker (1994) suggested that the information gathered on amino acid “His”, particularly, when it was located on the lateral side of the peptides is not quite enough to express the antioxidative function obtained, meaning that, this activity could be attributed to other chemical reaction like metal ion-chelating ability and lipid peroxy radical trapping or due to the chemical behavior of certain element group like imidazole group. Chen et al. (1996) indicated that the substitution or complete changing of amino acid “His” located on the C-terminal of the sequence chain lowered the biological function towards the inhibition of free radicals, whereas elimination of amino acid “Leu” located on the N-terminal sequence chain of peptide fragment did not show any effects. It is worthwhile to state that some correlations in between constant side of the active fragment and ACE-I-inhibitory peptides often in combination to the ACE enzyme could be established. For instance, the peptides structure and their antihypertensive effects were evaluated in the previous section (See Table 3) might raise new insights on understanding the relationship between structural characteristics of both constant and variable amino acids positioned on the lateral sides of the sequence chain of peptide fragments. It is suggested that the positively charge amino acids “Lys” and “Arg” as the ﬁxed side may donate to the inhibition efﬁcacies (Ondetti et al., 1977; Cheung et al., 1980; Ariyoshi, 1993a, 1993b; Meisel, 2003), “Leu” may donate the capacity of ACE-I, whereas, other branched chain aliphatic amino acids like “Ile” and “Val” are the prominent residues in peptide-rich ACE inhibitors (Gómez-Ruiz et al., 2004a, 2004b). It has been recognized that inactive peptide molecules did not show similarity pattern to the ACE-inhibitory peptides, which in turn inﬂuence their binding to ACE, particularly when the structure will adopt under speciﬁc environment of the binding site (Yamamoto et al., 1994; Meisel, 2003; Fitzgerald and Meisel, 2000). Based on the unique structure of biopeptides, which is highly depending on their amino acids types (aliphatic or aromatic) and nature (positively charged or negatively charged as well as hydrophobic or hydrophilic) their functionality can be determined and further ameliorated. For instance, an antimicrobial properties have been detected for most peptides having small size, polar and positively charged in nature (Hanco*ck

R O

in the presence of other isolated peptides. Further, it exhibits thermal stability under 80 °C during an incubation time of 30 min with an im1540 mune potency of 60%. Lactoferricin can be categorized among the potent 1541 peptides that could exhibit multifunctional activities such as anti-fungal, 1542 immunomodulating, and antimicrobial. The peptide showed to possess 1543 an antiviral property, which attributed to the amino residues Try/Arg in 1544 the sequence chain of the peptide, whereas the anti-inﬂammatory and 1545 immunomodulating properties were attributed to its amino residue 1546 with positively charged region located at the lateral of biopeptides frag1547 ments (Vogel et al., 2002). High chance for obtaining potent biological 1548 activities such as anti-cancer and immunomodulating possibly can be 1549 achieved using lactoferricin hydrolysates (Lòpez-Expòsito and Recio, 1550 2006). Most of the immunomodulating biopeptides were isolated from 1551 bovine caseins, the C-terminal hexapeptide was found to stimulate the 1552 Q104 macrophages (Fiat et al., 1989). The mechanism of action of these types 1553 of peptides is quite similar to that of opioid biopeptides, where the pos1554 itively charge like Arg at the C-terminal would have strong afﬁnity with 1555 cell receptors, thus provoking signiﬁcant physiological changes at tissue 1556 level such as stimulation the proliferation and maturation of immune 1557 system cells, macrophages phagocytic activities and antibody synthesis 1558 (Kayser and Meisel, 1996; Paegelow and Werner, 1986). 1559 Table 4 shows the selected immunomodulatory biopeptide frag1560 ments according to the different types of proteolytic enzymes used, in1561 cluding pepsin, and trypsin. It is pointed from Table 4 that the amino 1562 acids on the N-terminal of the immunomodulatory peptide fragments 1563 are Asn, Gly, Ser, Val, Tyr, and Trp. Further, the prominent amino acids 1564 positioned on the C-terminal of the lateral side of the sequence chain 1565 of active peptide are Pro, Arg, Tyr, and Trp. It is observed that the six1566 peptide fragments are categorized according to their molecular weight 1567 into three groups: IMBP1, IMBP3, and IMBP6 with low molecular weight, 1568 followed by IMBP2 and IMBP4 with middle molecular weight and ﬁnally 1569 IMBP5 with high molecular weight. The C-terminal sequence of β-casein 1570 showed to possess a direct effect on the immunomodulatory properties 1571 of the system (Coste et al., 1992; Sandre et al., 2001), whereas a potent 1572 antigenic epitope devoid of immunomodulatory function has been de1573 tected through the N-terminus of β-casein (Monetini et al., 2003; 1574 Youseﬁ et al., 2009). Wiśniewski et al. (1994) revealed that the incorpo1575 ration of peptide with small doses of 1 μg, 2 μg, and 5 μg lowered the psy1576 chom*otor activity of SHRs signiﬁcantly. Immunomodulatory peptides 1577 can enhance different immune cell functions, including cytokine regula1578 tion and lymphocyte proliferation (Horiguchi et al., 2005; Fitzgerald and 1579 Murray, 2006; Hartmann and Meisel, 2007). An effective result on 1580 immunomodulating activity has been noticed for the lower molecular 1581 mass and positively charged peptides isolated from soy proteins (Kong 1582 et al., 2008). Under certain circ*mstances of hydrolysis dipeptide may 1583 exhibit strong activity by its lateral amino acid residues, for instance 1584 Gly-Pro-Hyp bonds have been found more resistance against proteolytic 1585 enzymes resulting from the middle amino acid “Pro”, which is hypothe1586 sized to equilibrate the mechanism of action during the interaction 1587 (Wrόbel, 1992). Speciﬁc study on biopeptides drowned attention to 1588 the pharmacological activity of Gly (Zawadzka-Szeremeta, 1979), dem1589 onstrating that this amino acid lowered the psychom*otor activity of 1590 rats in Lat's test, diminishes catalepsy, promotes the apomorphine- and 1591 sustained the amphetamine stereotypy.

1538 1539

1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 Q105 1654 1655 1656 Q106 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667

Table 4 Relationship between biopeptides structure and immunomodulating biological function. IMBP5: [Tyr-X5-Thr]

✓

IMBP6: [Trp-X6-Thr] ✓

✓ ✓ ✓ ✓ ✓ ✓

✓

✓ ✓ ✓

✓

✓ ✓

✓

✓ ✓

✓

✓ Takahashi et al. (1994)

✓

✓ Hou et al. (2012)

✓

✓ H.M. Chen et al. (1995), J.R. Chen et al. (1995)

✓ ✓

✓ Parker et al. (1984)

✓ Fang et al. (2007)

✓ Hou et al. (2012)

Abbreviations: IMBP: immunomodulatory biopeptides; X1: Gly-Leu-Ala; X2: Thr-Pro-Met-Tyr-Pro-Leu-Pro; X3: Gly-Phe-Ala; X4: Glu-Pro-Ile-Pro; X5: X-Phe-Leu-Gly-Leu-Pro-Gly-X; X6: none.

t4:35

IMBP4: [Val-X4-Tyr]

Protein source: Alaska pollak (ﬁsh) frame Rice albumin Soybean protein Human casein Bovine placenta water soluble extract Enzyme applied: Pepsin Trypsin Puriﬁed technique used: Ultra-ﬁltration Chromatographic separation Active peptide fragment: Low molecular weight Middle molecular weight High molecular weight N-terminal amino acid: Non-polar amino acid (hydrophobic) Polar amino acid (hydrophilic) Basic amino acid (negatively charged) Acidic amino acid (positively charged) Aromatic amino acid Aliphatic amino acid C-terminal amino acid: Non-polar amino acid (hydrophobic) Polar amino acid (hydrophilic) Basic amino acid (positively charged) Acidic amino acid (negatively charged) Aromatic amino acid Aliphatic amino acid Reference name

IMBP3: [Ser-X3-Pro]

R O

t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18 t4:19 t4:20 t4:21 t4:22 t4:23 t4:24 t4:25 t4:26 t4:27 t4:28 t4:29 t4:30 t4:31 t4:32 t4:33 t4:34 Q19

IMBP2: [Gly-X2-Arg]

IMBP1: [Asn-X1-Pro]

Evaluation parameters

t4:3

t4:1 t4:2

S. Saadi et al. / Biotechnology Advances xxx (2014) xxx–xxx

1668

and Sahl, 2006; Korhonen and Pihlanto, 2006). In addition, signiﬁcant enhancement was noticed towards lymphocytes proliferation in the presence of isolated short peptides as it was reported by Pihlanto1671 Leppälä (2002). In fact, these parameters play a key role for their prima1672 ry sequence, which rarely could be developed into complicated special 1673 structure through the binding mechanism (Kaur et al., 2007).

1669 1670

Biopeptides with antimicrobial and anti-fungal properties

1675 1676

By deﬁnition an antimicrobial biopeptides are biological active fragments comprised of less than 50 amino acid residues with a molecular mass of less than 10 kDa. The majority of amino acid residues are hydrophobic in the nature. These types of bioactive peptides are typically produced using an enzymatic hydrolysis under in vitro conditions of microbial proteases (Bulet et al., 2004; Reddy et al., 2004). They can exhibit an efﬁcient role in the host defense against the most frequent pathogenic bacteria that interact directly with them and therefore scavenging them. Several methods have been reported to identify the biological role of bioactive peptides from protein hydrolysates and among these methods; we have in particular the agar diffusion assay, which is named as inhibition zone assay (Hickey et al., 2003). The basic of this method is the capability of formulated biological substances to scavenge and eliminate the bacterial growth in the formulated solid media by determining the minimum inhibitory concentration (MIC) after appropriate incubation time. Generally, the assay needs certain preparation parameters to be manipulated such as different concentrations of the biological substances and selection of appropriate bacteria strains. The migration of the biological substances towards the agarose medium will leave clear zones. However, the addition of an active compound such as antimicrobial peptides could exhibit a pivotal role in declining the risk of bacteria development (Bonev et al., 2008). Milk derived biopeptides is remain as the captivating liquid materials in

1681 1682 1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697

1679 1680

1677 1678

1674

exerting several biological effects than the antibacterial effects displayed by other defense proteins including immune and nonimmunoglobulin systems. Moreover, the natural occurring bactericidal biopeptides those generated from inactive protein precursors may have a direct effect to the overall activity (Clare and Swaisgood, 2000). For instance, Casecidin is a kind of the defense peptide generated by chymosin at neutral pH and usually applied against a particular number of microorganisms such as Bacillus subtilis, Streptococcus pyogenes, Sarcina, Diplococcus pneumoniae, and Staphylococcus (Lahov and Regelson, 1996). Bovine lactoferrin hydrolysates has been reported previously to be active against Gram-positive bacteria such as Streptococcus, Listeria and Bacillus and other kind of Gram-negative bacteria such as (Salmonella, Pseudomonas, Escherichia coli, Klebsiella, Proteus) (Tomita et al., 1991). Lactoferrin derived whey protein is an ironbinding glycoprotein that can exhibit multi-functional properties including immunomodulating, antioxidative, antihypertensive and in the host defense against microbial (Tomita et al., 1991). However, the digestion of lactoferrin that characterized with one disulﬁde bond using pepsin at the amino acids position of 17–41 gives the initiation of novel active fragments named as lactoferricin (Bellamy et al., 1992). The resulting biopeptides (lactoferricin) showed potent antibacterial properties much better than its original molecule of lactoferrin and therefore, may have a chance to cible exactly the preferred site on the surface of microbes on both Gram-positive and Gram-negative microorganisms (Jones et al., 1994; Tomita et al., 1991; Kuwata et al., 1998). The susceptibility of bacteria to lactoferricin peptides has proposed an objective on the mechanism of action of this biopeptides. Thus, the structural parameters and the nature of amino acids composed lactoferricin indicated that the cationic and amphipatic nature of this biopeptides is the key role in inducing the depolarization of bacteria membrane, and therefore its susceptibility (Hwang et al., 1998). Due to robust biological activity exerted by lactoferricin, particularly in the scavenging

1698 1699 1700 1701 1702 1703 1704 1705 1706 Q107 1707 1708 1709 1710 1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 Q108 1721 1722 1723 1724 1725 1726 1727 1728 1729

S. Saadi et al. / Biotechnology Advances xxx (2014) xxx–xxx

t5:5 t5:6 t5:7 t5:8 Q20 t5:9 t5:10 Q21 t5:11 t5:12 t5:13 t5:14 t5:15 t5:16 t5:17 t5:18 t5:19 Q22 t5:20 t5:21 t5:22

AOBP1 AOBP2 AOBP3 AOBP4 AOBP5 AOBP6 AHBP1 AHBP2 AHBP3 AHBP4 AHBP5 AHBP6 IMBP1 IMBP2 IMBP3 IMBP4 IMBP5 IMBP6

N-Terminal AA

Multifunction validity

His Asn Pro Pro Trp Asp ND ND ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND Asn Arg Asn Met Gly Tyr ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND Asn Gly Ser Val Tyr Trp

His Asn Pro Pro Trp Asp Asn Arg Asn Met Gly Tyr Asn Gly Ser Val Tyr Trp

C-Terminal AA AO

Leu Glu Val Thr Ile Tyr ND ND ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND Pro Leu Pro Asp Phe Asn ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND Pro Arg Pro Tyr Thr Thr

Multifunction validity

References

Leu Glu Val Thr Ile Tyr Pro Leu Pro Asp Phe Asn Pro Arg Pro Tyr Thr Thr

Mendis et al. (2005b) Zhang et al. (2011) You et al. (2010) Hsu et al. (2010) Ledesma et al. (2005) S.-H. Lee et al. (2010), S.-J. Lee et al. (2010) Jimsheena and Gowda (2011) Liu et al. (2010) Ghassem et al. (2011) Zhao et al. (2009) Saiga et al. (2006) Tsai et al. (2008) Hou et al. (2012) Takahashi et al. (1994) H.M. Chen et al. (1995), J.R. Chen et al. (1995) Parker et al. (1984) Fang et al. (2007) Hou et al. (2012)

t5:4

t5:3

Table 5 Bi- and multi-function validations of antioxidative, antihypertensive and immunomodulatory properties of biopeptides fragment.

R O

t5:1 t5:2

t5:23 t5:24

Abbreviations: AO: antioxidative; AH: antihypertensive; IM: immunomodulatory; BP: biopeptides; AOBP: antioxidative biopeptides; AHBP: antihypertensive biopeptides; IMBP: immunomodulatory biopeptides; AA: amino acid; ND: not determined.

1730 1731

1746

effects of early phases of viral infections (Gauthier et al., 2006). This biological function (anti-viral) has shown the ability to transform based on the existing virus types. The underlying mechanism of lactoferricin is associated (1) to its relative structure analogue that constituted of high contents of basic amino acids (8 of 25 amino acid residues are basic) and (2) to its rapid afﬁnity to bind either on host cell molecules or to viral particles (Andersen et al., 2004). Thus, the net positive charges with asymmetric clustering of their basic amino acids are considered among the key roles for the mechanism of action of lactoferricin that is usually expressed by its rapid attraction to the biological membranes as displayed by other efﬁcient cationic biopeptides having sensitive cell membrane permeability of microorganisms (Bellamy et al., 1993). The therapeutic application of this biopeptides revealed many biological roles including the inhibition of formation of lung metastatic colony in mice (Iigo et al., 1999), a signiﬁcant declining of lung metastases from murine tumor cells and signiﬁcant inducement of apoptosis in THP-1 human monocytic tumor cells (Yoo et al., 1997; Yoo et al., 1998).

1747

Biopeptides with anticoagulant activity

1742 1743 1744 1745

1748 1749

N C O

Many years ago, the commercial anticoagulants have been used for therapeutic purposes, particularly in medication and other thrombotic 1750 disorders. The side effects of these commercial anticoagulants including 1751 coumarine, warfarin, and heparin have been recognized such as 1752 thrombocytopenia, and hemorrhagic. Thus, the proposed issues have 1753 prompted the scientists to exploit natural products as alternative 1754 sources for commercial anticoagulants (Hylek et al., 2007). Due to the 1755 complexity of process of homeostasis, the pharmacologist and food sci1756 entists nowadays are rewording great efforts in the exploitation of nat1757 ural bioactive compounds derived food materials in order to impair 1758 particular biological function associated to blood coagulation. The role 1759 of biopeptides as natural protectants is one of the therapeutic agents 1760 that can be used to decline the bleeding in the same time to re1761 generate and reconstitute the damaged regions at the wall of the 1762 blood vessel. It is worth to state that more than 13 of plasma serine pro1763 teases are encrypted in the mechanism of blood coagulation (David and 1764 Thomas, 2007). Thus, in order to accomplish the clotting mechanism 1765 tenase and prothrombinase complexes are required (Blostein et al., 1766 Q109 2003). Many of the marine organisms such as Tegillarca granosa (Jung 1767 et al. (2007a, 2007b)); L. aspera (Rajapakse et al., 2005c), Mytilus edulis 1768 Q110 (Jung and Kim, 2009) and Scapharca broughtonii (Jung et al., 2002) have 1769 successfully used as starting materials in order to derive biopeptides as 1770 an anticoagulant agent. On the other hand, a potent anticoagulant

Biopeptides with opiate-like activity

1778

Opioid peptides by deﬁnition is kind of peptide that can be found in bovine milk and reported to play a central role in the nervous network within human body, which encoded by genes that are responsible for generating opiate-like activity and it can exhibit similarity mode of action of morphin (Pihlanto-Leppälä, 2001a, 2001b; Teschemacher et al., 1997; Sharma et al., 2011). This type of peptide acts by adhering with targeted cell receptor and among these receptors, we have for example μ-receptor, which can induce the emotional behaviors, and it found to suppress as well the motility of intestine. Another receptor associated to regulation of food intake is named “κ-receptor”, these types of biomarkers they are in most of cases active either by playing a role as antagonistic or agonistic according to their particular N-terminus involved in the sequence chain. The presence of tyrosine amino acid at the N-terminal sequence followed by aromatic amino acids in the third or fourth position of the sequence chain may the result for this structure prototype that has rapid afﬁnity with the opioid receptors (Hollt, 1983; Teschemacher, 1997; Paroli, 1988; Teschemacher et al., 1994).

1779

Biopeptides with anticancer and anti-tumor activities

1797

In cancer research therapy natural biomaterials such as protein hydrolysates might exhibit a pivotal biological role against cancer cells by targeting them and scavenging them from proliferation, particularly the tumor-associated antigens (Noguchi et al., 2012). A recent study has been successfully identiﬁed an anti-cancer food biopeptides having the following sequence of Ala-Phe-Asn-Ile-His-Asn-Arg-Asn-Leu-Leu from shellﬁsh Mytilus-coruscus by using pepsin hydrolysates (Kim et al., 2012). Biopeptides has become an alternative molecules compared to other efﬁcient drugs because of safety point of view that has shown towards human health. Peptides that have the ability of inducing the apoptosis in tumor cells are implicated to be as effective anticancer agents (Mader et al., 2007; Lehmann et al., 2006; Okumura et al., 2004).

1798

1771 1772

1740 1741

1738 1739

1736 1737

1734 1735

1732 1733

biopeptides have been isolated from marine macro and microalgae strains that is similar to that displayed by proteoglycan (Athukorala et al., 2007). Owing to its anticoagulant activity which is associated to sulfated polysaccharide and protein polysaccharide parts, proteoglycan may induce the function of anti-thrombin upon thrombin and target them to activate factor Xa for stimulating the clotting process and its formation (Samarakoon and Jeon, 2012).

1773 1774 1775 1776 1777

1780 1781 1782 1783 Q111 1784 1785 1786 1787 1788 1789 1790 1791 1792 1793 1794 1795 Q112 1796

1799 1800 1801 1802 1803 1804 1805 1806 1807 1808 1809

S. Saadi et al. / Biotechnology Advances xxx (2014) xxx–xxx

1828 1829 1830 1831 1832 1833 1834 1835 1836 1837 1838 1839 1840 1841 1842 1843 1844 1845 1846 1847 1848 1849 1850 1851 1852 1853 1854 1855 1856 1857 1858 1859 1860 1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 1874 Q113

Conclusion

1921

Biopeptides have a unique characteristic among other functional active substances such as protein, phenolic, and ﬂavonoid compounds. Due to biophysical and biochemical properties biopeptides are valued among the potential ingredients having the capability to regulating food intake, scavenging oxidative stress, reducing the risk of cardiovascular diseases and modulating the immune disorders once they exist with an adequate dosage in the living organisms. Moreover, it could be comprehended that the native structure of the amino acids positioned either on C-terminal or N-terminal are preferred to be on form aromatic or branched side chain. The presence of amino acid residues having linear chain, positively charged coupled at least with an amino acid possessing an aromatic cycle in the both penultimate positions of the C-terminal and N-terminal amino acid residues is more effective. While, the presence of Pro with an aromatic structure in combination to certain amino acids possessing aliphatic structures are favored in the both ultimate positions (C-terminal and N-terminal amino acid residues) as well. It has been

1922

1876 1877

1826 1827

1824 1825

1822 1823

1820 1821

1818 1819

1816 1817

1815

1813 1814

Food biopeptides are versatile materials for regulating human bodily functions by protecting them from different incidences and abnormal disorders, infection and maladies. The potentiality of these active materials is noted through their signiﬁcant contribution to the human health. Their research increased dramatically among food technologists, biotechnologists, medical doctors, pharmacologists and neurologists. Owing to their large nutritional platform that made them as alternative natural ingredients biopeptides exhibited similarity mechanism of action towards health beneﬁts as compared with other efﬁcient drugs. For these reasons extensive applications for these vital substances have been done either in vitro (at molecular and cell levels) or in vivo (by using animal trials like SHR). So far, many investigations have been conducted on biopeptides behavior under gastro intestinal tract conditions, but still potential exists to verify their bio-resistance in the enterocyte areas to ensure their intact absorption into portal circulation. This veriﬁcation in fact necessitates an extra works to conﬁrm the bioavailability of biopeptides fragment by conducting further in vivo examinations. Therefore, the best way to maintain this bio-resistance is by protecting the both ﬁxed (C-terminal amino acid) and variable (N-terminal amino acid) sides of biopeptides fragment via microencapsulation technique. In addition, it can couple by some ﬂuorescents active material to determine the exact biological target, particularly at the cell tissue. This method helps to assess the trajectory of biopeptides within the living system and their involvement in many of the biochemical transformation cycles. Currently, research progress of biopeptides is pronounced as compared to the last decade, but still not fully appreciated. It means the better understanding of the system (biopeptides) for determining their structure–biological functionality relationship is still going on. It is believed that many stages on biopeptides are clariﬁed, particularly on their domain efﬁcacies. However, most of the researchers have shown one intersect point that the N-terminal and C-terminal are the dominating poles for the biological activity of these fragments. Now the challenges are related to the amino acid properties positioned on the N-terminal and C-terminal of the peptides. Some of these amino acids are inert in the nature towards the biological functionalities, while some of them are signiﬁcant, depending highly on the type of enzyme used. This issue may open the door for more comprehending the enzymes speciﬁcity and their preferred catalyzing regions within the sequence chain of protein or polypeptides. To realize these, an extensive in vivo works with particular focus on the fundamental understanding of the molecular interactions are required. This might help to raise new insights on the mechanisms of action between biopeptides and the targeted biological diseases. Moreover, there is need to emphasize on the resulting metabolites (primary and secondary) effect and their communication at cellular level by coupling both of proteomic and of genomic sciences in combination with chemical engineering.

R O

It is well acknowledged that the scarcity of information and lack of evidences shown on the in vivo tests, made it very hard by the scientists to ensure the maximum bioavailability of peptides during their internal biochemical transformation. This is the reason why most of the recent researches are more focusing on an in vitro tests, rather than in vivo ones because in most of cases the efﬁcacy of biopeptides are reduced once they reach to the blood circulation domain, thereby, their desired biological effects will be not quite enough to display and record strong evidences on their mechanism of actions related to modulation, declining and also, inhibition against the most frequently maladies propagated within the living organisms. Segura-Campos et al. (2011) stated certain strategies related to the enhancement of the absorption of micronutrients by elimination of enzyme inhibitors and protecting the peptides in order to perform their natural resistance against enzyme biodegradation, thus extending gastric shelf life of these systems and their bioavailability in the blood domain, which in turn might exhibit robust biological functions against cardiovascular diseases. Another strategy is related to how to make protein with desired biological functions by improving their fragility structures to develop more resistance analogues. Further developed strategy is used to enhance the absorption capacity of proteins and peptides to cible the exact penetration route in order to develop more adapted system. Many efforts have been spent to design a genus complex system such as emulsions, liposomes and muco-adhesion models (Lee, 2002). Pegylation was applied since 1960s (Davis, 2002). It helps to assess the reactivity pathway of newly developed bioactive system having polytectic interaction sides, resulting from both X (e.g. lipid) and Y (e.g. peptide) poles and by joining both of X and Y side together (XY) with polyethylglycol (PEG) molecules. Basically this type of strategy is applied to modify the physicochemical properties of such molecule causing it to become more efﬁcient in declining the risk of toxicity (Pichereau and Allary, 2005; Biron et al., 2008). In the same way, other improvements can be realized by using the nitrogen methylation strategy to ameliorate the resulting biopeptides metabolites (primary and/or secondary). Gao et al. (2001) during an in vivo study of membrane transportation mechanism demonstrated that Caco-2 cell monolayers induced by N-methylation of the Ala-Phe peptide bond showed to increase the stability of molecule against protease degradation. Microencapsulation, an important alternative technique used to enhance the bioavailability of peptides and proteins in the enterocyte domain. This method has been widely applied in the assessment of novel developed active substances as peptides based active drugs and typically used in nutraceutical and pharmaceutical technologies. However, to realize the appropriate microencapsulation of such active system is an important thing to take into account the preparation parameters related to each processes such as solvent evaporation, interfacial polymerization and spray drying operation conditions. This strategy is getting much attention, particularly in food preservation associated to shelf life extension of newly developed active ingredients. However, the formulated microcapsule should be more stable under gastric and intestinal enzymes digestion, thus ensuring their intact absorption into blood circulation domain. Moreover, the physical aspect of the biopeptides and their mechanical resistance via micoencapsulation technique can lead to protect the morphology, granulity, encapsulation capacity, and the most important thing is the protection of both of the variable side (N-terminal amino acid) and constant side (C-terminal amino acid) of peptides fragment. In addition, the microsphere-based encapsulation method is one of the strategies that can be used to facilitate the manufacturing of newly formulated system containing proteins or peptides. It provides a good assessment for product quality in terms of safety, dosage, particularly during the kinetic release of the active substances (Saenz et al., 2007; Pihlanto-Leppälä, 2001a, 2001b).

1875

1812

Future prospects

Strategies needed for ameliorating biopeptides availability and potency

1810 1811

1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920

1923 1924 1925 1926 1927 1928 1929 1930 Q114 1931 1932 1933 1934 1935 1936 1937

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1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985

1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997

1986 Q115 Uncited references

Al-Mudallal et al., 1995 Boumgaertel, 1999 Gardner, 1998 Gold et al., 1998 Iroyukifujita et al., 2000 Suetsuna and Chen, 2002 Tsuge et al., 1991 Zhang and Zhang, 2010 Acknowledgment

Authors acknowledge the Malaysian Ministry of Science, Technology and Innovation for the grant awarded to Professor Dr. Nazamid Saari 1998 under project matriculation 05-01-04 SF1020.

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041 2042 2043 2044 2045 2046 2047 2048 2049 2050 2051 2052 2053 2054 2055 2056 2057 2058 2059 2060 2061 2062 2063 2064 2065 2066 2067 2068 2069 2070 2071 2072 2073 2074 2075 2076 2077 2078 2079 2080 2081 2082 2083

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remarked that the presence of amino acid with positive charge like “Arg” on the N-terminal of the sequence chain of peptide and also the amino acid with negative charge like “Met” on the C-terminal of peptide chain contributed signiﬁcantly to the ACE-inhibitory activity. Meanwhile, the immunomodulatory activity, Phe, Tyr and Pro are the most preferred amino acid residues, where the hydrophobic characteristic of Phe and hydrophilic afﬁnity of Tyr on the N-terminal amino acid residues showed to cause pronounced results towards immune response including stimulation of lymphocytes and modulating the cells growth. Owing to their structural properties, Pro is preferred to present on the ﬁxed side (C-terminal) of the sequence chain of biopeptides fragment due to its aliphatic structure and the absence of chain NH proton, which prevents its free rotation within the peptide sequence and intramolecular joining via hydrogen bonds as well. It has been noticed that certain structural parameters of amino acid residues play a key role in dominating the biological function of peptide fragment and among these parameters, we have for examples size and sequence of peptide, hydrophobicity, and nature of amino acid located on the variable and constant sides of peptide fragment. Thus, they can be implicated in order to understand the thermodynamic properties of amino acid residues and their behavior during biochemical transformation. Consequently, it is believed that many intrinsic and extrinsic obstacles are encrypted in the retardation of food biopeptides researches progress. This slow development could be attributed to several facts, such as: (a) the routine techniques and modes of screening that have been used to generate these active fragments, which made it hard by the researchers to achieve the exact biological target; (b) the slight intension that has been shown by the scientists towards these food sources and their scarcities towards these materials whether they are capable to liberate a potential food ingredients or not; (c) the lack of information on the end products of these compounds, which in most cases necessitates an in vivo study and this is also one of the barriers that delayed the recent investigation due to level of contamination, especially when it comes to cell culture; (d) the absence of sophisticated tools, made it impossible to discriminate the primary and secondary metabolite, which are considered as the main regulatory keys of human organism for maintaining a natural protective effect against numerous maladies and diseases; (e) the instability of peptides under gastrointestinal tract condition, and also, due to the lack of information on the advanced methods, particularly the nanotechnology processes, made it very difﬁcult by the scientist to conﬁrm the bioavailability of these systems by using in vivo tests, because usually the in vitro test is not quite enough to ensure the mechanism effect of such active substance; and (f) the insufﬁcient ﬁndings on immunomodulating properties compared to that done for antioxidative and antihypertensive activities. This is because of the lack of expertise in the area of immunology and due to the absence of a real linkage between major ﬁelds such as pharmacy, medicine, biotechnology, and chemical engineering.

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