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Chapter 20

Food-derived multifunctional bioactive proteins and peptides: Applications and recent advances

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Dominic Agyei, Ravichandra Potumarthi, and Michael K. Danquah

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20.1 Applications of multifunctional peptides

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Owing to their multipurpose functionalities, bioactive peptides can play a signi ficant role as leaders in the pharmaceutical, nutraceutical, and functional food industries as well as in the cosmetic industry. There is currently a booming market for bioactive peptides and a growing number of products are already on the market or under development by the food industries, exploiting the potential of food-derived bioactive peptides (Table 20.1).

20.1.1 Nutraceuticals and functional foods

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The potential for bioactive peptides to contribute to a healthier nutrition (e.g., by ingesting them with functional foods) has been widely discussed in the scientific community. Additionally, the discovery of bioactive peptides with potential health benefits has been the subject of growing commercial interest in the context of health-promoting functional foods. A number of reasons account for this trend. From a nutritional perspective, peptides are more bioavailable than their parent proteins or free amino acids (Hajirostamloo 2010). Also, due to the changes in the spatial three-dimensional structure of proteins upon hydrolysis, often the resulting peptides (especially those with low molecular weight) have been known to be less allergenic than their native proteins. This explains why milk protein hydrolysates are widely utilized in the formulation of hypoallergenic infant foods (Host and Halken 2004). The sales of dietary supplements and so-called functional foods and beverages are growing, suggesting an increasing belief in benefits of these “natural” approaches to disease prevention or management. Research continues to uncover novel bioactive peptides to reveal their possible functions and health benefits. In some developed countries, the marketing of food products with Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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508 Food-derived

multifunctional bioactive proteins and peptides

Table 20.1 A description of some commercially available food-derived bioactive peptides.

Whey-derived peptides

Prevention of dental caries, influence the clotting of blood, antimicrobial properties Blood pressure reduction

Whey-derived peptides

Fresh cheese whey protein isolate; BioPURE-GMP

Davisco Foods International Inc., USA Davisco Foods International Inc., USA Davisco Foods International Inc., USA

Hydrolyzed whey protein isolate; BioZate Whey protein isolate; BioPURE-GMP

Hydrolyzed whey protein isolate; Biozate Fermented low-fat hard cheese; Festivo

Aids mineral adsorption

Casein hydrolysates; Capolac® Sour milk; Calpis

Arla Foods Ingredients, Sweden Calpis, Co., Japan

Flavored milk drink, confectionary, or capsules; PRODIET F200/Lactium Casein-derived peptide; PeptoPro

Ingredia, France

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Val-Pro-Pro, Ile-Pro-Pro; Blood pressure reduction derived from β-casein and κ-casein in sour milk as1-casein f (91–100) (Tyr- Reduction of stress effects Leu-Gly Tyr-Leu-Glu-GlnLeu-Leu-Arg) Casein-derived peptide Improves athletic performance and muscle recovery Whey-derived peptide Aids relaxation and sleep

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Whey protein hydrolysate fortified with lactoferrin Whey protein extract

Casein-derived hydrolyzates

Milk peptides

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Blood pressure reduction

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αs1-casein f (1–9), αs1-caseinf (1–7), αs1-casein f (1–6) Caseinophosphopeptide

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Anticariogenic, antimicrobial, antithrombotic Reduction of blood pressure

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Glycomacropeptide (GMP), κ-casein f(106–169) β-lactoglobulin fragments

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Helps reduce acne

Helps reduce symptoms of mild to moderate psoriasis Helps regulate blood sugar peaks after a meal Reduces inflammation and promotes healing in the digestive tract

Whey-derived peptide; Vivinal Alpha

Lactoferrin-enriched whey protein hydrolysate; Praventin Whey protein extract; Dermylex

DSM Food Specialties, the Netherlands Borculo Domo Ingredients (BDI), the Netherlands DMV International, the Netherlands Advitech Inc., Canada

Casein hydrolysate; Insulvital

Wild Co., Germany

Milk peptides; Immunel

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Food-derived multifunctional bioactive proteins and peptides 509 Table 20.1 (Continued) Bioactive peptide

Properties/ biological roles

Available as commercial product Manufacturer

Supports and balances total immunity Reduction of stress effects

Milk peptides; Tegricel

Wild Co., Germany

Milk protein hydrolysate; Lactium®

Ingredia Nutritional, France

Helps sleep and memory

Whey protein isolate; α-lactalbumin

Caseinophosphopeptides

Anticariogenic


Helps mineral absorption


Anticariogenic


Anticariogenic

GC tooth mousse; water-based creme Caseinophosphopeptides; Kotsu Kotsu calcium Caseinophosphopeptides; Trident xtra care™ Caseinophosphopeptides; Recaldent™ Soy protein oligopeptides; HI-NUTE series whey proteins; Lacprodan® ALPHA-20

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Soy protein oligopeptides Helps prevent obesity and muscle fatigue α-lactoalbumin (60 %) Helps reduce peptic ulcers

Fish peptides (Leu-Lys-Pro-Asn-Met)

Hypotensive

Davisco Foods International Inc., USA GC Europe N.V., Belgium Asahi Soft Drinks Co. Ltd., Japan Cadbury Adams, USA Cadbury Enterprises Pte. Ltd., Singapore Fuji Oil Co. Ltd., Japan Arla Foods Ingredients Group P/S, Denmark Metagenics Inc., USA

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Milk peptides

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Product type; brand name

Bonito-derived peptide; Vasotensin®

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health claims is a well-established industry. For example, in Japan, there is a booming industry for the production and marketing of Food for Specified Health Uses (FOSHU), or “foods containing ingredients with functions for health and officially approved to claim its physiological effects on the human body.” Under such context, the safety assessment of the foods as well as effectiveness for the desired health functionality is required before approval can be sought for the marketing of food as FOSHU. FOSHU approval operates under three schemes: (1) qualified FOSHU (where food with demonstrated functionality is approved, although no scientific and established mechanism of action has been demonstrated); (2) standardized FOSHU (where foods are approved following the establishment of standards and specifications with sufficient accumulation of scientific evidence); and (3) reduction-of-disease-risk FOSHU (where food is approved when reduction-of-disease-risk is clinically and nutritionally established in an ingredient) (FOSHU 2013). Several reduction-of-disease-risk FOSHU products in Japan contain peptide-based ingredients.

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20.1.2 Clinical trials: Prospects and challenges

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A number of clinical trials have been conducted for some food having protein hydrolysates or bioactive peptides as ingredients and components (Table 20.2). It is observed from the literature that a large proportion of the clinical studies done explored the blood pressure–lowering (antihypertensive) activities of bioactive peptides. Not much human studies have been done for other biofunctionalities such as antioxidant, anticancer, and immunolomodulating properties, to mention a few (Agyei and Danquah 2012). Furthermore, the dosage amount seems to vary for each experiment. As well, the states (whether in powdered, capsule, or liquid form) in which products are administered vary for each experiment. The establishment of the dosage needed for a food-derived peptide to trigger physiological response is important information needed to help market a product health claims. Food components are known to have a chronic rather than an acute effect on health (Hartmann and Meisel 2007). As such, screening methods are needed for the measurement of both short- and long-term effects that follow the administration of certain food components that are proposed to promote good health (Hartmann and Meisel 2007). This will require a series of experiments at various levels, involving in vitro, animal models, and human studies. Valid bioindicators that represent an effect brought about by the bioactive food component must be developed and standardized. Such markers could be obtained in vivo (serum, fecal, urinary), in vitro (cytochemical), and also in situ (lesion samples) (Hartmann and Meisel 2007; Walker et al. 2009). Despite some advancement in this area, results from animal and human studies are quite contradictory. For example, whereas a number of research studies have demonstrated the multifunctional roles of CPP, studies conducted in connection with a European Union project (Caseinophosphopeptides: Nutra ceutical/Functional Food Ingredients for Food and Pharmaceutical Applications) have concluded that CPPs does not enhance calcium absorption in the gut of humans (López-Huertas et al. 2006; Teucher et al. 2006; Hartmann and Meisel 2007). Another product with questionable biofunctionality is α-casozepine, released during trypsin digestion of αS1-casein. It is a decapeptide (is αS1-casein (f91-100)) with the sequence Tyr-Leu-Gly-Tyr-Leu-Glu-Asn-Leu-Leu-Arg (Messaoudi et al. 2005; Kim et al. 2006; Cicero et al. 2011). α-casozepine is known for its anxiolytic properties (Kim et al. 2006) and products such Lactium® have α-casozepine as ingredients and are marketed with this health claim. However, the European Food Safety Authority (EFSA) has issued a report about a milk protein hydrolysate product supplemented with 1.7% of α-casozepine (product name Lactium® and claimed to “helps to calm mind,” “mental state and performance,” and “stress”). The report stipulates that “…a cause and effect relationship has not been established between the consumption of ‘αS1-casein tryptic hydrolysate’ and alleviation of psychological stress” (EFSA 2011).

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Food-derived multifunctional bioactive proteins and peptides 511 Table 20.2 Examples of clinical trials/human studies. Duration

Dosage

Results (Bioactivity claimed)

Reference

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Fermented milk containing tripeptides, IPP, and VPP

8 weeks

SBP and DBP ↓ vs. control (antihypertensive effects)

(Hata et al. 1996)

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Powdered fermented milk containing tripeptides, IPP, and VPP Fermented milk containing tripeptides, IPP, and VPP Fermented milk containing tripeptides, IPP, and VPP Skim milk

4 weeks

2 ml/kg body wt (i.e. 0.033 mg Val-Pro-Pro and 0.025 mg Ile-Pro-Pro/kg body wt). 12 g of powdered fermented milk was consumed for 4 weeks 150 mL of product twice daily

SBP and DBP ↓ vs. control (antihypertensive effects)

(Aihara et al. 2005)

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(Seppo et al. 2003)

(Buonopane et al. 1992)

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SBP and DBP ↓ vs. control (antihypertensive effects) 21 weeks 2.25 mg of Reduced SBP and Ile-Pro-Pro and DBP 2.55 to 3.75 mg (antihypertensive of Val-Pro-Pro effects) 8 weeks 2% solids-not-fat Reduced serum fortified skim milk triglycerides in the to the daily diet high-cholesterol subject Casein Consumption of Aids enamel phosphopeptides, 100 mL of CPP remineralization CPP mixed with fortified milk and bone milk; CPP has daily for calcification the structure 30 seconds for a (anticariogenic –Ser (P)-Ser (P)-Ser 15-day period properties); (P)-Glu-Glu# immunoenhancing (Recaldent) activity; antigenotoxic properties α-lactalbumin (a Two meals given Dosage per meal Increase in plasma whey protein) diet on two separate unspecified Trp-LNAA ratio rich in tryptophan 2-day periods and improvement in cognitive performance in high stress– vulnerable subjects 10 weeks

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(Markus et al. 2002)

SBP, systolic blood pressure; DBP, diastolic blood pressure; LNAA, large neutral amino acid; #, commercial product has CCP complexed with amorphous calcium phosphate as active ingredients.

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20.2 Pharmaceutical products

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A number of causes can be attributed to the discrepancies in results of foodderived bioactive peptide functional studies. Most of these clinical studies are carried out by or at least in conjunction with industry and thus not all data on the composition and manufacturing of these peptides are made available (Jäkälä and Vapaatalo 2010). With this failure to give such vital information, a repeat of their experiments is therefore highly likely to lead to different results. With the need to comply with strict regulatory standards, in vivo and clinical studies aimed at elucidating the mode of action and potency of bioactive peptides is increasingly becoming expensive. To forestall this, researchers have resorted first to conducting in vitro experiments to identify potential bioactive peptides before committing to in vivo and human trials (Agyei and Danquah 2012). Other authors such as Foltz et al. (2010), however, have argued that it is inappropriate to use results of in vitro studies as justification to test the in vivo effects of bioactive peptides. This is because this approach largely neglects other challenges such as the low bioavailability of most peptides resulting from poor absorption, distribution, metabolism, and excretion (ADME). As such, in vivo efficacy of bioactive peptides can only be tested when these peptides have exhibited reasonable and physiologically relevant ADME profiles (Foltz et al. 2010).

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Proteins and peptides play several important roles in living body systems. Thus, the diverse physiological roles of peptides make them suitable candidates for the development of therapeutic agents (Agyei and Danquah 2011; Lax 2012). A consi derably huge amount of scientific research data exists to demonstrate the therapeutic potential of bioactive peptides and these highlight the role food-derived peptides can play as active pharmaceutical ingredients (APIs) in therapeutic products. Due to a host of reasons such as heightened concerns with the side effects of small-molecule drugs and antibiotic resistance of several pathogenic microorganisms, there has been a search for a new generation of therapeutics that is safe and potent. Recently, there has been a surge of interest in peptide therapeutics within the pharmaceutical industry. It has been estimated that around 60 peptide drugs were approved and generated annual sales of around US $13 billion (about 1.5% of all drug products) in 2010. The numbers are increasing significantly with the current continuous annual growth rate between 7.5% and 10% (Lax 2012). Undoubtedly, most of these peptides are not food-derived; however, statistics show the potential and future of peptide-based therapeutics among consumers and regulatory bodies. Not only do bioactive peptides have prospects in the treatment of certain single-symptom diseases, but they can also be used in the control of syndromic ill-health conditions that are accompanied by several disorders. For example, the involvement of dietary bioactive proteins and peptides in the control and possible treatment of autism spectrum disorders (ASD) has recently been reviewed (Siniscalco and Antonucci 2013). This prospect rests

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Food-derived multifunctional bioactive proteins and peptides 513

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on the fact that most of the biochemical processes associated with ASD is addressed one way or the other by bioactive peptides. Some of these biochemical processes include oxidative stress; gastrointestinal impairments (intestinal increased permeability and dysbiosis); immune dysregulation; and immune activation of neuroglial cells (Siniscalco et al. 2012). Some properties of peptidebased therapeutics that highlights their use as APIs are: 1 Peptides are structurally diverse and have a wide spectrum of therapeutic action, low biodeposition in body tissues, and high biospecificity to targets (Agyei and Danquah 2011). Peptides are therefore effective in addressing a wide array of medical disorders including cancers and tumors, metabolic disorders, cardiovascular heath, and infectious diseases. 2 The acceptance of protein therapeutics by physicians, pharmaceutical corporations, and patients has increased over the past few decades. This increased acceptance is demonstrated by the fact that there is an over 20% probability of regulatory approval for peptide-based new chemical entities (NCEs), a rate that is double that of small molecules (Lax 2012). Also, for the past three decades in the United States alone, the number of peptide-based NCEs entering clinical study per year per decade has increased by about 1300% (from 1.2 per year in the 1970s, to 16.8 per year so far in the 2000s) (Reichert 2012). This increased acceptance has been partly attributed to the development of technological solutions to problems that hitherto affected the percolation of peptide therapeutics, namely, short half-life and difficulties in delivery (Saladin et al. 2009). Additionally, the use of peptide products is often unaccompanied by any side effects. Thus, side reactions are unanticipated or at best reduced for food-derived peptide drug candidates since food proteins and peptides have a long history of use and putatively are GRAS (Agyei and Danquah 2011). 3 Most peptides are composed of metabolically and allergenically tolerable amino acids. Thus, peptides are generally safe and nontoxic, considering their natural physiological roles in the body as peptide hormones, chemokines, and cytokines. For the most part, any known side effects with peptide drugs have often been related to dosage or local reactions at the injection site (Lax 2012). 4 Aside from their use as active ingredients, peptides also have the ability to be used as excipients in drug formulations for modification of biological activity and targeted delivery, or to aid transport across cellular membranes.

20.3 Dermopharmaceutical products Proteins and peptides play an active role in skin-care through a combination of several physiological activities (i.e., modulating cell proliferation, cell migration, cell and tissue inflammation, angiogenesis, and melanogenesis) (Fields et al. 2009). Multifunctional bioactive peptides have great potential for use as active cosmetic and dermopharmaceutical ingredients due to their ability to stimulate some of these physiological responses. Interestingly, a number of commercial

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20.4.1 Foodomics and other “omic” methodologies

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patents exist that describe peptide sequences that are used as ingredients in dermatological products. The sequence X-Thr-Thr-Lys-Y, where X = lysine and Y = serine has been patented (US Patent No. 6620419), as useful in stimulating skin healing, skin hydration, and combating wrinkles and consequences of skin ageing (Lintner 2000). Another disclosed invention (US Patent No. 8071555) is a tetrapeptide with the amino acid sequence Pro-Glu-Glu-X (where X can be either lysine or isoleucine). This tetrapeptide is effective in controlling inflammatory skin disorders (Zhang et al. 2011).

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A number of advances have been in many “omic” techniques that offer high through-put and an integrated approach to studying food protein peptides. Foodomics is a term that has been coined to describe a broad knowledge of food, covering the assessment of their composition, the and impacts of (bio)technological processes involved in their production, time-dependent changes in food composition, and the impact of food consumption on human health (Picariello et al. 2012). Thus, as a new global discipline, foodomics relies on the use of advanced omic tools including metabolomics, genomics, epigenomics, transcriptomics, and proteomics to address issues such as bioactivity, safety, quality, and food traceability, with the aim of improving consumer health, well-being, and confidence (Cifuentes 2013). Some of the foodomic tools that are of primary interest to the study and applications of food-protein-derived bioactive peptides are nutrigenomics, food proteomics, and food peptidomics. Food peptidomics seeks to unravel and also develop analytical strategies to study the peptidome of a given food product and establish the origin, evolution, organoleptic properties, and beneficial or adverse effects on human health (Carrasco-Castilla et al. 2012; Picariello et al. 2012). Advancement in “omic” techniques has been served greatly by the marriage of high-throughput mass-spectrometry-based methodologies (Mena and Albar 2013) and computational achievements in the development of new algorithms to analyse huge and bulky peptidomic data quickly and effectively. The proliferation of software programs, coupled with the use of tandem mass spectroscopic tools, provides a powerful and attractive tool for peptide quantification and characterization. Computational (in silico) peptidomics allow the prediction of bioactive p eptides that can be obtained from a food protein of known sequence, before actual wetlaboratory production is undertaken. With the rapid discovery, characterization, and deposition of bioactive peptide data in databases, it becomes increasingly possible to predict new peptides with improved bioactivities. This more or less bottom-up approach is captured in Figure 20.1 and can be adopted for the rational design of peptides with desired biofunctionalities. With this technology,

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6. Purification and activity testing of Peptides

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5. Wet laboratory synthesis of desired peptides with conventional method (solidphase peptide synthesis)

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4. In silico identification and characterization of peptide of interest

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3.In silicodigestion

Figure 20.1 The production of bioactive peptide

via the in-silico approach.

2.From protein sequence databases, select protein substrates and enzyme(s) that will yield the peptide of interest

1.Select peptide sequence with bioactivity of interest from literature

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an online protein database can be consulted to select protein sequences that encrypt specific peptides of interest. This is followed by in silico digestion with the use of enzyme(s) sources of characteristic specificity that will release (but not cleave) the desired peptide. Characterization of the “soft” peptides produced can also be done in silico after which laboratory synthesis may follow if the predicted properties of the peptides obtained are satisfactory. This approach to bioactive peptide production offers several advantages over the hitherto conventional trial-and-error method in that it is less labor intensive, relatively cheaper, and gives an indication of the bioactivities of the peptides with a high level of accuracy even before synthesis of the peptides (Agyei and Danquah 2011). As well, the study of the secondary structure and predictions of the physicochemical and physiological properties of peptides can also be done in silico. An example of peptide database application that can be used to perform in silico peptide studies is BIOPEP (www.uwm.edu.pl/biochemia/), which interlinks three databases of protein sequences, bioactive peptides, and proteolytic enzymes (Dziuba and Dziuba 2010). To date, there are 707 and 2,609 protein and peptide sequences and 25 proteolytic enzymes, with all three databases being regularly updated. Additionally, BIOPEP focuses on food-derived peptides

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and also has inbuilt programs for the prediction of allergenic and toxic pro perties of peptides and can also be interfaced with global databases such as SWISS–PROT (Minkiewicz et al. 2008; Dziuba and Dziuba 2010). Other peptide sequence applications/databases are PepBank (http://pepbank.mgh.harvard.edu), BioPD (http://biopd.bjmu.edu.cn), SwePep (www.swepep.org), and EROP-Moscow (http://erop.inbi.ras.ru), among others. Some in-silico digestion databases such as PeptideCutter (http://web.expasy.org/peptide_cutter/), POPS (http://pops.csse.monash. edu.au/pops-cgi/index.php) and NeuroPred (http://neuroproteomics.scs.illinois.edu/ neuropred.html) are proteolyse prediction databases (Carrasco-Castilla et al. 2012). A number of other Web-based peptide libraries are also being created. MilkAMP is a new database that contains information (i.e., microbiological and physicochemical data) on antimicrobial peptides obtainable from dairy products. This database is freely available at http://milkampdb.org and is a useful tool that can aid in forecasting the development and use of biologically active peptides in the pharmaceutical and food industries (Théolier et al. 2013). Other authors have proposed the development of a Wiki-like food database that utilizes advance bioinformatic tools in food and nutritional research (Holton et al. 2013).

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20.5 Quantitative structure activity relationship (qsar) models

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Another area of advancement is the use of models. Although model prediction of the behavior of biological systems has been known for several decades, recent great advancements in computational methods have made it possible and relatively easy to model the activities of biologically active molecules and compounds in an accurate manner (Nantasenamat et al. 2010). An example of this is the quantitative structure activity relationship (QSAR), which works on the principle that the activity of a biological molecule can be predicted based on its physicochemical or structural properties such as electronic charge, hydrophobicity, and steric properties, resulting in multivariate data (Nakai and Li-Chan 1993; Pripp et al. 2005; Hansch et al. 1995). The quantitative structure–property relationships (QSPR) model, on the other hand, is used to study and correlate the structural features of a compound with its respective chemical properties (Le et al. 2012). The basic assumption in QSAR modelling is that the biological activity is related to the structural variation of the compounds, and this relationship can be modelled as a function of molecular structure (Carrasco-Castilla et al. 2012). Often, a mathematical expression can be used to describe the relationship between the amount of a substance and the level of physiological response it triggers. In QSAR models, accurate characterization of the physicochemical properties of amino acids in the peptide sequence is very important since the model relies

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on such descriptors to predict and express chemical structure relevant for activity. In their pioneering work, Hellberg et al. (1987) established a system for describing the variation of amino acid sequences in peptides as three principal properties, also referred to as z-scores: hydrophilicity/hydrophobicity (z1), molecular size/bulkiness (z2), and electronic properties/charge (z3). These properties were obtained by principal components analysis of 29 physicochemical variables for the 20 natural amino acids (Kim and Li-Chan 2006). Other parameterization systems include the extended principal property scales (z′1, z′2, z′3), for both coded and noncoded amino acids (Jonsson et al. 1989); amino acid side-chain descriptors that use isotropic surface area (ISA) and electronic charge index (ECI) (Collantes and Dunn 1995); molecular surface-weighted holistic invariant molecular (MS-WHIM) 3D-descriptors (Todeschini et al. 1994; Zaliani and Gancia 1999); MARCH-INSIDE (Markovian Chemicals In Silico Design) methodology, based on the Markov chain theory (Ramos de Armas et al. 2004); and principal components that score Vectors of Hydrophobic, Steric, and Electronic properties (VHSE) (Mei et al. 2005). Such scoring systems are useful strategies for developing peptide QSARs. The use of QSAR model systems replaces the “trial-and-error” approach of selecting molecules with biological properties, therefore saving time, resources, and cost involved in screening large numbers of plausible molecule candidates. QSAR models are widely utilized in drug discovery and are equally applied in food-derived bioactive peptide research because a strong structure–activity relationship exists in most peptides, making secondary structure and physicochemical properties of peptides important factors that determine the bioactivities triggered by food-derived peptides. QSAR of several food bioactive peptides has been successfully used to estimate the structure–activity relationship of anti microbial, ACE-inhibitory (Norris et al. 2012; Gu and Wu 2013; Sagardia et al. 2013), and bitter-tasting peptides, as reviewed (Pripp et al. 2005). As well, in-silico digestion and QSAR model prediction have been used to identify potent angiotensin I converting enzyme (ACE) inhibitory peptides from several common food proteins (Majumder and Wu 2010; Gu et al. 2011).

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20.6 Research concerns and bottlenecks

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20.6.1 Allergenicity A food allergy is an adverse health effect as a result of an immune response that occurs reproducibly after exposure to a given food protein (Boyce et al. 2010). Although peptides have been known to be less allergenic than their native proteins (Host and Halken 2004), it is also true that several native proteins act as precursors for both bioactive peptides and allergenic peptides, giving both toxic and nontoxic effects. For example, whereas cow’s milk is a rich source of bio active proteins and peptides (Korhonen and Marnila 2013), it also acts as a

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storehouse for several protein- or peptide-based allergenic molecules, and constitutes the leading cause of food allergies in infants and young children under age 3 years (Koletzko et al. 2012; Ludman et al. 2013). All the constituents that are responsible for both pollen and food allergies are proteinaceous in nature. Some protein hydrolyses may retain part of the allergenicity of the native protein and thus can also be considered as allergens (Hartmann et al. 2007). Foodderived bioactive peptides must therefore be subjected to comprehensive safety assessment in order to rule out any cytotoxic and allergenic concerns that will render the products unsafe for human consumption. In many cases, such safety assessments are a requirement by food standards, for food labeling and advertisement purposes. Immune reactions of food allergens may be immunoglobulin (Ig)E mediated, non-IgE mediated, or mixed. Also, symptoms of food allergies are sometimes masked by other gastrointestinal problems such as dyspepsia and abdominal pain. Thus, appropriate diagnostic mechanisms are needed to clarify, for the average consumer, the difference between food allergies and food intolerance phenomena (which may be enzymatic, pharmacological, or toxin-mediated) (Hartmann et al. 2007; Koletzko et al. 2012). To this end, the diagnosis of food allergies in food proteins and its hydrolytic products have been reported in the findings of some studies (Boyce et al. 2010; Waserman and Watson 2011; Koletzko et al. 2012). Additionally, since αs1 casein is the major allergen of cow’s milk, Elsayed et al. (2004) have used synthetic peptides and derivatives from this protein to develop a sensitive technique for detecting masked cow-milk protein epitopes in processed food. Although their method was effective in showing latent allergenic properties, it is, however, laborious, tedious, and relatively expensive since it required pure peptide samples. Research is therefore needed to elucidate the allergenicity and antigenicity of bioactive peptides in a quick and robust manner, particularly when the peptides are obtained from allergenic food proteins.

20.6.2 Possibility of microbial resistance of peptides

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The possibility of pathogenic microbial strains developing resistance to bioactive peptides is a research concern that needs to be addressed. This is exemplified in the bioactivities of lactoferrin (LF). Owing to their antimicrobial potential, LF has been granted a GRAS status (notification number 67), to be used as an electrostatic spray to a concentration of less than 2% by weight, in the control of microbial contamination of raw meat (Taylor et al. 2004). However, it has also been reported that some pathogenic microorganisms show resistance or can develop resistance to LF, especially at certain physical conditions and metabolic states (Arnold et al. 1981; Bortner et al. 1989). It has therefore been suggested that the widespread use of LF or lactoferricin should be done with caution since it could lead to the development of resistant pathogens, which, in

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the case of LF or lactoferricin, also indicates the possibility of the pathogens to resist molecules of human innate immunity (Korhonen and Marnila 2013). On the other hand, other studies have shown conclusions that are contrary to this notion. For example, Chen et al. (2013) have demonstrated that several probiotic bacterial strains are resistant to LF and its hydrolysates. The probiotic strains included L. rhamnosus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus coryniformis, L. acidophilus, Bifidobacterium infantis, Bifidobacterium bifidum, and Pediococcus acidilactici. On the other hand, the growth of foodborne pathogens such as Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Enterococcus faecalis was inhibited by LF and its hydrolysates. It follows, therefore, that more experimental research is needed to clarify the discrepancies and evaluate the possibility and conditions that could favor microbial resistance of LF intended for food application.

20.6.3 Stability and bioavailability

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Multifunctional bioactive peptides, in order to exert any bioactive effects after oral ingestion, have to reach the target system in an active form. The challenge here is that proteins and peptides are highly susceptible to protease degradation en route to the body. Also, the low solubility and relatively bulky nature of multifunctional proteins and peptides leads to difficulty in transport and delivery across membranes. These leads to low stability that results in decreased oral bioavailability and short half-life (Agyei and Danquah 2011). Some multifunctional peptides such as LF have been shown to retain bio activities and resist trypsin and chymotrypsin treatment in amounts com parable to duodenal contents in human infants and the presence of high concentrations of a specific trypsin inhibitor could account for this observation (Brines and Brock 1983). On the other hand, the presence of specific amino acid types is responsible for the ability of some peptides to resist proteolytic attacks. For example, it is known that (hydroxy)proline-containing peptides are generally resistant to degradation by digestive enzymes (Vermeirssen et al. 2004) so that the incorporation of proline groups in peptides help enhance their proteolytic stability. Although a number of simulated gastrointestinal digestion studies have been done (Hernández-Ledesma et al. 2004; Lo and Li-Chan 2005; Cinq-Mars et al. 2007; Escudero et al. 2010), most of these studies described ACE inhibitory peptides. Research information (on the other biofunctionalities) is therefore still scarce or and in some cases the results on the resistance of milk protein to enzymatic attack along the digestive tract are conflicting (Picariello et al. 2010). The mystery of peptide resistance to gastrointestinal digestion, intestinal absorption, and stability in blood needs to be unraveled. Thus, more detailed clinical studies are needed to establish the biological potency of multifunctional peptides that have proven to be effective in in vitro experiments.

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20.7 Conclusions and future projections

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Increasingly, there is a blurring of the boundaries between food and classic pharmaceutical products. Food products containing multifunctional peptides are a potent alternative to achieving therapeutic effects in human health by the use of nonpharmacological, food-derived biomolecules. The sequencing and prediction of the biological roles of peptides is escalating, owing to recent robust and highthroughput “omic” techniques. However, to be fully exploited, a number of research studies aimed at elucidating unknown toxicity and stability of peptides need to be undertaken. Once these bottlenecks are removed, we project that there will be an escalation of interest among consumers, researchers, and clinicians since multifunctional peptides constitute a more “natural” approach to disease prevention and management.

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