Trends in Food Science & Technology 23 (2012) 62e69

Review

Rethinking food-derived bioactive peptides for antimicrobial and immunomodulatory activities

Dominic Agyei* and Michael K. Danquah
Bio Engineering Laboratory, Department of Chemical Engineering, Monash University, Wellington Road, Clayton, Victoria 3800, Australia (Tel.: D61 (0)3 9905 1867; fax: D61 (0)3 9905 5686; e-mail: dominic.agyei@monash.edu)
Bioactive peptides from food proteins have been studied over the past decade to elucidate their biological potency in the major systems of the body such as the digestive, cardiovascular, nervous, and immune systems. Some bioactive peptides have been established for their antimicrobial roles as well as humoral and cell-mediated immune functions and thus have prospects of being incorporated as ingredients in functional foods, nutraceuticals and pharmaceuticals where their biological activities may assist in the control and prevention of diseases. However, further insightful research on the pharmacokinetics of immunomodulatory peptides in vivo and clinical studies are needed to rmly establish their therapeutic potency.

Background The demand for functional foods and nutraceuticals has increased signicantly in response to the increasing awareness of the inuence of diet on health. The therapeutic potentials of some food components have drawn the attention
* Corresponding author.
0924-2244/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2011.08.010

of clinicians, food manufacturers, researchers and consumers, thus becoming a major endeavour. It appears the contemporary world is coming to terms with the ancient quote of Hippocrates Let food be thy medicine and medicine be thy food than ever before. The therapeutic potentials of functional foods and nutraceuticals are attributable to the presence of specic functional groups and their molecular derivatives released during food metabolism. Dietary proteins, aside their ability to supply calories and amino acids, have also been known to offer health benets in vivo and in vitro either in the intact form or as hydrolysates. Food protein hydrolysates that induce benecial biological functionalities are called bioactive peptides. Bioactive peptides are produced by microbial fermentation, enzyme digestion or proteolysis by enzymes in vitro, and can perform physiological activities in the major body systems (Korhonen & Pihlanto, 2006). Such functionalities include antioxidative, antimicrobial, antihypertensive, cytomodulatory and immunomodulatory effects (Hartmann & Meisel, 2007; Yang et al., 2009). Fig. 1 shows some physiological effects of bioactive peptides in the human body. Bioactive peptides have been produced from a wide range of food materials including those from animal sources; [milk (Gill, Doull, Rutherfurd, & Cross, 2000), egg, cheese, beef (Jang & Lee, 2005), pork (Jang, Jo, Kang, & Lee, 2008), seafood (Kim & Wijesekara, 2010), sh (Kim & Wijesekara, 2010; Yang et al., 2009), chicken]; plant sources [rice, corn, soy and soy products (Chiang, Tsou, Tsai, & Tsai, 2006, Brassica carinata (Pedroche et al., 2007), wheat, broccoli (Hartmann & Meisel, 2007), pulse crops]; microalgae (Chlorella vulgaris 87/1) (Morris et al., 2009); and fungi [mycoproteins from Fusarium venenatum (Brownsell, Williams, & Andrews, 2001), mushroom (Sun, He, & Xie, 2004), Brewers yeast (Kanauchi, Igarashi, Ogata, Mitsuyama, & Andoh, 2005)]. Recent studies have demonstrated that unhealthy lifestyles practices and stress may have a cumulative effect on the immune system increasing the risk for various cardiovascular diseases, cerebrovascular disorders and cancer (Nakano et al., 2001). On the other hand, research on the immunomodulatory effects of different bioactive peptides and their potential for human health promotion and disease risk reduction is also well documented (Biziulevi cius,  _ , & Zukait _ , 2006; Gill et al., Kislukhina, Kazlauskaite e 2000; Morris et al., 2009).

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Fig. 1. Bioactivities of food protein-derived peptides and the body system inuenced.

Of particular interest are the bioactive peptides of whey proteins which have been shown to modulate both specic and non-specic immune responses in vitro (Gill et al., 2000) and in mice models (Gauthier, Pouliot, & SaintSauveur, 2006). Further, oral administration of Chlorella vulgaris 87/1 hydrolysate produced from pancreatin has been shown to enhance the stimulation humoral and cell-mediated immune functions in undernourished mice (Morris et al., 2009). Antimicrobial and immunomodulatory peptides therefore have the prospects of use as a new generation of drug candidates. However, there exist some limitations to the full exploitation of immunomodulatory peptides to enhance human nutrition and health. Review of literature reveals that the action of immunomodulatory peptides is relatively nonspecic and this may account for the reason why the exact mechanism of action as well as the in vivo fate of these peptides is largely unknown. Further, there are no high throughput methods for the production of large quantities of these peptides. In this commentary, we discuss research advances in immunomodulatory and antimicrobial bioactive peptides, production platforms and immunological assessment, and some challenges that preclude the largescale production of bioactive peptides.

Antimicrobial and immunological potency of bioactive peptides Many peptides, mainly milk-derived, have shown multifunctional properties, where specic peptide sequences may possess two or more different biological activities (Pihlanto-Lepp al a, 2002). Peptides with antimicrobial and/or immunomodulatory activities are ubiquitous in virtually all life forms. The mechanism of action of bioactive peptides in disease control and prevention is by interacting with microbial host invaders as antimicrobial agents or by suppressing or stimulating certain immune responses (Hancock & Sahl, 2006) (Fig. 2). Recent studies have uncovered a wide range of bioactive peptides obtainable from food protein materials. Mammalian milk has been shown to contain a number of very potent immunomodulatory peptides which regulate immune functions by suppressing or stimulating certain immune factors (Gauthier et al., 2006; Gill et al., 2000; Pihlanto-Lepp al a, 2002). Both immunosuppression and immumostimulation nd use in various areas of disease control. Immunosuppressive peptides may be applied during medical conditions such as graft and transplant rejection as well as inammations induced by autoimmune disorders whilst immunostimulation

Fig. 2. Biological roles of host defense peptide.

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processes boost overall immune potency (Gauthier et al., 2006). Some immunomodulatory peptides (such as casein phosphopeptides) are already being marketed commercially. Casein phosphopeptides (CPP) are casein derived peptides released in the gastrointestinal tracts which, among other functions also stimulate immunoglobulin A (IgA) production in mice (Ferraretto, Signorile, Gravaghi, Fiorilli, & Tettamanti, 2001; Otani, Nakano, & Kawahara, 2003). Several studies have also shown microbial or viral inhibitory peptides. Zeng et al. (2008) obtained some 5e10 kDa peptide hydrolysates from Pacic oysters (Crassostrea gigas) and the peptides showed herpes virus growth inhibition properties. In another study, oyster protein extracts have been shown to enhance the proliferation of immunocytes in human immunodeciency virus (HIV-1). The oyster protein extract enhanced the interleukin (IL-2) dependent activation of peripheral blood mononuclear cells (PBMC) and the extract induced effect was marked in HIV seropositive asymptomatic individuals. Thus, oyster protein extract could prevent or delay the occurrence of IL-2 dependent immune deciency including AIDS (Achour et al., 1997).

Further, Yust et al. (2004) identied two fractions of rapeseed protein hydrolysates which inhibited HIV protease in E. coli BL21. The E. coli BL21 cells contained the plasmid, PT5, which carries a cDNA coding for HIV protease. These peptides were detected as they could enter the E. coli cells and inhibit the HIV protease thus allowing cell growth. Protein hydrolysates with both antimicrobial and cancer cell cytotoxic effects have also been shown from several studies (Falciani, Pini, & Bracci, 2009; Jang et al., 2008; Korhonen & Pihlanto, 2006). Table 1 shows the protective effect and immune responses triggered by some bioactive peptides. The activity of immunomodulatory peptides is dependent on the structure of the peptide molecule. The functional property of a bioactive peptide is determined by its unique three-dimensional (3D) structure which is dependent on the type (e.g. cysteine forms disulphide bridges) and the nature (e.g. charge and hydrophobicity) of key amino acids in the primary sequence. However, binding characteristics of bioactive peptides have been attributed largely to the secondary structure rather than the tertiary structure (Kaur, Garg, & Raghava, 2007).

Table 1. Immune activity of some protein fractions and bioactive peptides from different food materials. Food Product Bovine milk Protein as1-casein Isracidin Identied Peptide Enzyme treatment used Pancreatin Chymosin Immune effect Y lymphocytes proliferation Protect mice against infection by Staphylococcus aureus. [ phagocytic response in mice infected with Candida albicans. Protect cows and sheep against mastitis Y lymphocytes proliferation Y lymphocytes proliferation, [ mice resistance to Klebsiella pneumonia infection [ antibody formation and [ phagocytosis in vitro [ mice immune response to SRBCs and HRBCs activates murine splenocytes, increasing their proliferation and gamma-interferon (IFNg) secretion in vitro Murine splenic lymphocyte proliferation, [ phagocytic effect of peritoneal macrophages Antimicrobial activities, immunomodulatory, anti-cancer, and anti-hypertensive activities [ Natural killer cell activityy Ref. (Gill et al., 2000) (Gill et al., 2000)

N e terminal sequence 1e23

b-casein b-casomorphin

Para-k-casein a-Lactalbumin Mushroom Auricularia polytricha hemoagglutinative protein (APP) Unidentied soy proteins Ovalbumin

Tyr-Pro-Phe-Pro-GlyPro-Ile-Pro-Asn-SerLeu (60e70) Phe-Phe-Ser-Asp-Lys (17e21) Hydrolysed a-lactalbumin uncharacterized peptides of molecular weight 13.4 kDa

Pancreatin Trypsin

(Gill et al., 2000) (Gill et al., 2000)

Trypsin

(Gill et al., 2000) (Gill et al., 2000) (Sheu et al., 2004)

Soy beans

Alcalase

(Kong, Guo, Hua, Cao, & Zhang, 2008) (Mine & KovacsNolan, 2006) (Horiguchi, Horiguchi, & Suzuki, 2005) (Morris et al., 2009)

Egg

Peptides not specied Immunopeptides

Wheat

Gluten

Enzymatic digestion Pancreatin (following ethanol treatment of Chlorella biomass)

Chlorella vulgarian 87/1

Unidentied algae proteins

uncharacterized peptides of molecular weight 2e5 kDa

stimulation of humoral immune functions, haemopoiesis, monocyte-macrophage system activation,

[, increase; Y, decrease; yHuman studies.

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The structural characteristics, hydrophobicity, basicity as well as the composition and sequence of amino acids play a crucial role in determining the biological activities triggered by bioactive peptides (Hancock & Sahl, 2006; Korhonen & Pihlanto, 2006). Most peptides with antimicrobial properties are short, hydrophobic and cationic in nature (Hancock & Sahl, 2006). Further, the presence of key amino acids has been known to impart specic functionalities. For example, all the most potent angiotensin-I-converting enzyme (ACE) inhibitory peptides, namely, valine-prolineproline and isoleucine-proline-proline, contain at least one proline residue. Also, glycine is a key amino acid in the synthetic peptides (Tyr-Gly and Tyr-Gly-Gly) which have been shown to enhance the proliferation of human peripheral blood lymphocytes (Pihlanto-Lepp al a, 2002). Bioactive peptides as therapeutics The conventional use of drugs such as antibiotics and vaccines as a means to control diseases suffers several demerits leading to consequences on both manufacturers and consumers. Factors such as high cost of manufacture of vaccine, bureaucratic procedures for certication, changes in government and government regulations, and requirements for adjuvants and ancillary medical supplies often lead to periodic production interruptions and shortage of therapeutics. Within the rst half of the last decade there were nationwide shortages of six recommended childhood vaccines targeting nine diseases, and the supply of adult inuenza vaccine was interrupted three times in the US alone (Coleman, Sangrujee, Zhou, & Chu, 2005; Obaro & Palmer, 2003). It is therefore pertinent that measures are implemented for the sustainability of pharmaceutical products by developing alternative sources of these biologics. Immunomodulatory peptides, due to their hormonal or neuronal functions can be used as therapeutic agents. However, unlike bioactive peptides, the effects of vaccines on the immune system are highly specic inducing protective adaptive immune responses and immunological memory. Thus, bioactive peptides cannot be used as effective substitutes for vaccines. Nevertheless, due to the physiological effects they trigger in the body system, immunomodulatory peptides could act as safe substitutes for antibiotics and other pharmacological agents. The use of antibiotics may be accompanied by side effects which could compromise consumer health through drug-to-drug interactions, fever, and allergenic reactions. Development of resistance by microorganisms is another important issue that hinders the use of many small molecule drugs. Immunomodulatory peptides have the potential to address these setbacks. As peptides, they accumulate less in body tissues and thus their use may be accompanied by little or no side effects. Further, resistance by microorganism has not been known to accompany the use of immunomodulatory peptides, yet still this is not anticipated because these peptides do not interact directly with the

microorganism but rather through diverse mechanisms in innate immune system responses (Hancock & Sahl, 2006; Kim & Wijesekara, 2010). Moreover, the use of bioactive peptides to generate therapeutic products for disease control is economically more attractive compared with the use of and antibiotics or vaccines. Many of the diseases (such as HIV, malaria and measles) for which vaccines are needed, exist principally in third world countries with low standards of living. This coupled with the high cost of biological raw materials required makes development of such vaccines costineffective which discourages most drug manufacturing companies, being prot driven rms (Obaro & Palmer, 2003). Bioactive peptides, on the other hand, can be produced from by-products of food protein bio-processing. Fish skin, bones, cakes from pulse crops and residual minced meat are relatively cheap and the utilization of such by-products may reduce production cost with an added advantage of an efcient waste disposal (Agyei & Danquah, 2011; Yang et al., 2009). Moreover, immunomodulatory bioactive peptides are encrypted in a wide range of food materials proteins (Table 1) and most of these foods are abundant in nature. Some commercial food products containing bioactive peptides have already found their place in Foods for Specied Health Use (FOSHU) products (Hartmann & Meisel, 2007). Additionally, a host of bioactive peptides can be produced from a single food source by the use of a combination of enzymes. Gill et al. reported about 20 immunomodulatory peptides of casein and whey proteins in milk alone (Gill et al., 2000). Many of the milk-derived peptides also show multifunctional properties. Thus a single peptide may perform more than one function in the body systems. Conclusively, peptide based therapeutics offer several advantages over conventional drugs as a results of their high bioactivity and biospecicity to targets, and the wide spectrum of their therapeutic action (Marx, 2005). Bioactive peptides as therapeutics - hindrances and suggested solutions Although immunomodulatory, antihypertensive and antimicrobial peptides have good prospects of use in the allied health and food industries some potential drawbacks need to be addressed before the bioactive peptides can be optimally exploited to that end. Some of the major drawbacks and suggested strategies to overcome them have been highlighted in Table 2. The very potent antimicrobial peptides have been known to be cationic with hydrophobic amino acid residues (Hancock & Sahl, 2006). These properties, together with their large hydrodynamic size therefore lead to a compromise in their solubility and membrane permeability. This setback can be overcome through specialized delivery strategies such as peptide encapsulation with micro- and nano-sized coordination polymers or particles such as dendrimers, liposomes, and polyectrolyte microspheres.

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Table 2. The cons of peptide based therapeutics and strategies to address them. Challenge Low oral bioavailability Low stability Strategies Use of parenteral delivery routes Chemical modication of peptides (with retention of activity) Use of peptidomimetics Use or incorporation of unnatural or D-amino acids Toxicological studies in animal models Encapsulation in coordination polymers or particles (such as dendrimers, and polyectrolyte microspheres) Use of micro- or nano-sized liquid marbles Encapsulation in liposomes Use of cheap food protein sources and bi-products of protein processing as substrates Use of natural sources such as bacteriocins

potent peptides from food proteins can be used to overcome the high cost setback in peptide manufacture. Breakthroughs in this area include the use of bi-products from food protein processing operations as substrates for enzymatic and/or microbial fermentation. Also, naturally produced peptides sources such as bacteriocins which do not require synthetic manufacture can also be used. Bioactive peptides e bioprocessing Bioactive peptides are produced from proteins by the use of one or more of the following methods: (1) enzymatic hydrolysis with digestive enzymes, (2) fermentation of food proteins with proteolysis starter cultures and (3) proteolysis by enzymes derived from plants or microorganisms (Korhonen & Pihlanto, 2006). However, some intact proteins have been known to possess bioactive properties. Lysozyme and a-lactalbumin have been shown to possess antibacterial properties (Exp ositoa & Recio, 2006). However, enzymatic hydrolysis of whole molecules is known to be the most commonly used method for bioactive peptides. The enzymes used could be of gastrointestinal origin, or from bacterial, fungal and plant sources. The use of proteolytic enzymes or their precursors from recombinant DNA technology has also been reported (Korhonen & Pihlanto, 2006). Commonly used enzymes are pepsin, trypsin, alcalase, chymotrypsin, papain and pancreatin, which are either used alone or in combination with other enzymes. Production of bioactive peptides by enzymatic hydrolysis is performed at the optimal conditions of enzyme(s), and the type of peptides generated is dependent on the hydrolytic specicity of the enzyme(s). Moreover with this method, specic enzyme combinations could be used to simulate the fate of a protein molecule when it passes through the digestive system. For example Pedroche et al. (2007) sequentially digested B. carinata protein isolates in vitro with trypsin, chymotrypsin and carboxypeptidase A and obtained peptides with antioxidant and angiotensin converting enzyme (ACE) inhibitory activities. Bioactive peptides generated with this order of enzymes combinations is believed to be mimic those produced by the human digestive system (Pedroche et al., 2007). Moreover, a combination of different enzyme treatments usually leads to the production of many novel peptides. The use of immobilized enzymes over the conventional soluble enzymes for the production of bioactive peptides is gaining momentum. In a study by Pedroche et al., bioactive peptides were produced from B. carinata hydrolysed by immobilized enzymes on a glioxyl-agarose support (Pedroche et al., 2007). Fermentation of food proteins with proteolytic starter cultures is another method of bioactive peptide production. The proteolytic system of lactic acid bacteria (LAB), for example, involves a plethora of proteases that offer varied enzymatic specicities. LAB possess cell envelope associated proteinases (CEP) as well as intracellular peptidases such as endopeptidases, aminopeptidases, tripeptidases and dipeptidases for possible application in the production of bioactive peptides. Probiotics bacteria of the genera

Toxicity and effect on body systems Difculty in delivery

Difculty in transport across membranes High cost of synthesis

Further, the susceptibility of peptides to protease degradation is another major setback which can shorten the in vivo half-life of the peptide drug and may also lead to undesirable pharmacological reactions and potentially toxic end-products. To this, interventions such as chemical modication of peptides and the incorporation of unnatural amino acids (including D-amino acids) have been known to enhance the stability of peptides. The presence of certain key biomolecules enhances the stability of certain peptides. For example, the presence of a proline residue at the carboxyl terminal end of a peptide has been shown to increase the peptides resistance to degradation by digestive enzymes (Yamamoto, Ejiri, & Mizuno, 2003). Also, the sequence, ser(P)-ser(P)-ser(P)-glu-glu (P being phosphate) present in casein phosphopeptides constitutes a motif unto which cationic minerals can bind. The resulting complex is resistant to proteolysis en route the gastrointestinal system (Ferraretto et al., 2001). Other approaches include the use of peptidomimetics, or cyclic peptides and research in this area is receiving attention (Hancock & Sahl, 2006). Shen et al., have developed a new chemical method of peptide synthesis which can be used to incorporate unnatural amino acids into a peptide chain as well as custom design the chirality of the end product (Shen, Makley, & Johnston, 2010). However, such interventions will involve a trade-off between enhanced stability without signicant loss in activity. Moreover, in peptide therapeutics manufacture, the single largest setback is the high cost of manufacturing peptides (Hancock & Sahl, 2006). This is because the conventional manufacturing approaches such as transgenic, recombinant, or synthetic methods are relatively expensive and thus prohibitive for scale-up operations (Marx, 2005). However, the plausibility of manufacturing physiologically

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Lactobacillus and Bidobacterium have been most widely studied for the production of probiotics and bioactive peptides (Alhaj, Kanekanian, Peters, & Tatham, 2010). The activities of peptidases are affected by growth conditions of the producing microorganism hence the formation of peptides by this method can be manipulated to some extent (Korhonen & Pihlanto, 2006). The development of optimized production methods for the commercial production of bioactive peptides is an ongoing research endeavour. Advances in bio-separation technologies used in isolating and purifying biomolecules coupled with chemical engineering convective mass transfer principles could offer a cost-effective and scalable production platform for immunomodulatory peptides. To date, the methods that have been used for peptide fractionation and enrichment are those that enable recovery of bioactive peptides with minimal destruction. Briey, they involve ultraltration, ion exchange and gel ltration technologies. Coupled methodologies such as enzymatic hydrolysis followed by ultraltration in membrane reactors, and ion exchange membrane chromatography have also been used (Chabeaud et al., 2009; Li, Chen, Wang, Ji, & Wu, 2007; Pedroche et al., 2007; Sheu, Chien, Chien, Chen, & Chin, 2004). The use of a combination of methods for the production, separation, purication and identication of bioactive peptides is also becoming popular. Such methods lead to products of high purity and/or yield. Electro-membrane ltration is a method used for the isolation and enrichment of bioactive peptides (Bargeman et al., 2002). This method combines electrophoresis and membrane ltration and thus very efcient l for strongly charged biomolecules such as bioactive peptides. Additionally, key operating parameters such as the type of membrane, electrical eld strength, salination of hydrolysate and hydrolysate concentration can be manipulated to improve the product transfer and separation rates (Bargeman et al., 2002). Bottlenecks to advancements Even though bioactive peptides have numerous potentials, there exist some limitations to the production of large quantities to satisfy the growing market demands. Presently, the production and utilization of bioactive peptides has not realized its full potential due to two major challenges namely scientic and relating techno-economic setbacks and regulatory issues. Scientic and techno-economic challenges There is a dearth of reports that address the production of bioactive peptides from a bioprocess engineering and economics perspective in order to assess commercial viability. Laboratory-scale preparations are not optimised and routinely results in low volumetric titres. The lack of viable bioprocesses, transferable to industrial scale, is a major hindrance to the rapid percolation of bioactive peptides into the therapeutic consumer markets.

The scaling up of production methods for bioactive peptide requires optimization of certain key steps in the raw material conditions and process operations. Novel separation and enrichment technologies to enrich bioactive peptide fractions are needed (Pihlanto-Lepp al a, 2002). Some advances have been made in the use of continuous reactor and membrane ultraltration processes for the continuous production and separation of peptides (Korhonen & Pihlanto, 2006) however, most of these technologies are not scalable. The issue of fouling is also a major technical challenge that accompanies the use of membrane-ultraltration for peptides. The lack of scalable purication and enrichment methodologies can result in an overall increase in the cost of peptide manufacture. It has been estimated that the separation and purication stages in industrial biotechnology processes can account for up to 70% of the capital and operating costs (Brady, Woonton, Gee, & OConnor, 2008). The achievement of high-yield bioactive peptides manufacturing hinges on detailed optimisation raw materials and process operations essential for the generation of specic peptides of distinct bioactivities under economic conditions. Another bottleneck is that in vivo scientic data from toxicological studies of bioactive peptides is lacking. This is a major conclusion drawn from all the studies made on immunomodulatory peptide in the last decade. Due to their relatively small size, bioactive peptides, unlike their parent proteins, may react with other food components especially during processing conditions producing reaction products that may be detrimental to health (Korhonen & Pihlanto, 2006). Unlike the ACE inhibitory peptides which often have very small peptide sequences (usually di- and tripeptides) the antimicrobial and immunomodulatory peptides generally have longer sequences with larger molecular weights which raise safety and allergenic concerns. Thus, to be safely used as therapeutic products clinical tests have to be conducted to ascertain the safety of bioactive peptides, their mode of delivery, and dosage. Moreover, the exact mechanism of action of immunomodulatory and antimicrobial peptides is still unclear although they have been studied extensively. Transmembrane pore formation, inhibition of cell wall and/or nucleic-acid synthesis, and activation of the host autolytic enzyme system are some of the proposed mechanisms (Biziulevicius, 2004). Biziulevi cius et al. (2006) also indicated that the immunostimulatory activity of food protein hydrolysates is a consequence of their antimicrobial activity. According to their hypothesis, some food-borne peptides act as antimicrobials by activating the autolytic processes of the intestinal microora in situ and thus cause the release of microbial lysis products. These lysis products which are immunoenhancers account for the immunostimulatory effects resulting from the consumption of certain food proteins. In vivo and human interventional studies with bioactive peptides are expensive and thus in vitro experiments are often performed to identify potential bioactive peptides before committing to complete human trials.

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Foltz et al., however, have argued in their review that it is inappropriate to use the in vitro stimulatory or inhibitory effects as the justication to test the in vivo effects of bioactive peptides because the approach largely neglects the low bioavailability of peptides resulting from poor absorption, distribution, metabolism, and excretion (ADME). In their view, it is only valid to propose in vivo efcacy for bioactive peptides when the peptide exhibits reasonable proteolytic stability and physiologically relevant ADME proles (Foltz, van der Pijl, & Duchateau, 2010). Moreover, major conclusions drawn from most studies on immunomodulatory peptides have been contradictory. Such differences have in part been attributed to variations in methodologies, raw materials, and models used however, they need to be addressed. Further, possible systemic side effects have to be considered through in vivo studies (Gauthier et al., 2006). These technological issues therefore have to be settled before bioactive peptides can be exploited optimally for human nutrition and health. The efcacy and safety of bioactive peptides should be investigated further by carrying out animal studies to verify benecial effects and to clarify adverse affects. Another area requiring intensive research is elucidating the fate of bioactive peptides during gastro intestinal transit as well as the evaluation of peptide stability in the living body systems. Walsh et al. (2004) have demonstrated that the antihypertensive peptide obtainable from b-lactoglobulin [b-LG f(142e148)] is not sufciently stable to gastrointestinal and serum proteases. Thus, this peptide may be helpless to act as a hypotensive agent in humans following oral ingestion. Findings from studies on the fate of peptides en-route the body systems will provide useful information needed in the design of technologies aimed at formulating peptides into forms capable of surviving the harsh conditions created by the stomach and pancreatic juices. Regulatory related challenges It is highly prognostic that in the next few decades, the bioactive peptides market will become a self-standing industry with its own professional associations promoting its interests due to the recent expansion of the areas of application of bioactive peptides. Government and consumer protection bodies must therefore come out with regulations that govern the production, marketing and utilization of such products. Information needed for design of regulatory measures will depend largely on the advances made in scientic studies on the toxicity, mode of action in the body and nutritional intake recommendations. Conclusions Food proteins have been shown to offer health benets beyond serving the nutritional needs of the body. Such nutritional intervention can be of immense benet in enhancing the health of immune compromised as well as healthy individuals. In humans, studies have established the effect of certain lifestyle related practices on some parameters of the

immune system. By implication, peptides with antihypertensive, antimicrobial and immunomodulatory properties may be used as preventive or control aids against lifestyle related-diseases such as hypertension, cancers, cardiovascular diseases, diabetes, osteoporosis, stress and obesity. Immunomodulatory peptides may also be incorporated into food and drug formulations that can be consumed as supplements. However, to realize the full therapeutic potential of these peptides in vivo models and clinical studies are needed to clarify concerns such as the mode of action, toxicity, dosage, mode of delivery, and possible systemic effects that these peptides might have on the body. Also, completely optimized bioprocesses which are transferable to large-scale operations need to be developed for the production, and purication of these important biopeptides. References
Achour, A., Lachgar, A., Astgen, A., Chams, V., Bizzini, B., Tapiero, H., et al. (1997). Potentialization of IL-2 effects on immune cells by oyster extract (JCOE) in normal and HIV-infected individuals. Biomedecine & Pharmacotherapy, 51(10), 427e429. doi:10.1016/s0753-3322(97)82320-7. Agyei, D., & Danquah, M. K. (2011). Industrial-scale manufacturing of pharmaceutical-grade bioactive peptides. Biotechnology Advances, 29(3), 272e277. doi:10.1016/j.biotechadv.2011.01.001. Alhaj, O. A., Kanekanian, A. D., Peters, A. C., & Tatham, A. S. (2010). Hypocholesterolaemic effect of Bidobacterium animalis subsp. lactis (Bb12) and trypsin casein hydrolysate. Food Chemistry, 123(2), 430e435. doi:10.1016/j.foodchem.2010.04.061. Bargeman, G., Koops, G. H., Houwing, J., Breebaart, I., van der Horst, H. C., & Wessling, M. (2002). The development of electromembrane ltration for the isolation of bioactive peptides: the effect of membrane selection and operating parameters on the transport rate. Desalination, 149(1e3), 369e374. doi:10.1016/ s0011-9164(02)00824-x.  _ J., & Zukait _ V. Biziulevi cius, G. A., Kislukhina, O. V., Kazlauskaite, e, (2006). Food-protein enzymatic hydrolysates possess both antimicrobial and immunostimulatory activities: a cause and effect theory of bifunctionality. FEMS Immunology & Medical Microbiology, 46(1), 131e138. doi:10.1111/j.1574-695X.2005. 00019.x. Biziulevi cius, G. A. (2004). How food-borne peptides may give rise to their immunostimulatory activities: a look through the microbiologists window into the immunologists garden (hypothesis). British Journal of Nutrition, 92(06), 1009e1012. doi:10.1079/BJN20041270. Brady, R., Woonton, B., Gee, M. L., & OConnor, A. J. (2008). Hierarchical mesoporous silica materials for separation of functional food ingredients e a review. Innovative Food Science & Emerging Technologies, 9(2), 243e248. doi:10.1016/ j.ifset.2007.10.002. Brownsell, V. L., Williams, R. J. H., & Andrews, A. T. (2001). Application of the plastein reaction to mycoprotein: II. Plastein properties. Food Chemistry, 72(3), 337e346. doi:10.1016/s03088146(00)00234-x. Chabeaud, A., Vandanjon, L., Bourseau, P., Jaouen, P., ChaplainDerouiniot, M., & Guerard, F. (2009). Performances of ultraltration membranes for fractionating a sh protein hydrolysate: application to the rening of bioactive peptidic fractions. Separation and Purication Technology, 66(3), 463e471. doi:10.1016/j.seppur.2009.02.012. Chiang, W., Tsou, M., Tsai, Z., & Tsai, T. (2006). Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate

D. Agyei, M.K. Danquah / Trends in Food Science & Technology 23 (2012) 62e69 and produced by using membrane reactor. Food Chemistry, 98(4), 725e732. doi:10.1016/j.foodchem.2005.06.038. Coleman, M. S., Sangrujee, N., Zhou, F., & Chu, S. (2005). Factors affecting U.S. manufacturers decisions to produce vaccines. Health Affairs, 24(3), 635e642. doi:10.1377/hlthaff.24.3.635.  sitoa, I. L., & Recio, I. (2006). Antibacterial activity of peptides Expo and folding variants from milk proteins. International Dairy Journal, 16(11), 1294e1305. doi:10.1016/j.idairyj.2006.06.002. Falciani, C., Pini, A., & Bracci, L. (2009). Oligo-branched peptides for tumor targeting: from magic bullets to magic forks. Expert Opinion on Biological Therapy, 9(2), 171e178. doi:10.1517/1471259080 2620501. Ferraretto, A., Signorile, A., Gravaghi, C., Fiorilli, A., & Tettamanti, G. (2001). Casein phosphopeptides inuence calcium uptake by cultured human intestinal HT-29 tumor cells. The Journal of Nutrition, 131(6), 1655e1661. Foltz, M., van der Pijl, P. C., & Duchateau, G. S. M. J. E. (2010). Current in vitro testing of bioactive peptides is not valuable. The Journal of Nutrition, 140(1), 117e118. doi:10.3945/jn.109.116228. Gauthier, S. F., Pouliot, Y., & Saint-Sauveur, D. (2006). Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins. International Dairy Journal, 16(11), 1315e1323. doi:10.1016/j.idairyj.2006.06.014. Gill, H. S., Doull, F., Rutherfurd, K. J., & Cross, M. L. (2000). Immunoregulatory peptides in bovine milk. British Journal of Nutrition, 84(Suppl. 1), S111eS117. Hancock, R. E. W., & Sahl, H.-G. (2006). Antimicrobial and hostdefense peptides as new anti-infective therapeutic strategies. Nature Biotechnology, 24(12), 1551e1557. doi:10.1038/nbt1267. Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: from research to food applications. Current Opinion in Biotechnology, 18(2), 163e169. doi:10.1016/ j.copbio.2007.01.013. Horiguchi, N., Horiguchi, H., & Suzuki, Y. (2005). Effect of wheat gluten hydrolysate on the immune system in healthy human subjects. Bioscience, Biotechnology, and Biochemistry, 69(12), 2445e2449. Jang, A., Jo, C., Kang, K.-S., & Lee, M. (2008). Antimicrobial and human cancer cell cytotoxic effect of synthetic angiotensinconverting enzyme (ACE) inhibitory peptides. Food Chemistry, 107(1), 327e336. doi:10.1016/j.foodchem.2007.08.036. Jang, A., & Lee, M. (2005). Purication and identication of angiotensin converting enzyme inhibitory peptides from beef hydrolysates. Meat Science, 69(4), 653e661. doi:10.1016/ j.meatsci.2004.10.014. Kanauchi, O., Igarashi, K., Ogata, R., Mitsuyama, K., & Andoh, A. (2005). A yeast extract high in bioactive peptides has a bloodpressure lowering effect in hypertensive model. Current Medicinal Chemistry, 12, 3085e3090. doi:10.2174/092986705774933461. Kaur, H., Garg, A., & Raghava, G. P. S. (2007). PEPstr: a de novo method for tertiary structure prediction of small bioactive peptides. Proteins & Peptide Letters, 14, 626e631. Kim, S.-K., & Wijesekara, I. (2010). Development and biological activities of marine-derived bioactive peptides: a review. Journal of Functional Foods, 2(1), 1e9. doi:10.1016/j.jff.2010.01.003. Kong, X., Guo, M., Hua, Y., Cao, D., & Zhang, C. (2008). Enzymatic preparation of immunomodulating hydrolysates from soy proteins. Bioresource Technology, 99(18), 8873e8879. doi:10.1016/ j.biortech.2008.04.056. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: production and functionality. International Dairy Journal, 16(9), 945e960. doi:10.1016/j.idairyj.2005.10.012. Li, B., Chen, F., Wang, X., Ji, B., & Wu, Y. (2007). Isolation and identication of antioxidative peptides from porcine collagen

69

hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry. Food Chemistry, 102(4), 1135e1143. doi:10.1016/j.foodchem.2006.07.002. Marx, V. (2005). Watching peptides grow up. Chemical and Engineering News, 83(11), 17e24. Mine, Y., & Kovacs-Nolan, J. (2006). New insights in biologically active proteins and peptides derived from hen egg. Worlds Poultry Science Journal, 62(1). doi:10.1079/wps200586.  Bermu dez, R. C., Morris, H. J., Carrillo, O. V., Almarales, A, Alonso, M. E., Borges, L., et al. (2009). Protein hydrolysates from the alga Chlorella vulgaris 87/1 with potentialities in immunonutrition. Biotecnolog a Aplicada, 26(2), 162e165. Nakano, Y., Ando, K., Nakamura, S., Hirata, M., Yoshida, T., Matunaga, I., et al. (2001). Relationships between lifestyle-related factors and immune parameters in middle-aged male workers. Journal of Occupational Health, 43(6), 321e330. Obaro, S. K., & Palmer, A. (2003). Vaccines for children: policies, politics and poverty. Vaccine, 21(13e14), 1423e1431. doi:10.1016/s0264-410x(02)00634-5. Otani, H., Nakano, K., & Kawahara, T. (2003). Stimulatory effect of a dietary casein phosphopeptide preparation on the mucosal IgA response of mice to orally ingested lipopolysaccharide from Salmonella typhimurium. Bioscience, Biotechnology, and Biochemistry, 67(4), 729e735. n-Calle, J., Pedroche, J., Yust, M. M., Lqari, H., Megias, C., Giro Alaiz, M., et al. (2007). Obtaining of Brassica carinata protein hydrolysates enriched in bioactive peptides using immobilized digestive proteases. Food Research International, 40(7), 931e938. doi:10.1016/j.foodres.2007.04.001. Pihlanto-Lepp al a, A. (2002). Milk proteins j bioactive peptides. In R. Hubert (Ed.), Encyclopedia of dairy sciences (pp. 1960). Oxford: Elsevier. Shen, B., Makley, D. M., & Johnston, J. N. (2010). Umpolung reactivity in amide and peptide synthesis. Nature, 465(7301), 1027e1032. doi:10.1038/nature09125. http://www.nature.com/ nature/journal/v465/n7301/suppinfo/nature09125_S1.html Sheu, F., Chien, P.-J., Chien, A.-L., Chen, Y.-F., & Chin, K.-L. (2004). Isolation and characterization of an immunomodulatory protein (APP) from the Jews Ear mushroom Auricularia polytricha. Food Chemistry, 87(4), 593e600. doi:10.1016/j.foodchem.2004.01.015. Sun, J., He, H., & Xie, B. J. (2004). Novel antioxidant peptides from fermented mushroom Ganoderma lucidum. Journal of Agricultural and Food Chemistry, 52(21), 6646e6652. doi:10.1021/jf0495136. Walsh, D. J., Bernard, H., Murray, B. A., MacDonald, J., Pentzien, A. K., Wright, G. A., et al. (2004). In vitro generation and stability of the lactokinin [beta]-lactoglobulin fragment (142e148). Journal of Dairy Science, 87(11), 3845e3857. doi:10.3168/jds.S0022-0302(04)73524-9. Yamamoto, N., Ejiri, M., & Mizuno, S. (2003). Biogenic peptides and their potential use. Current Pharmaceutical Design, 9(16), 1345e1355. Yang, R., Zhang, Z., Pei, X., Han, X., Wang, J., Wang, L., et al. (2009). Immunomodulatory effects of marine oligopeptide preparation from Chum Salmon (Oncorhynchus keta) in mice. Food Chemistry, 113(2), 464e470. doi:10.1016/j.foodchem.2008.07.086.  n-Calle, J., Alaiz, M., Yust, M.d. M., Pedroche, J., Meg as, C., Giro Mill an, F., et al. (2004). Rapeseed protein hydrolysates: a source of HIV protease peptide inhibitors. Food Chemistry, 87(3), 387e392. doi:10.1016/j.foodchem.2003.11.020. Zeng, M., Cui, W., Zhao, Y., Liu, Z., Dong, S., & Guo, Y. (2008). Antiviral active peptide from oyster. Chinese Journal of Oceanology and Limnology, 23(3), 307e312. doi:10.1007/ s00343-008-0307-x.