426_200600246_Stanton.qxd Biotechnology Journal 02.04.2007 10:09 Uhr Seite 426 DOI 10.1002/biot.200600246 Biotechnol. J. 2007, 2, 426–434 Review Putting microbes to work: Dairy fermentation, cell factories and bioactive peptides. Part I: Overview Maria Hayes1, 2, R. Paul Ross1, 3, Gerald F. Fitzgerald2, 3 and Catherine Stanton1, 3 1Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland of Microbiology, University College, Cork, Ireland 3Alimentary Pharmabiotic Centre, Cork, Ireland 2Department A variety of milk-derived biologically active peptides have been shown to exert both functional and physiological roles in vitro and in vivo, and because of this are of particular interest for food science and nutrition applications. Biological activities associated with such peptides include immunomodulatory, antibacterial, anti-hypertensive and opioid-like properties. Milk proteins are recognized as a primary source of bioactive peptides, which can be encrypted within the amino acid sequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria (LAB) is an attractive approach for generation of functional foods enriched in bioactive peptides given the low cost and positive nutritional image associated with fermented milk drinks and yoghurt. In this review, we discuss the exploitation of such fermentation towards the development of functional foods conferring specific health benefits to the consumer beyond basic nutrition. In particular, in Part I, we focus on the release of encrypted bioactive peptides from a range of food protein sources, as well as the use of LAB as cell factories for the de novo generation of bioactivities. Received 12 December 2006 Revised 7 March 2007 Accepted 7 March 2007 Keywords: Casein · Whey · Proteolysis · Lactobacilli · Bioactive peptides 1 Introduction The fermentation or souring of milk by microorganisms can be traced back some 8000–10 000 years, to the Middle East and the country known today as Iraq, where it is thought that initial domestication of animals began and the first edible cheese was developed [1]. In Europe, especially in the Nordic countries, a long tradition of using fermented dairy products exists [2]. Seasonal variations in weather and concurrently, milk production, led to a need for preservation of milk in its fermented form as buttermilk or other sweet milks known as ‘villi’ (Finland) and ‘Skyr’ Correspondence: Dr. Catherine Stanton, Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland E-mail: [email protected] Fax: +353-2542340 Abbreviations: ACE, angiotensin-1-coverting enzyme; FOSHU, food for specified health use; GM, genetically modified; LAB, lactic acid bacteria; PLGA, poly (D,L-lactic-co-glycolic acid); UF, ultrafiltration 426 © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim (Iceland) [2, 3]. However, it was not until the early 1950s that the health promoting or “functional” properties, independent of the nutritional properties, of fermented dairy products were realized. It is unlikely that the Bulgarian peasants, whose long life expectancy was observed by Metchnikoff almost 100 years ago [4], and attributed by him to their daily intake of yoghurt, knew much of the potential bioactive nature and associated health benefits of this fermented dairy product. Despite a reliance on this preservation technique, fermentation was a sporadic, uncontrolled process where improvements were empiric and based on trial and error. The earliest dairy fermentations were optimized through back-slopping; inoculation of the raw material with a small quantity of a previously performed successful fermentation [5]. Today, the benefits of fermented dairy products in the diet are well accepted, and the science of food and nutrition has evolved towards ‘nutrition for optimal health’ [6]. The central role of microorganisms in fermentation, especially lactic acid bacteria (LAB), is now widely acknowledged, and it is accepted that these microorganisms can 426_200600246_Stanton.qxd 02.04.2007 10:09 Uhr Seite 427 Biotechnol. J. 2007, 2, 426–434 exert beneficial effects through two mechanisms: direct effects of the live microbial cells, known as the ‘probiotic effect’ [7] or indirect effects during fermentation where these microbes act as cell factories for the generation of secondary metabolites with health-promoting properties [8]. For the latter, the term ‘biogenics’ has been coined, and among the most important biogenic compounds in fermented milk are bioactive peptides [9] released from milk proteins by members of the genera Lactobacillus (Lb.), Bifidobacterium and other LAB. For the purpose of this review, the essential link between fermentation and bioactive peptides are proteolytic LAB, capable of releasing milk-derived bioactive peptides, demonstrated to exert positive effects on human health. Indeed, such processes undoubtedly occur to an extent in the human gut itself through the action of the gut flora on dairy substrates. The commercial and technological exploitation of bioactive peptides as novel functional food ingredients are discussed. In addition, the future of food-grade cultures and the influence that genetic engineering may play to advance the future discovery and verification of novel bioactive peptides are addressed. 2 Overview of bioactive peptides Bioactive peptides are described as ‘food-derived components (genuine or generated) that, in addition to their nutritional value, exert a physiological effect in the body’ [10]. In 1950, Mellander [11] first described bioactive peptides when he reported that ingestion of casein-derived phosphorylated peptides led to enhanced vitamin D-independent calcification in rachitic infants. Since then, fundamental studies have opened a new field of research related to the generation of bioactive peptides from a variety of food proteins, with milk proteins currently being the primary source. Bioactive peptides can be latent (or encrypted) within the primary or parent proteins, where proteolysis is required for their release and activation to exert a physiological response [12] on the various systems in the body. Some peptides also act as biocarriers by sequestering calcium and other minerals, thereby enhancing bioavailability [13]. For a detailed description of some examples of known bioactive peptides, see Part II of this review [14]. 3 Liberation of bioactive peptides from milk via the proteolytic systems of LAB Industrially used dairy LAB strains useful for the generation of bioactive peptides through microbial fermentation are proteolytic in nature. The enzymes involved in milk protein degradation are: (i) cell-envelope proteinases (CEP), and (ii) intracellular peptidases [15, 16]. The exploitation of milk proteins by proteolytic LAB usually commences with degradation of the protein by a CEP into © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com oligopeptides, which are further degraded into shorter peptides and amino acids by the collaborative proteolytic actions of intracellular peptidases [17]. LAB also possess a transport system for amino acids, and di-, tri- and oligopeptides. As a result of this system, the residual level of peptides with bioactivity, such as angiotensin-1coverting enzyme (ACE)-inhibitory activity, increases in fermented milks such as: Calpis® and Evolus®, Japanese and Finnish fermented milk drinks with anti-hypertensive properties attributed to the tripeptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) corresponding to β-casein f (84–86) and β-casein f (74–76), respectively, and associated dairy products, for example, naturally ripened cheeses such as ‘Festivo’, a low-fat cheese developed with Lb. acidophilus and Bifidobacterium spp., which contains anti-hypertensive peptides [18] and yoghurts. Despite detailed knowledge of the proteolytic systems of LAB as outlined above, information on the production of bioactive peptides through milk fermentation and the specific proteinases and peptidases responsible for bioactive peptide release during dairy fermentation is lacking, with only a few reports available. For example, Algaron et al. [19] fermented milk with mutant Lactococcus (Lc.) lactis strains lacking either aminopeptidase N, PepX or tripeptidase to test these mutants for the ability to produce peptides with anti-hypertensive or immunomodulatory activities, and to relate the resultant bioactivity to the resulting mutation. They reported that, in some cases, the modified proteolytic system of Lc. lactis gave rise to a significant difference in the mixture of peptides produced, indicating that the specific peptidase activity of LAB affects the bioactive nature of the peptides produced. Recently, a gene corresponding to an endopeptidase was sequenced from Lb. helveticus CM4 and cloned into Escherichia coli [20]. The designated strain, E. coli CM4EP, transformed with the endopeptidase gene pepO, expressed endopeptidase activity capable of releasing the anti-hypertensive peptides VPP and IPP from oligopeptide parent sequences [20]. These data indicate that the pepO gene product plays a role in processing the anti-hypertensive peptides derived from Lb. helveticus fermentation of milk proteins [20]. The gene pepO has previously been reported in Lb. helveticus CNRZ32, suggesting that there are some differences in the endopeptidases responsible for the generation of IPP and VPP [21]. Additionally, Ueno and Yamamoto [22] purified and characterized an endopeptidase from Lb. helveticus CM4 and verified that this peptidase can generate the tripeptides IPP and VPP using propeptides as substrate. Undoubtedly, identification of the links between the proteolytic machinery of cultures used in dairy fermentation and resulting bioactivity profiles of milk will benefit the development of ‘designer’ milk fermentations with specific, tailored and proven health promoting compounds targeted at niche sections of the food and possibly pharmaceutical industries. 427 426_200600246_Stanton.qxd 02.04.2007 10:09 Uhr Seite 428 Biotechnology Journal 4 Bioavailability of bioactive peptides and functional food development For bioactive peptides such as ACE-inhibitory peptides to exert their effects in vivo, they must reach their target organ intact. This represents a major challenge to the success of bioactive peptides as functional food ingredients, as proteolysis during gastric transit can destroy activity, previously detected using in vitro assay techniques. Digestion of polypeptides begins in the stomach by the action of pepsin at pH 2–3 (Fig. 1). In the more alkaline conditions of the small intestine, polypeptides are further degraded to smaller, more potent peptides or deactivated by the action of the pancreatic proteases trypsin, α-chymotrypsin, elastase and carboxypeptidase A and B. Oligopeptides and free amino acids are then absorbed into the enterocytes across the brush border membrane via distinct transport systems [10]. In addition, they may be hydrolyzed further by the action of brush border peptidases, principally aminopeptidases N and A [23] that cleave N-terminal neutral and anionic amino acids. The peptides are also subjected to a battery of brush border endopeptidase and dipeptidase activities. Natural β-casomorphins can resist digestion due to their high Pro content [24]. The ACE-inhibitory peptides IPP and VPP [25] most likely resist digestion in vivo due to the presence of the C-terminal Pro-Pro, which is resistant to the action of Pro-specific peptidases [26]. Different transport systems for delivery of oligopeptides have been described, i.e., paracellular, passive diffusion, endocytosis, and carrier-mediated transport [10, 11]. Biotechnol. J. 2007, 2, 426–434 Bioactive peptides may be absorbed via carrier-mediated transport or via paracellular diffusion, which is the main mechanism for transport of intact peptides across the cell monolayer [27, 28]. A specific peptide transport system exists in the brush border membrane to facilitate the transport of peptides consisting of di-, tri- or tetra peptides across the brush border membrane [10, 29]. Bioactive peptides are subjected to the proteolytic activities of iminodipeptidases or prolidases, and can be activated or deactivated before reaching the portal circulation [10]. Blood contains substantial activities of peptidase enzymes and angiotensin II degradation occurs within seconds [30]. Only ACE inhibitors that are not affected by the action of angiotensin II and gastrointestinal enzymes, or ACE inhibitors that are converted to stronger ACE inhibitors (called pro-drug inhibitors) exert anti-hypertensive effects in vivo [10]. The anti-hypertensive tripeptides IPP and VPP were detected in the aorta of spontaneously hypertensive rats, following oral administration of fermented milk [31]. In contrast, β-casomorphins rapidly degrade once they enter the blood stream, and it is suggested that their activity results from interaction with opioid receptors in the gastrointestinal tract [32]. This contributes further to the evidence favoring the resistance of tripeptides such as IPP, VPP and Val-Ala-Pro (VAP) to intestinal and circulatory peptides. The resistance of bioactive peptides to degradation during digestion may be enhanced by modification, thereby improving bioavailability, and a number of methods of peptide modification have been described (Table 1). Chemical modifications of peptides introduced at po- Figure 1. Bioavailability of bioactive peptides. Fate of milk-derived bioactive peptides “in vivo”: potential activation and inactivation sites. Milk has both native and latent bioactivities. Latent bioactivities require proteolysis for release of bioactive peptides from the parent milk protein precursors. Digestion of proteins begins in the stomach with pepsin at acidic pH. In the lumen of the small intestine polypeptides are cleaved by an array of pancreatic proteases at alkaline pH conditions. Oligopeptides and free amino acids are absorbed in the brush border membrane via amino acid transport systems. Oligopeptides may be absorbed through tight junctions along with active transport of sugars and amino acids. Additionally, oligopeptides may be further hydrolyzed by the brush border peptidases resulting in di-, tri- and tetra peptides along with free amino acids. These peptides may then be subject to proteolysis by serosal and blood peptidases. If the peptides survive this proteolysis, they can then target specific organs of the body where they can exert various physiological effects. 428 © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 426_200600246_Stanton.qxd 02.04.2007 10:09 Uhr Seite 429 Biotechnol. J. 2007, 2, 426–434 tential cleavage sites may increase the in vivo stability of peptide drug candidates. Identification of potential weak sites in the peptide sequence is a requirement for the rational design of chemical modifications to improve stability [33]. Microencapsulation provides a simple and costeffective way to enclose bioactive materials such as drugs or peptides within a semi-permeable polymeric membrane for the purpose of protecting the biomaterial and releasing the enclosed substance in a controlled fashion at the target site [34]. The bioactive peptide or drug can be incorporated within a capsule, approximately 300 µm in diameter, which consists of any of the following: sugars, gums, proteins, natural and modified polysaccharides, lipids or synthetic polymers [35]. Additionally, liposomes (vesicles in which an aqueous inner volume is enclosed by a phospholipid bilayer) have been used as carriers for drugs, toxins, proteins and peptides [36–40]. Attention has also been focused on the use of microspheres (spheres sized from about 0.5 to 100 µm) composed of the biodegradable biocompatible polymer, poly(D,L-lactic-coglycolic acid) (PLGA) as antigen delivery systems. Studies have shown that protein-loaded PLGA microspheres are capable of eliciting T cell-proliferative responses [41, 42]. Mucins are large (>200 kDa) complex glycoproteins composed of large amounts of carbohydrate (>50 wt%), Olinked to a protein core through Ser and Thr, and expressed on the surface of normal and malignant epithelial cells [43]. The human cancer-associated MUC1 mucin or polymorphic epithelial mucin frequently exhibits aberrant glycosylation, exposing normally cryptic peptide sequences, and these cancer-associated epitopes are the basis of vaccine design for carcinomas expressing MUC1. Additionally, it was demonstrated that a MUC1 24merpeptide-loaded PLGA microsphere elicits T cell-specific immune responses in mice, and this was enhanced by the presence of monophosphoryl lipid A [44]. In addition to the approaches described in Table 1, peptides can be chemically modified to increase their oral delivery by accessing combinatorial libraries [45]. More recently, mixture-based libraries, i.e., large collections of thousands to millions of compounds systematically arranged as mixtures, have been developed that allow high-throughput screening of millions of compounds [45, 46]. These libraries when arranged in a positional scanning format provide extensive structure-activity information in any given assay, and are favored by pharmaceutical companies for the development of new therapeutics [46]. Several attempts to increase the pharmacological activity of opioid peptides using combinatorial libraries have been made. For example, a synthetic combinatorial library containing 52, 128 and 400 D-amino acid hexapeptides was used to identify a ligand for the µ opioid receptor [45]. The peptide Ac-D-Arg-D-Phe-D-Trp-D-Ile-D-Asn-DLys-NH2 was shown to be a potent agonist at the µ opioid receptor and also induced analgesia in mice [45]. This peptide could cross the blood-brain barrier as, unlike pep- © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com tides comprised of L-amino acids, the D-amino acid peptide was not degraded by proteases, remained intact when administered intraperitoneally and produced a greater analgesic effect than the L-amino acid [45]. In addition, a considerable increase in analgesic activity in dogs was obtained by substitution of L- with D-amino acids and by C-terminal amidation [45]. Examples of chemically modified opioid peptides with increased activities include morphiceptin (β-casomorphin-4-amide) and casokefamide (D-Ala2, 4, Tyr5-β-casomorphin-5amide) [10]. To overcome serum inactivation in vivo and toxicity towards non-tumor cells, the conversion of L-amino acid cytomodulatory peptides to their D-diastereomer form has proved successful [47]. Furthermore, database screening can show sequences and the appropriate position of bioactive fragments in proteins as well as changes in bioactive fragments due to natural or artificial mutations. Therefore, when the aim is to detect peptides with known activity in products of protein hydrolysis, using such methods as mass spectrometry, it may be possible to omit the bioactivity assay screening stage [48]. Examples of such databases include BIOPEP (www.uwm. edu.pl/biopep), which contains over 527 sequences of bioactive peptides with anti-hypertensive, opioid, immunomodulatory and other activities [48], and the Active Sequence Collection (ASC) database (http://crisceb.unina2.it/ASC/), which contains over 650 entries in its bioactive peptide (BAC) section. A search of BAC in the ASC database can be helpful when an active region is to be identified within a whole protein sequence or when designing bioactive peptides. In the latter case, sequences sharing homology with known active peptides are sought, as well as negative control peptides sharing no homology with any peptides [49]. Furthermore, a bioactive peptide database has been developed that can be accessed at http://aps.unmc.edu/AP/main.html [50], and contains detailed information on approximately 525 peptides [50]. More recently, a Fourier transform-based screening method was developed that can mine data in the available genomic and proteomic databases and identify potential antimicrobial peptides [51]. ACE-inhibitory peptides can also be produced using genetic engineering techniques. For example, the ACEinhibitory peptides IPP and VPP were synthesized and designed to be multimerized to isoschizomer sites. Subsequently, the cloned gene ap3, was multimerized up to six times in a plasmid and expressed as a fusion protein in E. coli [52]. The peptide (AP3) was easily purified from E. coli and subsequent cleavage with chymotrypsin (a pancreatic enzyme), and resulted in a digest with an IC50 value (the concentration required to yield 50% inhibition) of 18.53 mM. Therefore, a potent ACE inhibitor may be released upon oral ingestion, as chymotrypsin is designed to release the ACE-inhibitory peptide [52]. In addition, DNA recombination technology was used to express the anti-hypertensive peptide FFVAPFPEVFGK in E. coli 429 426_200600246_Stanton.qxd 02.04.2007 10:09 Uhr Seite 430 Biotechnology Journal Biotechnol. J. 2007, 2, 426–434 Table 1. Modes of peptide modification to enhance bioavailability Chemical modification strategies Advantages Disadvantages References Structural modification to enhance stability Increased stability Potential increase in membrane transport Potential increase in receptor affinity Increased membrane permeability Potential decrease in receptor affinity Potential decrease in receptor transport [78] Lipidisation Increased membrane permeability Increased plasma protein binding Intracellular sequestration Non-specific targeting Receptor interference [78] Cationisation Increased membrane permeability Increased serum half-life Increased plasma protein binding Non-specific targeting Rapid elimination of charged moieties [33, 78] Polymer conjugation Increased stability Increases serum half-life Decreases elimination rate Decreases immunogenicity/toxicity Decreases protein-binding Potential for control release Hydrophobic polymers reduce membrane permeability Decreases receptor binding Non-specific targeting [33, 78] Prodrug Increases pharmacokinetics Increases permeability Potential target specificity “Redox” pro-drugs lock peptide in tissue Charged „redox“ pro-drugs are eliminated rapidly [33, 78] Glycosylation Increases membrane permeability Increases stability Increases serum half-life Non-specific targeting Receptor interference [33, 78] Nutrient transport Potentially specific targeting Limited capacity of carriers [33, 78] Genetic modification strategies Cross-linking of target peptide to protein transduction domains Lack of toxicity Lack of sensitivity to serum [24] Genetically engineering microbes for delivery in situ utilising signal peptides Stability in physiological buffer Stabilise the conformation of the bioactive peptide [52, 61] JM109 [53]. DNA technology allows the precise alteration of the DNA sequence of an organism and, therefore, it is possible to change a single nucleotide or a single codon in the chromosome of a microbe [54]. Genetic engineering for microbial production and delivery of bioactive peptides in situ via the enteric microflora is an alternative approach for delivery of bioactive peptides [55]. Probiotics, such as bifidobacteria and lactobacilli, adhere to the intestinal mucosa preventing the subsequent attachment of pathogens, a phenomenon known as competitive exclusion. Additionally, metabolic end-products of lactobacilli present in cell-free culture supernatant inhibit adhesion or invasion of pathogenic bacteria. They also promote accelerated epithelial repair, preventing translocation of the epithelium by pathogens. LAB strains are anaerobic, gram positive and possess generally recognized as safe (GRAS) status. Additionally, many LAB 430 © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim stimulate the immune system of the host as adjuvants [56, 57], making them ideal candidates for the production and delivery of bioactive peptides in the digestive tract [10, 58]. LAB modified to deliver antigens derived from infectious agents are currently undergoing European Commission-supported studies [57]. The delivery of allergens by orally administered genetically modified (GM) lactobacilli was found to inhibit T helper (Th) cells (white blood cell produced in the thymus), Th1 (which produce IFN-γ and IL-12) and Th2 (which produce IL-4, IL-5 and IL-13) subset responses, suggesting the potential of molecular vaccines to allergic disorders [59]. Additionally, peptides obtained following hydrolysis of β-lactoglobulin with Lb. paracasei NCC2461 peptidases repressed lymphocyte stimulation and up-regulated IL-10 production, while down-regulating IFN-γ and IL-4 secretion in vitro. These data suggest that Lb. paracasei may induce oral tolerance 426_200600246_Stanton.qxd 02.04.2007 10:09 Uhr Seite 431 Biotechnol. J. 2007, 2, 426–434 to β-lactoglobulin in vivo by degrading acidic peptides and releasing immunomodulatory peptides [60]. Such engineered LAB can elicit both mucosal and systemic immune responses. Several reports of the use of Lactobacillus sp. and Lactococcus sp. as vectors are available [61–63]. Efficient expression systems have already been developed for controlled and targeted production of the desired antigen for exposure to the gastrointestinal mucosal immune system. Lc. lactis, used for cheese production, can be genetically engineered to secrete effective amounts of bioactive cytokines [61]. For example, Steidler and colleagues [63] reported the delivery of the cytokine IL-10, using the GM Lc. lactis strain, engineered to produce IL-10, in two mouse models of colitis. Trefoil factors (TFFs) are nonmitogenic peptides that are important in the protection and repair of the intestinal lumen, and are promising tools for the treatment of acute colitis [64]. GM Lc. lactis strains also produced and secreted biologically active murine TFFs and oral application of these strains resulted in production of TFFs in situ in mice [65]. Lc. lactis has also been genetically engineered to enhance lipid digestion. The Staphylococcus hyicus lipase was expressed in the cytoplasm of Lc. lactis, and when this GM strain was fed to pigs with ligated pancreatic ducts, fat absorption was enhanced [62]. In the same way that therapeutic compounds are delivered in situ by GM LAB, bioactive peptides could be delivered to the small intestine where they would be exported and cleaved by peptidases resulting in activation. The bioavailability of bioactive peptides may also be improved by cross-linking the target peptide to protein transduction domains or by means of specific peptide carriers [24]. For example, a strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1 [66] was devised, which was able to efficiently deliver a variety of peptides and proteins to several cell lines in a biologically active form, without the need for prior chemical covalent coupling or denaturation steps. This carrier system also presented advantages such as lack of toxicity, stability in physiological buffer and lack of sensitivity to serum [66]. Development of efficacious functional foods enriched in bioactive peptides involves consideration of a variety of factors, including optimized fermentation conditions for release of the bioactive peptides, bioactive peptide enrichment (see below) and their stabilization in the food matrix formulation, as discussed [67]. Bioactive peptide enrichment via ion-exchange membrane chromatography in combination with in situ hydrolysis has been used to enrich cationic antibacterial peptides derived from αs2casein and lactoferricin B (LFcin-B) corresponding to lactoferrin f (17-14) [68]. The isolation of a fraction enriched in LFcin-B can be achieved directly from cheese whey, without the need for intermediate isolation of lactoferrin. Membrane-based processes also have the advantage of speed as they are more rapid than bead-based sys- © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com tems by allowing a higher flow rate (about 20-fold faster). Application of an ultrafiltration (UF) membrane reactor for continuous extraction of permeate enriched with small bioactive fragments has been described for production of antithrombotic peptides [69, 70]. Recently, electrodialysis combined with an UF membrane (EDUF) was found to be a selective method for separation and enrichment of βlactoglobulin-derived peptides with the added benefit that UF membrane integrity was maintained, and EDUF minimized fouling of the UF membrane [71]. 5 Market potential for functional foods based on bioactive peptides The exploitation of bioactive peptides and their use in the manufacture of fermented functional food products, i.e., foods that promise the consumer an “added benefit” over and above the nutrient content, has the potential to contribute to the optimal health of populations and to reduce the risk of chronic disease [8]. Indeed, given the increasing consumer demand for such foods, functional foods are now a multi-billion dollar market with high growth rates and recent estimates indicating up to a $50 billion annual share [72]. The largest segment of this market in Europe, Japan and Australia comprises foods containing probiotics, prebiotics and synbiotics. In recent years, a few functional food products, based on bioactive peptides have been launched on the market (Table 2) [73]. For example, ACE-inhibitory peptides produced during the fermentation of milk are already the basis for health claims associated with some functional foods such as Calpis® sour milk, which is certified as a ‘food for specified health use’ (FOSHU) in Japan with the appropriate health claim on the label [74] and Evolus® launched by Valio in Finland. The FOSHU system is based on a list of approved foods and food ingredients that the Japanese Ministry of Health and Welfare deems as having enough scientific evidence to endorse health claims, and since its establishment in 1993, over 400 foods have been approved and bear the FOSHU label [32, 75]. The development of any fermented functional food containing bioactive peptides requires a detailed understanding of the technological development and scientific aspects of claimed health benefits following consumption. Additionally, the regulatory issues regarding the claimed health benefits in relation to marketing of such foods must be addressed. 6 Conclusion As the perception of nutrition expands to encompass the idea of disease prevention and treatment of specific health conditions, diet has become an important element of the mainstream self-care movement [76]. Much of the appeal of bioactive peptide-containing food products 431 426_200600246_Stanton.qxd 02.04.2007 10:09 Uhr Seite 432 Biotechnology Journal Biotechnol. J. 2007, 2, 426–434 Table 2. Bioactivities produced by hydrolysates and fermentates of dairy products Hydrolysed or fermented product Observed bioactivity Reference Sour milk Sour milk Yoghurt Yoghurt Yoghurt Yoghurt Yoghurt Yoghurt Quarg Dahi Parmesan, Reggiana cheeses Comte cheese Cheddar cheese Mozzarella, Italico cheeses Crescenza, Gorgonzola cheeses Edam, Emmental, ‘Festivo’ cheeses Feta, Swiss, Cheddar, Edam, Camembert cheeses Feta, Swiss, Cheddar, Edam, Camembert cheeses Feta, Swiss, Cheddar, Edam, Camembert cheeses Feta, Swiss, Cheddar, Edam, Camembert cheeses Gouda cheese, Havarti cheese Calpis® sour milk, Calpis Co. Japan Evolus® sour milk, Valio, Finland Whey Protein Hydrolysate (Biozate 1), Davisco, USA. Casein hydrolysate containing the C12 peptide DMV , Holland Casein hydrolysate Casein DP containing the C12 peptide. Kanebo Ltd., Japan Milk protein trypsin milk hydrolysate ING 911 enriched with Alpha S casein called LactiumTM, Cedex, France Phosphopeptides Antihypertensive properties ACE-inhibitory activity Immunomodulatory Antihypertensive properties Antiamnesic Microbiocidal Antithrombotic ACE-inhibitory activity ACE-inhibitory activity Opioid activity Phosphopeptides Phosphopeptides ACE-inhibitory activity ACE-inhibitory activity ACE-inhibitory activity ACE-inhibitory activity Immunomodulatory Antiamnesic Opioid activity ACE-inhibitory activity ACE-inhibitory activity, antihypertensive ACE-inhibitory activity, antihypertensive Antihypertensive properties Antihypertensive properties Antihypertensive properties Anxiolytic-like activity [13] [26] [58] [58] [75] [75] [75] [75] [58] [58] [58] [58] [58] [58] [12] [18] [26] [26] [26] [26] [32] [32] [32] [58] [13] [58] [58] manufactured through dairy fermentation by LAB is the perception that their natural origin equates to a ‘healthier option’. Taking the presented information into account, the future of bioactive peptides, food-grade cultures for over-expression of bioactive peptides, and functional fermented foods looks bright, and it may well be that the words of Chandra ‘the era of nutritional manipulation of the immune system has finally dawned and it brings with it the promise of using diet and nutrition as innovative powerful tools to reduce illness and death caused by infection’ have come of age [77]. However, for successful future expansion of this aspect of the functional food market, there is a need to further investigate the range of bioactivities associated with peptides in food, development of new separation and enrichment technologies, and better techniques for stabilization of the bioactive peptide in foods. Molecular studies are also required to assess the mechanisms by which bioactive peptides exert their activities in vivo. 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