Marine Proteins and Peptides: Biological Activities and Applications

By Se-Kwon Kim

Food proteins and bioactive peptides play a vital role in the growth and development of the body’s structural integrity and regulation, as well as having a variety of other functional properties. Land animal-derived food proteins such as collagen and gelatine carry risks of contamination (such as BSE). Marine-derived proteins, which can provide equivalents to collagen and gelatin without the associated risks, are becoming more popular among consumers because of their numerous health beneficial effects. Most marine-derived bioactive peptides are currently underutilized. While fish and shellfish are perhaps the most obvious sources of such proteins and peptides, there is also the potential for further development of proteins and peptides from sources like algae, sea cucumber and molluscs. Marine-derived proteins and peptides also have potential uses in novel products, with the possibility of wide commercialization in the food, beverage, pharmaceutical and cosmetic industries, as well as in other fields such as photography, textiles, leather, electronics, medicine and biotechnology.

Marine Proteins and Peptides: Biological Activities and Applications presents an overview of the current status, future industrial perspectives and commercial trends of bioactive marine-derived proteins and peptides. Many of the industrial perspectives are drawn from the food industry, but the book also refers to the pharmaceutical and cosmetics industries. There have recently been significant advances in isolating functional ingredients from marine bio-resources and seafood by-products for use in these industries, but little has been published, creating a knowledge gap, particularly with regard to the isolation and purification processes. This book is the first to fill that gap.

Marine Proteins and Peptides: Biological Activities and Applications is a valuable resource for researchers in marine biochemistry field as well as food industry managers interested in exploring novel techniques and knowledge on alternative food protein sources. It will become a standard reference book for researchers involved in developing marine bio-resources and seafood by-products for novel nutraceutical, cosmetics, and pharmaceutical applications. It will also appeal to managers and product developers in the food, pharmaceutical and cosmetics industries, particularly those looking to use marine-derived proteins and peptides as substitutes or replacements for unfashionable or outdated food components.

• Language: English
• Publisher: Wiley
• Release date: Mar 18, 2013
• ISBN: 9781118375112

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Chapter 1

Marine-derived Peptides: Development and Health Prospects

Se-Kwon Kim¹,² and Isuru Wijesekara²

¹Marine Bioprocess Research Center, Department of Chemistry, Pukyong National University, Busan, Republic of Korea

²Department of Chemistry, Pukyoung National University, Nam-Gu, Busan, Republic of Korea

1.1 Introduction

The role of protein in the human diet has been acknowledged recently worldwide. Dietary proteins have become a source of physiologically active components, which have a positive impact on the body's function after gastrointestinal digestion. Bioactive peptides may be produced by one of three methods: solvent extraction, enzymatic hydrolysis and microbial fermentation of food proteins. Marine-derived bioactive food proteins and biopeptides are often effective in promoting health and lead to a reduction in the risk of disease. Recently, much attention has been paid by consumers to natural bioactive compounds as functional ingredients. Hence, it can be suggested that marine-derived bioactive food proteins and biopeptides are alternative sources for synthetic ingredients that can contribute to consumers' well-being, as a part of functional foods, pharmaceuticals and/or cosmetics. Furthermore, they can be utilized in other industries such as medicine, animal feed, printing, textile and so on. This chapter presents an overview of the development, health effects, industrial perspectives and commercial trends of marine-derived bioactive food proteins and biopeptides used in the food, pharmaceutical and cosmetic industries.

1.2 Development of Marine Peptides

Enzymatic hydrolysis of marine-derived proteins allows preparation of bioactive peptides, which can be obtained by in vitro hydrolysis of protein substrates using appropriate proteolytic enzymes. The physicochemical conditions of the reaction media, such as the temperature and pH of the protein solution, must then be adjusted in order to optimize the activity of the enzyme used. Proteolytic enzymes from microbes, plants and animals can be used for the hydrolysis process of marine proteins in order to develop bioactive peptides. Enzymatic hydrolysis is carried out under optimal conditions to obtain a maximum yield of peptides. For example, α-chymotrypsin, papain, Neutrase and trypsin have been applied to the hydrolysis of tuna dark muscle under optimal pH and temperature conditions for each by Qian et al. (2007).

One of the most important factors in producing bioactive peptides with desired functional properties for use as functional materials is their molecular weight (Deeslie & Cheryan, 1981). Therefore, for efficient recovery and in order to obtain bioactive peptides with a desired molecular size and functional property, an ultrafiltration membrane system can be used. This system's main advantage is that the molecular-weight distribution of the desired peptide can be controlled by adoption of an appropriate ultrafiltration membrane (Cheryan & Mehaia, 1990). In order to obtain functionally active peptides, it is normal to use three enzymes in order to allow sequential enzymatic digestion. Moreover, it is possible to obtain serial enzymatic digestions in a system using a multistep recycling membrane reactor combined with an ultrafiltration membrane system to separate marine-derived bioactive peptides (Jeon et al., 1999). This membrane bioreactor technology has recently emerged for the development of bioactive compounds and has potential for the utilization of marine proteins as value-added neutraceuticals with beneficial health effects.

1.3 Health Benefits of Marine Peptides

Marine-derived antihypertensive peptides have shown potent antihypertensive effect with angiotensin-I-converting enzyme (ACE)-inhibition activity. The potency of these marine-derived peptides has been expressed as an IC50 value, which is the the ACE-inhibitor concentration that inhibits 50% of ACE activity. The inhibition modes of ACE-catalyzed hydrolysis of these antihypertensive peptides have been determined by Lineweaver–Burk plots. Competitive ACE-inhibitory peptides have been reported most frequently (Lee et al., 2010; Zhao et al., 2009). These inhibitors can bind to the active site in order to block it or to the inhibitor-binding site remote from the active site in order to alter the enzyme conformation such that the substrate no longer binds to the active site. In addition, a noncompetitive mechanism has been observed in some peptides (Qian et al., 2007; Suetsuna & Nakano, 2000). Numerous in vivo studies of marine-derived antihypertensive peptides in spontaneously hypertensive rats have shown potent ACE-inhibition activity (Fahmi et al., 2004; Zhao et al., 2009).

Recently, a number of studies have observed that peptides derived from different marine-protein hydrolysates act as potential antioxidants; these have been isolated from marine organisms such as jumbo squid, oyster, blue mussel, hoki, tuna, cod, Pacific hake, capelin, scad, mackerel, Alaska pollock, conger eel, yellow fin sole, yellow stripe trevally and microalgae (Kim & Wijesekara, 2010). The beneficial effects of antioxidant marine bioactive peptides in scavenging free radicals and reactive oxygen species (ROS) and in preventing oxidative damage by interrupting the radical chain reaction of lipid peroxidation are well known. The inhibition of lipid peroxidation by marine bioactive peptide, isolated from jumbo squid, has been determined by a linoleic acid model system; its activity was much higher than α-tocopherol and was close to the highly active synthetic antioxidant BHT (Mendis et al., 2005b).

Marine-derived antimicrobial peptides have described in the hemolymph of many marine invertebrates (Tincu & Taylor, 2004), including the spider crab (Stensvag et al., 2008), oyster (Liu et al., 2008), American lobster (Battison et al., 2008), shrimp (Bartlett et al., 2002) and green sea urchin (Li et al., 2008). Antibacterial activity has been reported in the hemolymph of the blue crab, Callinectus sapidus; it was highly inhibitory to Gram-negative bacteria (Edward et al., 1996). Although there are several reports of antibacterial activity in seminal plasma, few antibacterial peptides have been reported in the mud crab, Scylla serrata (Jayasankar & Subramonium, 1999).

The anticoagulant marine bioactive peptides have rarely been reported, but have been isolated from marine organisms such as marine echiuroid worm, starfish and blue mussel. Moreover, marine anticoagulant proteins have been purified from blood ark shell and yellow fin sole. The anticoagulant activity of these peptides has been determined by prolongation of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TP) assays and compared with heparin, the commercial anticoagulant.

Biologically active marine peptides are food-derived peptides that exert a physiological, hormone-like effect beyond their nutritional value, and have a possible role in reducing the risk of cardiovascular diseases by lowering plasma cholesterol level and show anticancer activity through a reduction in cell proliferation on human breast-cancer cell lines. Moreover, calcium-binding bioactive peptides derived from pepsin hydrolysates of the marine fish species Alaska pollock (Theragra chalcogramma) and hoki frame (Johnius belengerii) can be introduced to Asians with lactose indigestion and intolerance as an alternative to dairy products (Kim & Wijesekara, 2010).

1.4 Conclusion

Marine-derived proteins and bioactive peptides have potential for use as functional ingredients in neutraceuticals and pharmaceuticals due to their effectiveness in both prevention and treatment of diseases. Moreover, cost-effective and safe drugs can be produced from marine bioactive proteins and peptides. Further studies and clinical trials are needed for these bioactive proteins and peptides.

References

Cheryan, M., Mehaia, M. A. (1990). Membrane bioreactors: enzyme process. In: Schwartzberg, H., Rao, M. A. eds. Biotechnology and Food Process Engineering. Marcel Dekker: New York.

Deeslie, W. D., Cheryan, M. (1981). Continuous enzymatic modification of proteins in an ultrafiltration reactor. Journal of Food Science, 46, 1035–1042.

Jeon, Y. J., Byun, H. G., Kim, S. K. (1999). Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes. Process Biochemistry, 35, 471–478.

Lee, S. H., Qian, Z. J., Kim, S. K. (2010). A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats. Food Chemistry, 118, 96–102.

Qian, Z. J., Je, J. Y., Kim, S. K. (2007). Antihypertensive effect of angiotensin I converting enzyme-inhibitory peptide from hydrolysates of bigeye tuna dark muscle, Thunnus obesus. Journal of Agricultural and Food Chemistry, 55, 8398–8403.

Chapter 2

Bioactive Proteins and Peptides from Macroalgae, Fish, Shellfish and Marine Processing Waste

Pádraigín A. Harnedy and Richard J. FitzGerald

Department of Life Sciences, University of Limerick, Limerick, Ireland

2.1 Introduction

The marine environment, which makes up more than 70% of the earth's surface, represents a vast, relatively untapped resource for biofunctional compound mining. To date, numerous nitrogenous components (protein, peptides and amino acids) with diverse biological activities have been identified in macroalgae, fish and shellfish. Furthermore, macroalgae, fish, shellfish and marine processing waste contain significant quantities of high-quality protein (10–47% (w/w)), which represents a good candidate raw material for further biofunctional peptide mining.

Significant quantities of waste are generated annually from onshore processing of fish and shellfish and during the processing of aquacultured fish and shellfish. For example, in Norway 800 000 metric tonnes of byproducts were generated by fish processing industries in 2009 (Rustad et al., 2011). It has been estimated that up to 25% of fish and shellfish can end up as waste. In general, this waste material consists of trimmings, viscera, fins, bones, head, skin, undersized fish and shellfish, damaged shellfish and shells. These waste components contain significant levels of protein with potential biofunctional and technofunctional properties. The mining and subsequent exploitation of marine byproducts/waste streams for components with bioactive properties represents a specific strategy for added-value generation. Furthermore, it provides a solution to the legal restrictions, high costs and environmental problems associated with disposal of such waste material. However, regulations concerning the treatment, storage and transport of fish and shellfish byproducts must be carefully adhered to if these raw materials are to be used as sources of functional food ingredients.

2.2 Macroalgal, Fish and Shellfish Proteins: Potential Sources of Bioactive Hydrolysates and Peptides

Macroalgal, fish and shellfish proteins represent a vast resource for the mining of novel biofunctional peptides with specific or multifunctional activity. To date, numerous bioactive peptides have been characterised from these protein-rich marine sources. Furthermore, given their high structural diversity, proteins produced by macroalgae, fish and shellfish potentially contain a range of as yet undiscovered novel bioactive peptides encrypted within their primary structure(s).

2.2.1 Macroalgal Proteins

Marine macroalgae, or seaweeds as they are commonly known, are a diverse group of marine organisms that have developed complex biochemical pathways to survive in highly competitive marine environments. Macroalgae are classified into three groups according to their pigmentation: Phaeophyceae (brown), Rhodophyta (red) and Chlorophyta (green). To date, seaweed has mainly been exploited as a source of food in Asian countries and as a source of technofunctional polysaccharides (agar, carrageenan and alginates) in the Western world. However, macroalgae have begun to emerge as an alternative dietary source of protein and as a reservoir of potential bioactive proteinaceous components.

The protein content of marine algae varies to a large extent with species and season. In general, the highest levels of protein are found in the red species (maximum 47% (w/w) dry weight), with moderate to low levels found in green (9–26% (w/w) dry weight) and brown (3–15% (w/w) dry weight) phyla (Fleurence, 2004). In some red seaweed, protein levels can be as high as 35% (w/w) (Palmaria palmata (dulse)) and 47% (w/w) (Porphyra tenera (nori)), while the green alga Ulva pertusa (anori) can contain up to 26% (w/w) protein (Fleurence, 2004; Wong & Cheung, 2000). Most brown seaweed, with the exception of Undaria pinnatifida (wakame) and Alaria esculenta, which contain protein levels in the range 11–24 and 9–20% (w/w) respectively, has a maximum protein content of 15% (w/w) (Burtin, 2003; Fleurence, 2004; Morrissey et al., 2001). In general, macroalgal proteins contain all essential amino acids, high levels of glutamic and aspartic acid (7.9–44.0% (w/w)) and low levels of threonine, lysine, tryptophan, histidine and the sulfur amino acids cysteine and methionine (Fleurence, 2004). However, the concentrations of those amino acids found at low levels are in general higher than the levels found in terrestrial plants (Galland-Irmouli et al., 1999). Furthermore, some macroalgal species contain high levels of specific amino acids, such as Palmaria palmata, glycine; Porphyra sp. and Chondrus crispus, arginine; Ulva armoricana, proline and Ulva pertusa, arginine; Laminaria digitata, alanine; Undaria pinnatifida, alanine, glycine, arginine, leucine, valine, lysine and significant levels of methionine (Augier & Santimone, 1978; Dawczynski et al., 2007; Fleurence, 2004; Fleurence et al., 1995; Fujiwara-Arasaki et al., 1984; Galland-Irmouli et al., 1999; Morgan et al., 1980; Young & Smith, 1958).

The levels of protein and the corresponding amino acid profiles in macroalgae vary significantly with season. These fluctuations are believed to be linked with a number of variables, such as: geographical location, nutrient supply, environmental conditions (such as light irradiation, water temperature and salinity) and fluctuations in carbohydrate levels (Galland-Irmouli et al., 1999; Marinho-Soriano et al., 2006; Martínez & Rico, 2002; Morgan & Simpson, 1981a,1981b). Furthermore, variations in seaweed protein amino acid profiles indicate variations in the types of proteins or enzymes present in algal tissues at different times of the year. For example, the storage phycobiliprotein, phycoerythrin, was shown to increase in Palmaria palmata during the winter months and early spring—times when seawater nitrogen resources were plentiful—and to decrease during the summer and early autumn months—when seawater nitrogen levels were at their lowest and nitrogen was required for growth (Galland-Irmouli et al., 1999).

2.2.2 Fish and Shellfish Proteins

The protein content of raw fish flesh and shellfish can range from 17 to 22% (w/w) and from 7 to 23% (w/w), respectively (Murray & Burt, 2001). Myofibrillar, sarcoplasmic and stroma proteins are the three main groups of fish and shellfish muscle proteins. In general, myofibrillar proteins (structural proteins) account for 65–75% (w/w) of the total protein in fish and shellfish muscles (Venugopal, 2009). These high-salt soluble proteins consist primarily of myosin, actin, tropomyosin, m-protein, alpha-actinin, beta-actinin, c-protein and troponin T, I and C, of which myosin and actin account for 65–78% (w/w) (Vareltzis, 2000). Additionally, invertebrate muscle contains paramyosin, a protein not found in vertebrate myofibrils (Vercruysse et al., 2005). The level of paramyosin can vary significantly from one shellfish myofibril to another. Levels in the range 3–19% (w/w) have been reported for scallops, squid and oysters, and 38–48% (w/w) for oyster and clam smooth-muscle adductor muscles (Venugopal, 2009). Furthermore, paramyosin contains significant quantities of glutamic acid, arganine and lysine, and low levels of proline (Belitz et al., 2004; Venugopal, 2009).

Sarcoplasmic (water or low-salt buffer soluble) proteins constitute approximately 15–35% (w/w) of the total muscle protein. In general, this protein fraction consists primarily of myoglobin, haemoglobin, cytochrome proteins and a wide variety of endogenous enzymes. However, the level of each protein can vary significantly between species. Some molluscs, for instance, contain no haemoglobin (Belitz et al., 2004). Furthermore, compositional differences have been reported between fish- and mammalian-derived myoglobin (Belitz et al., 2004). Fish-derived myoglobin was shown to contain cysteine, whereas the mammalian equivalent lacked this residue.

Stroma or connective-tissue proteins consist primarily of collagen and elastin (Belitz et al., 2004; Venugopal, 2009). In general, muscle proteins contain approximately 3% (w/w) stroma proteins. However, in some fish, such as shark, ray and skate, stroma proteins can account for 10% (w/w) of total muscle protein. Collagen is the single most abundant protein found in fish species (Kim & Mendis, 2006). It is present in bone, skin, tendons, cartilage and muscle. This triple-helix protein and its partially hydrolysed coiled form, gelatin, contain repeated glycine-proline-hydroxyproline-glycine-X-X amino acid sequences and in addition to glycine, proline and hydroxyproline are rich sources of valine and alanine (Kim & Mendis, 2006; Vercruysse et al., 2005). Currently, the collagen and gelatin used in functional foods, cosmetics and pharmaceutical applications come from bovine and porcine sources (Benjakul et al., 2009b; Venugopal, 2009). However, marine-derived gelatin is an important alternative source for religious reasons and it combats consumer concerns associated with the emergence of bovine spongiform encephalopathy (BSE) and foot and mouth diseases (Kim & Mendis, 2006).

Waste derived from the fish and shellfish industries contains significant quantities of protein. In general, this waste consists of trimmings, fins, frames, heads, skin and viscera, undersized fish and shellfish, and shellfish that have been rejected during grading, due for example to broken shells or excessive fouling (Kim & Mendis, 2006). It has been estimated that 10–20% (w/w) of total fish protein can be found in waste components (Kristinsson, 2008). In addition to trimmings, frames and heads contain residual meat and thus are a good source of muscle proteins. Furthermore, frames, fins and skin are an excellent source of collagen and gelatin (Venugopal, 2009). Rejected shellfish, shells with attached meat and head components are the main waste products arising from the shellfish industry. On average, this waste can account for up to 30–45% (w/w) of the unprocessed weight and represents an excellent source of protein.

The conversion of waste into high-value functional ingredients not only can add value to existing marine resources but also provides industry with a method for dealing with the high cost and legal restrictions associated with the disposal of such waste.

2.3 Enzymatic Hydrolysis of Macroalgal, Fish and Shellfish Processing Waste Proteins: Bioactive Protein Hydrolysates and Peptides

Dietary proteins have been shown to contain peptide sequences that can influence physiological parameters in the body. Some of the parameters modified include blood pressure, insulin and glucose homeostasis, blood cholesterol level and immune function. In general, bioactive peptides are 2–20 amino acids in length and are released from the parent protein during gastrointestinal digestion and/or food processing.

While a number of proteolytic enzymes have been characterised in macroalgae, the endogenous proteolytic system appears to be less developed than that in other marine organisms such as fish and shellfish. Peptides are produced from fish and shellfish muscle proteins during normal post mortem storage by the action of inherent proteolytic enzymes such as calcium-activated calpains and lysosomal cathepsins (Bauchart et al., 2007). Fermented fish products are a popular food source in many Asian countries. During this fermentation process (up to 18 months) fish proteins are hydrolysed by intrinsic muscle proteinases, digestive-tract proteinases and proteinases produced by halophilic bacteria. This fermentation process can result in products with varying consistencies and qualities. Furthermore, due to legal criteria set for potential hazards such as halophilic pathogen bacteria, high salt content and formation of biogenic amines (histamine), domestic and international marketing of these products is becoming difficult. Furthermore, fish proteins can also be hydrolysed using acid or alkali. However, products produced by chemical hydrolysis have limitations on their use as food ingredients. Enzymatic hydrolysis of macroalgal, fish and shellfish processing waste proteins with proteolytic preparations from plant, animal or microbial sources also has the capability to release an array of peptides with potential biofunctional properties in a highly controlled environment.

To date, fish protein hydrolysates have primarily been used for the production of low-value animal feeds, aquaculture (fish and shellfish) feeds, flavours and ingredients for food supplementation (Thorkelsson & Kristinsson, 2009; Venugopal, 2009). Furthermore, fermented fish sauces and pastes are used as staples or condiments in South East Asian, Scandinavian and Eskimo cultures (Fitzgerald et al., 2005). However, a growing body of scientific evidence demonstrates that many marine-derived protein hydrolysates and peptides, including macroalgal, fish and shellfish processing waste byproducts, may play a role in the prevention and management of certain chronic diseases, such as cardiovascular disease (CVD), diabetes, cancer and obesity-related chronic conditions, and thus can be used as functional food ingredients (Harnedy & FitzGerald, 2011; Kim et al., 2008; Kim & Wijesekara, 2010). A summary of macroalgae-derived protein hydrolysates and peptides exhibiting biofunctional activity is given in Table 2.1. These include angiotensin-I-converting enzyme (ACE)-inhibitory, antihypertensive, antioxidant, antitumour, antityrosinase, anticoagulant, calcium precipitation-inhibitory, antimutagenic and plasma and hepatic cholesterol-reducing, blood sugar-lowering and superoxide dismutase (SOD)-like activities. Tables 2.2 and 2.3 summarise bioactive hydrolysates and peptides as reported in the literature from fish waste and shellfish, respectively. Fish waste-derived hydrolysates and peptides display antioxidant, ACE-inhibitory, antihypertensive, anticoagulant and calcium-binding activity. Shellfish-derived hydrolysates and peptides display antioxidant, antihypertensive, antimicrobial, ACE-inhibitory, appetite-suppressant and human immunodeficiency virus (HIV)-1 protease-inhibitory activity.

Table 2.1 Biological activity associated with macroalgal protein hydrolysates and peptides. Adapted from Harnedy & FitzGerald (2011).

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Table 2.2 Biological activity associated with fish waste-derived protein hydrolysates and peptides. Adapted from Harnedy & FitzGerald (2012). Copyright 2012, with permission from Elsevier.

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Table 2.3 Biological activity associated with shellfish-derived protein hydrolysates and peptides. Adapted from Harnedy & FitzGerald (2012). Copyright 2012, with permission from Elsevier.

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2.3.1 In Vitro and In Vivo Cardioprotective Activity

Hypertension, or high blood pressure, is one of the major independent controllable risk factors for CVD. It contributes to an estimated 7.1 million deaths annually, and with increasing longevity worldwide the prevalence of hypertension in people aged 20 years and older is estimated to reach 1.56 billion by 2025 (Kearney et al., 2005). Furthermore, individuals with hypertension are at a greater risk of stroke, coronary artery disease, heart failure, vascular disease and chronic renal failure.

Peripheral blood pressure is regulated by a number of biochemical processes in the body. These include the renin–angiotensin system (RAS), the kinin–nitric oxide system, the neutral endopeptidase system and the endothelin-converting enzyme system (FitzGerald et al., 2004). Treatment of hypertension and heart failure has focused on therapeutic manipulation of the RAS pathway, and in particular inhibition of ACE (EC 3.4.15.1). This carboxydipeptidase converts the inactive decapeptide angiotensin I to angiotensin II, a potent vasoconstrictor (Meisel et al., 2006). Furthermore, ACE inactivates the vasodilator bradykinin. Synthetic ACE inhibitor drugs, such as captopril, enalapril, alcacepril and lisinopril, are widely used for the treatment and prevention of hypertension. Although these synthetic inhibitors are remarkably effective as antihypertensive drugs, they can cause adverse side effects, such as cough, allergic reactions, taste disturbances and skin rashes, among others. Therefore, the use of naturally occurring components as therapeutic agents for the treatment and management of hypertension has gained great interest in recent years. A wide variety of ACE-inhibitory peptides with potent activity in vitro have been identified and characterised from macroalgae, shellfish and marine processing waste proteins. The majority of the ACE-inhibitory peptides shown in Tables 2.2 and 2.3 have relatively short sequences and low molecular masses. Although the structure–activity relationship of ACE-inhibitory peptides has not yet been fully established, several structural features have been identified which appear to influence the ACE-inhibitory action of peptides. It seems that competitive inhibitory peptides with hydrophobic (aromatic or branched side chain) residues in the last three C-terminal positions preferentially bind to ACE (Murray & FitzGerald, 2007). The majority of ACE-inhibitory di- and tripeptides in Tables 2.2 and 2.3 contain Tyr, Phe, Trp, Lys, Val and Ile, and when present Val and Ile are at the N-terminal position. In general, these peptides display ACE IC50 values in the range 1.5–45.0 µM. The in vitro potency of these peptides is comparable to the potency of the ACE inhibitory peptides derived from other food protein sources (Meisel et al., 2006). The potency of an ACE-inhibitory peptide is usually expressed as an IC50 value, which is equivalent to the concentration of peptide that inhibits ACE activity by 50%. Careful control and reporting of enzyme units, in addition to reporting of IC50 values for positive controls such as captopril, is essential for comparison of the different ACE-inhibitory values that have been reported from different laboratories.

Information obtained from in vitro inhibition and simulated gastrointestinal studies is only an indicator of a peptide's potential to act as an hypotensive agent in vivo. Usually the first step in investigating the antihypertensive potential of an ACE-inhibitory protein hydrolysate or peptide in vivo is an animal study using spontaneously hypertensive rats (SHR). A number of macroalgal, shellfish and marine-derived waste protein hydrolysates and peptides have shown hypotensive effects in SHR. In general, the reduction in systolic blood pressure (SBP) following oral administration of 10 mg/kg shellfish and marine-derived peptides was on average of the order of 25 mmHg compared to controls (Je et al., 2005c; Jung et al., 2005a; Lee et al., 2010; Nii et al., 2008). This reduction in SBP was similar to that of captopril. ACE-inhibitory tetrapeptides (YKYY, KFYG and YNKL) derived from the macroalgal species Undaria pinnatifida were shown to significantly reduce SBP at an ingestion level of 50 mg/kg (Suetsuna & Nakano, 2000). Furthermore, dipeptides from the same species were shown to reduce SBP by 14 mmHg (VY, FY and IW) and 21 mmHg (IY) following oral administration at a dose of 1 mg/kg (Suetsuna et al., 2004; Suetsuna & Nakano, 2000). Protein hydrolysates and peptide fractions derived from oyster proteins, sea bream scale collagen and Porphyra yezoensis proteins have also shown antihypertensive activity in SHR (Fahmi et al., 2004; Katano et al., 2003; Saito & Hagino, 2005; Suetsuna, 1998a; Suetsuna & Saito, 2001; Wang et al., 2008). However, variations in sample type, dosage and duration of administration make it difficult to compare these hydrolysates in terms of SBP reduction. While a number of shellfish and marine-derived ACE-inhibitory peptides have shown hypotensive effects in SHR studies, there is a lack of information in relation to human intervention studies. However, macroalgal protein-derived hydrolysates and peptides exhibited promising cardioprotective effects in two human studies. Undaria pinnatifida protein hydrolysates in a jelly format were fed at two doses to two groups of mildly hypertensive subjects over an 8-week period. A significant reduction in SBP compared to controls was observed after 8 weeks for the group consuming 300 mg/day and after 6 weeks for the group consuming 500 mg/day (11 mmHg) (Kajimoto et al., 2002). In a second study, a significant antihypertensive effect was observed after 8–12 weeks when 1.6 g Nori (Porphyra yezoensis) oligopeptides was consumed per day for 12 weeks by subjects with high-normal blood pressure (Kajimoto et al., 2004).

Inhibition of blood clot formation and the lowering of plasma and hepatic cholesterol levels can significantly reduce the risk associated with CVD. Blood clotting or coagulation is a complex biochemical pathway triggered to prevent excessive bleeding when a blood vessel is injured (Schaffer et al., 1991). Once the injury has healed, the body naturally dissolves the clot. Unwanted clot formation is however detrimental and may result in stroke, heart attack, pulmonary embolism or deep vein thrombosis. Heparin, a highly sulfated polysaccharide, is one of the most common anticoagulant agents used for the treatment of such blood clots (Wijesinghe & Jeon, 2012). However, there are some well-documented side effects, such as excessive bleeding, associated with the clinical use of heparin as an anticoagulant agent. A number of protein hydrolysates and peptides derived from macroalgae and marine-derived waste components exhibit anticoagulant activity (Tables 2.1 and 2.2), (Athukorala et al., 2007; Rajapakse et al., 2005a). However, to date no research appears to have been carried out to assess the anticoagulant efficacy of these proteinaceous components in vivo. In contrast, a number of animal studies have shown that feed containing Porphyra yezoensis protein hydrolysates can significantly lower total cholesterol, free cholesterol, triglyceride and phospholipid levels in rats and reduce plasma total cholesterol, triglycerides and low-density lipoprotein (LDL) levels in mice (Suetsuna & Saito, 2001). As with soy protein, where according to an approved health claim an intake of 25 g of soybean per day can lower serum cholesterol levels by 5–10%, these sea vegetable-derived protein hydrolysates may promote cardiovascular health by aiding in the treatment or prevention of hyperlipidaemia or hypercholesterolaemia (Nagaoka, 2006).

2.3.2 Oxidative Stress

Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) such as superoxide anion (O−2) and hydroxyl (OH−1) radicals and the inability of a biological system's endogenous antioxidant defence mechanisms (enzymatic and non-enzymatic) to readily detoxify and deal with reactive intermediates. Oxidative stress has implications in many chronic diseases, including heart disease, stroke, arteriosclerosis, diabetes and cancer (Dávalos et al., 2004). Furthermore, deterioration in food quality, arising from oxidation of unsaturated fatty acids, is a major concern for the food industry. Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate are added to food products to retard lipid oxidation. However, the utilisation of these synthetic antioxidants must be rigorously controlled due to potential adverse side effects (Shahidi & Zhong, 2005). Currently, there is a growing interest in the use of natural antioxidant agents with little or no known harmful health effects to combat oxidative stress and prolong the quality of food.

Macroalgae, as previously mentioned, are a diverse group of marine organisms that have developed complex biochemical pathways to survive in highly competitive marine environments. Harsh environmental factors, such as high irradiation, desiccation, freezing, low temperature, heavy metals and salinity fluctuations, promote the production of excess ROS within macroalgal cells (Cornish & Garbary, 2010). In response, seaweeds synthesise intrinsic antioxidant components and stimulate the production of endogenous antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). The range of natural antioxidant components with potential functional food applications produced by seaweed is reviewed elsewhere (Cornish & Garbary, 2010; Tierney et al., 2010). Numerous protein hydrolysates generated by direct hydrolysis of macroalgal cells in aqueous solutions via a range of food-grade proteolytic enzyme preparations exhibit antioxidant activity in vitro (Table 2.1). The mechanisms by which macroalgal hydrolysates exert antioxidant activity include radical scavenging, transition metal chelating, lipid peroxidation inhibition and H2O2-induced DNA damage protective effects.

While the precise relationship between peptide structure and antioxidant activity has not yet been elucidated, the type, position and hydrophobicity of amino acids present in the peptide are thought to play essential roles. Several amino acids, such as histidine, leucine, tyrosine, methionine and cysteine, are generally accepted as antioxidants. These improve radical scavenging activity by donating protons to electron-deficient radicals (Mendis et al., 2005a; Sarmadi & Ismail, 2010). As shown in Tables 2.2 and 2.3, several shellfish and fish waste-derived peptides containing such amino acids exhibit radical scavenging activity (Je et al., 2005d; Kim et al., 2007; Mendis et al., 2005a,b; Rajapakse et al., 2005b,c). Furthermore, the gelatin-derived peptides HGPLGPL and GE(Hyp)GP(Hyp)GP(Hyp)GP(Hyp)GP(Hyp)G exhibit potent lipid peroxidation-inhibiting activity (Kim et al., 2001; Mendis et al., 2005b). Both of these peptides contain the characteristic repeating glycine–proline sequence associated with gelatin. Hydrophobic amino acids such as glycine, valine, alanine, proline and hydroxyproline are found in abundance in gelatin, and it is the presence of these amino acids that is believed to exert such a potent lipid peroxidation inhibition effect (Kim & Wijesekara, 2010). Hydrophobic amino acids have high affinity for lipid systems. Therefore, oil-soluble radicals (e.g. hydrophobic peroxyl radicals) generated during oxidative attack of unsaturated fatty acids are believed to be neutralised by hydrophobic amino acid-containing antioxidant peptides. Moreover, two squid muscle-derived peptides containing the same dipeptide (GP) repeating sequence have also been shown to inhibit free radical-mediated oxidation of linoleic acid (Mendis et al., 2005a).

Protein hydrolysates and peptides derived from macroalgal and marine waste have also shown antioxidant activity in a number of cell model systems. Hydrolysates generated from Laminaria japonica stimulated the production of the endogenous antioxidant enzymes CAT, GPx and glutathione S-transferase (GST)) in H2O2-treated cells (Park et al., 2009). Furthermore, cellular antioxidative enzyme (SOD, CAT and GPx) levels were significantly elevated in a human hepatoma cell model following induction with a purified antioxidant peptide, HGPLGPL, from a Hoki skin hydrolysate (Mendis et al., 2005b).

Oxidative stress may also play a role in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. In particular, reactive quinones in the brain produced by tyrosinase are believed to be linked to Parkinson's disease (Schurink et al., 2007). Tyrosinase (EC:1.14.18.1) is a multifunctional copper-containing enzyme present in plant and animal tissues that catalyses the hydroxylation of a monophenol (tyrosine) and the oxidation of o-diphenols to the corresponding o-quinone (Chang, 2009). This is the key enzyme involved in the biosynthesis of the pigment melanin and in the browning that occurs in vegetables and fruit upon bruising or long-term storage. Various dermatological disorders result in the accumulation of excessive levels of epidermal pigmentation and a number of cosmetic preparations contain chemical antityrosinase inhibitors for the treatment of hyperpigmentation. Partially purified hydrolysates derived from the macroalgae Enteromorpha prolifera and Porphyra tenera have been reported to inhibit tyrosinase in vitro (Lee et al., 2005).

A need currently exists to demonstrate the efficacy of antioxidant and antityrosinase protein hydrolysates and peptides derived from macroalgae, shellfish and marine waste in carefully controlled human-intervention studies. Furthermore, studies are needed to assess the potential of marine-derived peptides as natural lipid peroxidation-inhibiting agents or natural biopreservatives for the prevention of browning in food systems.

2.3.3 Other Biofunctionalities

As shown in Tables 2.2 and 2.3, protein hydrolysates and peptides with appetite-suppressing, blood sugar-reducing, calcium-binding, calcium precipitation-inhibiting, antimicrobial and HIV protease-inhibiting activity have also been identified in macroalgae, shellfish and marine processing waste.

Cholecystokinin (CCK) is an important hormone which regulates appetite and gastric emptying. Quantification of CCK can be used as a biomarker for assessing satiety within the body. Low-molecular-weight peptides (1.0–1.5 kDa) from shrimp-head protein hydrolysates have been identified as having a significant stimulatory effect on the release of CCK in intestinal endocrine cells (Cudennec et al., 2008). Furthermore, Porphyra yezoensis protein hydrolysates incorporated in feed aided in the reduction of plasma glucose levels in rats (Suetsuna & Saito, 2001). It is commonly accepted that intervention is needed to combat the rise in obesity and associated chronic diseases. The prevalence of obesity has reached epidemic proportions worldwide. In the USA and UK, levels are predicted to increase by 65 million and 11 million, respectively, by 2030. Chronic obesity-related diseases such as type II diabetes, CVD and cancer are also predicted to escalate within this timeframe. Products containing proteinaceous biofunctional ingredients targeted at prevention may have application in the management of obesity.

Components which bind and solubilise minerals such as calcium can be considered physiologically beneficial in the prevention of osteoporosis, dental caries, hypertension and anaemia (Korhonen & Pihlanto, 2006). Hoki and pollock frame-derived peptides and a semipurified hydrolysate derived from Porphyra yezoensis aid in calcium binding and inhibition of calcium precipitation in vitro (Jung & Kim, 2007; Jung et al., 2005b, 2006a; Suetsuna & Saito, 2001). Furthermore, fish meal rich in hoki frame phosphopeptides has been shown to increase calcium bioavailability in osteoporosis model rats to the same level as a commercially prepared casein oligophosphopeptide preparation (Jung et al., 2006b). Potential applications of such marine-derived phosphopeptides include dairy-free functional ingredients for people who are lactose intolerant or for the prevention and management of caries and osteoporosis.

The unstoppable, ongoing emergence of resistant microbes worldwide has rapidly accelerated the search for novel antimicrobial compounds to replace and/or supplement conventional antibiotics. Furthermore, the food industry is looking for additional safe biopreservatives to replace existing synthetic antimicrobial components. Nisin, a polycyclic antibacterial peptide, is an example of such an agent used in the food industry. Marine organisms by their very nature have innate defence mechanisms to deal with water-based pathogens. In general, nitrogenous components produced by macroalgae, fish and shellfish to combat such an attack come in the form of endogenous non-protein-derived peptides (Table 2.4). In most cases, these peptides have similar structural characteristics, being cationic and amphiphilic. However, a novel cysteine-rich antimicrobial peptide, CgPep33, exhibiting activity against Gram-positive and Gram-negative bacteria and fungi, has been isolated from enzymatic hydrolysates of Crassostrea gigas oyster protein (Liu et al., 2008). To date, no in vivo or food-stability studies have been performed to determine the therapeutic and food-preservation efficacy of marine-derived antimicrobial peptides.

Table 2.4 Endogenous bioactive peptides derived from marine sources. Adapted from Harnedy & FitzGerald (2012). Copyright 2012, with permission from Elsevier.

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Finally, HIV-1 protease-inhibitory peptides, LLEYSI and LLEYSL, have been identified from thermolysin digests of oyster proteins (Lee & Maruyama, 1998). These peptides have potential application as antiviral agents for the control of HIV-1 infection by inhibiting HIV-1 protease, an enzyme central to retrovirus HIV-1 replication.

2.4 Endogenous Bioactive Peptides from Macroalgae, Fish and Shellfish

Several linear, cyclic peptides, depsipeptides and peptide derivatives have been characterised from marine resources including macroalgae, fish and shellfish. In addition to the antimicrobial peptides mentioned in Section 2.3.3, peptides with antioxidant, mitogenic, anticancer, antiproliferative, antiviral, antiprotozoan, calcium-binding, cytotoxic, opioid, antidiabetic, anticoagulant, haemolytic and agglutinating activity have also been reported. The specific details concerning these peptides are summarised in Table 2.4. Carnosine, anserine and glutathione are antioxidant peptides found in high quantities in fish muscle (Bragadóttir, 2001). Furthermore, the histidyl dipeptide carnosine and the tripeptide glutathione have been identified in macroalgae. The latter peptide is involved in the inherent enzymatic antioxidant defence system (ascorbate–glutathione cycle) (Fleurence, 2004; Shiu & Lee, 2005).

Galaxamide 1 and Kahalalide F are two endogenous peptides produced by macroalgae that exhibit potential anticancer activity. Galaxamide 1, a peptide isolated from Galaxaura filamentous, has shown antiproliferative activity against human renal-cell carcinoma GRC-1 and human hepatocellular carcinoma HepG2 cell lines (Xu et al., 2008). Furthermore, Kahalalide F, a cyclic depsipeptide isolated from the mollusc Elysia rufescens but originated from its foodstuff, the green alga Bryopsin sp., has been shown to display potent cytotoxic activity against cell lines from solid tumours, including prostate, breast and colon carcinomas, neuroblastoma, chondrosarcoma and osteosarcoma (Hamann & Scheuer, 1993; Hamann et al., 1996; Suárez et al., 2003). The anticancer activity of this cyclic depsipeptide has been assessed in three phase II clinical trials. While the compound was show to stabilise the tumours, the studies were discontinued after the first stage due to lack of significant antitumour activity (Galan et al., 2006; Martín-Algarra et al., 2009; Paz-Ares et al., 2006; Provencio et al., 2006). However, in one of the trials an advanced lung-cancer tumour in one of the patients showed a partial response to Kahalalide F (Provencio et al., 2006).

2.5 Bioactive Proteins from Macroalgae, Fish and Shellfish

Poor permeability through biological membranes due to molecular size, physical and chemical instability, degradation by intrinsic proteolytic enzymes, aggregation, adsorption and immunogenicity are some of the limitations that affect the bioavailability of bioactive proteins and peptides. In order for a bioactive component to exert a modulating effect in vivo, it needs to reach its target site in an intact form. In general, peptides with two to six amino acids are more readily absorbed across the gastrointestinal tract than are whole proteins (Grimble et al., 1986). On ingestion, proteins and peptides may be hydrolysed by gastrointestinal enzymes such as pepsin, trypsin and chymotrypsin. Peptides can then be further degraded by brush-border peptidases and intracellular peptidases during their passage across intestinal epithelial cells into the bloodstream. There are three major routes by which this takes place (Fig. 2.1): (1) PepT1 carrier-mediated transport; (2) tight-junction paracellular diffusion; and (3) endocytosis–exocytosis (Young & Mine, 2009). PepT1 specifically transports di- and tripeptides from the apical side of the epithelial membrane into the cell by H+ coupling. Once inside the cell, peptides may be degraded to amino acids by cytoplasmic peptidases and amino acids are transported across the basolateral membrane into the bloodstream by amino acid transporters. In some cases, peptides may remain intact if their structural characteristics render them resistant to attack by cytoplasmic peptidases. In paracellular diffusion, oligopeptides are transported passively via pores at the tight junctions in an intact form. Peptides can also be transported in and out of the cell by endocytosis and exocytosis. This route involves engulfing the peptide at the apical side of the epithelial membrane inside a vesicle and transporting it to the basolateral membrane, where the peptide is expelled. However, peptides can be hydrolysed to amino acids within this vesicle. In general, basic and hydrophobic peptides are transported by this method (Young & Mine, 2009). PepT1 carrier-mediated transport and tight-junction paracellular diffusion offer the best mode of transport of intact peptides across intestinal epithelial cells into the bloodstream. Furthermore, peptides can be degraded in the blood by serum peptidases. As a result, peptides, and more specifically proteins, may not reach their target site in an intact form.

Fig. 2.1 Peptide transport routes across intestinal cells.

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However, some marine-derived proteins have shown biological activity in animal models following oral administration. It may be that these active agents are broken down into fragments. Such protein molecules include lectins from the macroalgal species Eucheuma sp., Bryothamnion triquetrum, Bryothamnion seaforthii and Amansia multifida. Lectins are a structurally diverse group of carbohydrate-binding proteins found in a wide range of organisms (Hori et al., 2000). They are involved in a number of biological processes, such as host–pathogen interactions, cell–cell communication, induction of apoptosis, cancer metastasis and differentiation (Calvete et al., 2000; Ziółkowska & Wlodawer, 2006). ESA-2, a lectin isolated and characterised from Eucheuma serra, has been shown to suppress colonic carcinogenesis in mice following oral administration (Hori et al., 2007). Furthermore, lectins from this species of macroalga have been shown to exhibit strong mitogenic activity against mouse and human lymphocytes and are reported to be cytotoxic against several cancer cell lines, such as colon cancer (Colo201) and cervical cancer (HeLa) cells—they inhibited the growth of 35 human cancer cell lines (Hori et al., 2007; Kawakubo et al., 1997, 1999; Sugahara et al., 2001). Lectins with mitogenic activity have also been isolated from Soleria robusta (Hori et al., 1988). These monomeric glycoproteins have been shown to be active against mouse spleen lymphocytes and inhibited the growth of mouse leukaemia cells L1210 and mouse FM3A tumour cells (Hori et al., 1988). Lectins from several species of marine alga—Bryothamnion triquetrum, Bryothamnion seaforthii and Amansia multifida—have shown acetic acid-induced abdominal contraction inhibitory activity in Swiss mice (Neves et al., 2007; Viana et al., 2002). Other bioactive properties exhibited by macroalgal lectins include antibiotic, anti-inflammatory, antiadhesion, anti-HIV activity and human platelet aggregation inhibition activities (Harnedy & FitzGerald, 2011).

The phycobiliproteins (phycoerythrin, phycocyanin, allophycocyanin and phycoerythrocyanin) are another group of interesting bioactive proteins found in red algae. These molecules are highly fluorescent compounds. As a result, phycobiliproteins, in particular phycoerythrin, have wide-ranging applications in biotechnology, being utilised in fluorescent immunoassays, fluorescent immunohistochemistry assays, biomolecule (protein, antibody, nucleic acid) labelling and fluorescent microscopy (Aneiros & Garateix, 2004; Sekar & Chandramohan, 2008). In addition, phycobiliproteins are used as natural colourants for foods such as chewing gum and dairy products and for cosmetics such as lipsticks and eyeliners (Sekar & Chandramohan, 2008). While most research has been performed with phycobiliproteins from the blue-green microalga Spirulina and the unicellular red alga Porphyridium, it is possible that phycobiliproteins from red macroalgae may have similar activities. Phycobiliproteins from Spirulina and Porphyridium exhibit antioxidant, anti-inflammatory, neuroprotective, hypocholesterolaemic, hepatoprotective, antiviral, antitumour, liver-protecting, atherosclerosis-treatment, serum lipid-reducing and lipase-inhibition activities (Harnedy & FitzGerald, 2011; Sekar & Chandramohan, 2008). Bioactive proteins with anticoagulant, antimicrobial, immunostimulatory, anticancer and hypocholesterolaemic properties have also been identified in fish and shellfish (Harnedy & FitzGerald, 2011; Jung et al., 2002; McFadden et al., 2003; Nagashima et al., 2003; Patat et al., 2004; Riggs et al., 2002; Smith et al., 2000). The majority of these proteins exhibit antimicrobial activity and are believed to be produced as part of an organism's defence mechanism against microbial attack. However, if marine-derived bioactive proteins are to be incorporated into foods as biofunctional ingredients, a critical requirement exists that the biological activity of these proteinaceous components be assessed in human studies.

2.6 Commercial Products Containing Marine-Derived Bioactive Protein Hydrolysates and Peptides

Substantial scientific validation of the health-promoting effects of biofunctional ingredients is required before a health claim can be made that has European Food Safety Authority (EFSA), Food and Drug Administration (FDA) and Foods for Specified Health Use (FOSHU) approval. Numerous products containing fish-protein hydrolysates/peptides as functional ingredients have been given FOSHU status in Japan (Table 2.5), including Valtyron® and Lapis Support™, both of which are marketed as having hypotensive effects. The ‘Sardine Peptide product’ Valtyron®, generated using a commercially available food-grade alkaline protease from Bacillus licheniformis, has been incorporated into 33 different products, including soft drinks, jelly, powdered soup and dietary supplements (EFSA Panel on Dietetic Products, 2010). These products, in particular the dipeptide VY, have been shown to significantly reduce SBP in mildly hypertensive patients (Kawasaki et al., 2000). Furthermore, Valtyron® has recently been passed as safe by the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) for use as a novel food ingredient at a level of 0.6 g/serving (EFSA Panel on Dietetic Products, 2010).

Table 2.5 Commercially available marine protein, protein hydrolysate and peptide products. Adapted from Harnedy & FitzGerald (2012). Copyright 2012, with permission from Elsevier.

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A hypotensive ‘Peptide Soup’ product generated from katsuobushi (bonito) muscle has also been awarded FOSHU status. The bioactive peptide therein, LKPNM, has been shown to exhibit antihypertensive effects in mildly hypertensive subjects (Fujita et al., 2001; Fujita & Yoshikawa, 2008). This product is commercially available in Japan in beverage format (soup and tea), as a powdered ingredient and in tablet format, in which it is marketed as Peptide ACE 3000 (http://www.nippon-sapuri.com/english/index.html, last accessed 08/10/12). Furthermore, the tablet format is sold in the USA under the trade names Vasotensin™ and PeptACE™ and in Canada as Levenorm™ (Thorkelsson & Kristinsson, 2009). Two other marine-derived peptides with FOSHU-approved antihypertensive claims are the Wakame peptide (YNKL), which is sold as a peptide jelly, and the Nori peptide (AKYSY), which is sold under the trade name Peptide Nori S (http://en.item.rakuten.com/kenkoex/noripepu30/, last accessed 08/10/12).

Several marine-derived proteinaceous components are sold in Europe and North America as food supplements without approved health claims. These include Stabilium® 200, Protizen®, Collagen HM, Glycollagen®, Marine Cartilage Powder, Protein M+, PeptiBal™, AntiStress 24, Nutripeptin™, Seacure® and Fortidium Liquamen®. A blue ling autolysate Stabilium® 200 and the fish hydrolysates Protizen® and AntiStress 24 claim to have relaxing effects (Crocq et al., 1980; Guerard et al., 2010; Le Poncin & Lamproglou, 1996; Thorkelsson & Kristinsson, 2009). In addition to Protizen®, Copalis Sea Solutions markets products containing marine-derived collagen hydrolysates that aid joint health. Collagen HM, which contains fish skin collagen-derived oligopeptides (<3.6 kDa), is recommended as a food supplement for the promotion of cartilage, bone and skin regeneration, while Glycollagen®, Marine Cartilage Powder and Protein M+ are sold as supplements to soothe joint pain. Glycollagen®, Marine Cartilage Powder and Protein M+ also contain chondroitin sulfate and glucosamine. These two mucopolysaccharides have been shown to aid joint regeneration in vivo (Reginster et al., 2001; Wildi et al., 2011). PeptiBal™, an ultrafiltrated shark-protein hydrolysate product, has been reported to have immunomodulatory effects in human clinical trials. A statistically significant increase in IgA production was observed in a randomised double-blind placebo-controlled study (Boutin et al., 2012). Nutripeptin™, Seacure® and Fortidium Liquamen® are also sold as food supplements. Nutripeptin™ is marketed as having postprandial blood glucose-lowering activity, Seacure® is sold as a supplement for the improvement of gastrointestinal health and Fortidium Liquamen® is commercialised as having multifunctional effects including antioxidant, antistress and glycaemic index-lowering activities (Guerard et al., 2010).

A number of factors need to be taken into account when producing marine-derived protein hydrolysates/peptides at an industrial scale. Two strategies can be adopted for the hydrolysis of marine proteins: hydrolysis of fish and shellfish muscle and marine waste directly with food-grade proteolytic enzymes, such as Alcalase®, Neutrase®, Flavourzyme® and Protamex®, and pre-extraction of protein prior to hydrolysis (Giménez et al., 2009; Guerard et al., 2010; Hai-Lun et al., 2006). Membrane-separation techniques such as ultrafiltration and nanofiltration seem to provide the most suitable industrially relevant technology for the enrichment of peptides within specific molecular-weight ranges and are currently used for the industrial production of ingredients containing bioactive peptides (Korhonen, 2009; Korhonen & Pihlanto, 2007). Membranes with molecular mass cut-off values in the range 1–10 kDa are primarily used for the fractionation of peptides (Hai-Lun et al., 2006; Je et al., 2005a; Jeon et al., 1999; Rajapakse et al., 2005b). Furthermore, electromembrane filtration, which involves the use of charged membranes and membrane bioreactor technology combining enzymatic hydrolysis of marine proteins and peptide separation by ultrafiltration, is being considered as a potential method for the generation and fractionation of marine-derived bioactive peptides (Kim & Wijesekara, 2010). In general, in vitro bioactivity assay-directed purification is the approach taken to fractionate and purify peptides with specific biofunctional activities. However, if specific peptides are to be purified, specific chromatographic techniques may need to be coupled with membrane processing (Pouliot et al., 2006). The extent to which a peptide needs to be purified is dependent on the potency of the bioactive component, and on an industrial level the cost of purifying specific bioactive peptides must be weighed against the value of the purified or semipurified product. As a result, high-cost semi- and preparative-scale chromatography may only be utilised if highly purified peptides are required for commercialisation (Pouliot et al., 2006).

2.7 Conclusion

Marine-derived waste, fish and shellfish are a relatively untapped source of new bioactive proteinaceous compounds, and more efforts must be made to fully exploit their potential for use and for delivery to consumers in food products. However, in order for marine bioactive peptides or protein hydrolysates to be utilised as health-promoting functional food ingredients, aspects such as large-scale production, compatibility with different food matrices, gastrointestinal stability, bioavailability and long-term stability must be addressed. Carefully controlled human-intervention studies are needed to demonstrate the efficacy of bioactive components in vivo. Furthermore, there is a need for more detailed understanding of the mechanisms by which different peptides/hydrolysates may mediate their physiological effects. In order to fully understand the relationship between exposure in the body and physiological effect, metabolomic and nutrikinetic studies may have to be performed. Finally, marketing of bioactive health-promoting functional ingredients requires scientific validation before a health claim can be made with European Food Safety Authority (EFSA), Food and Drug Administration (FDA) and Foods for Specified Health Use (FOSHU) approval.

Acknowledgement

This project (Grant-Aid Agreement No. MFFRI/07/01) is carried out under the Sea Change Strategy with the support of the Marine Institute and the Department of Agriculture, Food and the Marine, funded under the National Development Plan 2007–2013.

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