Marine protein hydrolysates: their present and future perspectives in food chemistry – a review

Abstract

Marine protein hydrolysates are usually prepared by the enzymatic digestion of different proteases at controlled pH and temperature. The important biological activities of biologically potential peptides and essential amino acids have been scientifically proved. Now, marine bio-diversity can be considered in the utilization of protein hydrolysates, which can provide nutritional benefits and play a significant role as functional ingredients for food industries. This manuscript reviews the preparation, purification and bioavailability of various marine based protein hydrolysates and bioactive peptides using recent technology tools. Fractionated peptides with biological activities have potential use in major health issues and as functional ingredients for food processing.

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Vijaykrishnaraj M.

Mr Vijaykrishnaraj M. is an emerging researcher, pursuing his Doctoral studies under the supervision of Dr P. Prabhasankar at the CSIR-CFTRI. He is a Marine Biotechnology graduate from Bharathidasan University. He was the university first rank holder in his batch. His area of interest includes biochemistry, marine biology and molecular nutrition. He has experience in port biological sampling and ballast water sampling as a project staff in the CSIR-NIO. Currently, he has initiated work in food technology and the nutritional enrichment of marine protein hydrolysates for people in need. He has published peer-reviewed research articles in well-known journals and presented at several national/international symposia.

Prabhasankar P.

Dr P. Prabhasankar is a well-known person in the area of Food chemistry, Food processing and Immunochemical methods. His research publications can be seen in highly reputed peer-reviewed journals. He has twenty-three years of research experience consisting of fourteen years research experience in the above mentioned area and seventeen years of teaching experience in various academic areas. He has three US patents and four international patents, and has also developed seven processes on different types of pasta. He is the Editor-in-chief of the Journal of Food Science and Technology and is an editorial board member of many other reputed journals.

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Introduction

The surface of our planet is covered with 70% saline water and rest filled with its remaining resources. The structure of the Earth is separated into different layers with the hydrosphere entirely covered with marine water. Marine sources play a vital role in our ecosystem as well as the food web in the oceanic community. Marine biodiversity is one of the largest biodiversities on the earth based on the adaptive mechanism of huge variants of living organisms, which are abundant from the micro to macro levels.¹ Recently, studies have been focused on marine biomolecules, which have biological potential in healthcare, drug molecules and functional food ingredients. The Greek philosopher Aristotle gave the definition for marine sources as “the ladder of life” and described 500 species of which several were identified from marine sources.² Spatially, marine environment is divided into different zones, and mainly pelagic and benthic zones. The pelagic zone covers from the surface of the ocean layer to the photic zone. The benthic zone is the deeper area of the ocean layer and light cannot penetrate to reach this zone. Overall, these two different regions are habitats for various living organisms.³

Marine ecosystems have a vast abundance of living organisms, which include those from estuaries and wetland ecosystems. Life in the sea has been fascinating for thousands of years. The study of these organisms and their importance in food science and nutrition are very rare; now, it is important to find marine sources that will be beneficial to human health. Many microorganisms are responsible for the majority of atmospheric oxygen fixation on the Earth. These tiny organisms can also be responsible for the primary production of the marine food web. The microscopic organisms found in seas, ponds, and lakes help to recycle nutrition. According to their size, they are classified from micro to mega planktons. Those with the size in the range of 20–200 μm have been categorized as micro planktons, while those having size more than this level have been classified as mega planktons. An overview of marine life and its impacts on earth has been depicted in Fig. 1. The secondary metabolites of these organisms play a vital role in physiological functions. Mega planktons are highly influenced by the sea as well as by the humans.³

Fig. 1 A schematic representation of marine life and its impacts.

The zones of the oceans depend on the depth of the floor and sea. The surface layer of the sea is called the epipelagic zone. It covers up to 200 m from the surface layer of the water. Most tiny living organisms are abundant in this area because they need sunlight and energy to proliferate. The mesopelagic and bathypelagic zones cover up to 1000 m from the pelagic zone, this zone consists of abundant floating organisms and swimming organisms.⁴ The bio-actives present in marine and other aquatic resources can rescue and treat the adverse health effects of chronic diseases. Fish is one of the major marine foods consumed all over the world because of its nutritional benefits.⁵ The discards from seafood processing account for approximately three-quarters of the total weight of catch and include trimmings, fins, frames, heads, shells, skin and viscera.^6–8 Large quantities of fish are collected worldwide every year with approximately 50% of the protein rich processed by-products from fish discarded and used as animal feed and fish meal.⁹ The use of marine food and its by-products as substrates leads to a novel approach for the potential discovery of high-value bio-actives.⁷ Fish and fish by-product hydrolysates and active ingredients are the “big-dream” of the marine biotechnology industry: though these products are in low quantity, their value is high, and they also possess tremendous potential as innovative bio-molecules.¹⁰

Marine bioprocessing industries have a high potential to convert and utilize marine food products and their by-products as valuable functional ingredients.¹¹ Seafood from both fisheries and aquaculture are supplied to the world markets, providing approximately 2.9 million people with at least 15% of the protein of their average per animal protein intake.¹² World aquaculture production of fishes, crustaceans, mollusks, etc. have increased yearly. According to the FAO,¹³ Asia is the largest producer of aquaculture (580 million), followed by Africa (1.4 million) and Europe (2.8 million). Marine organisms provide functional compounds such as PUFA (polyunsaturated fatty acids), protein and its bioactive peptides, minerals, vitamins and polysaccharides.¹⁴

The human body undergoes physiological imbalance and an exposure to extrinsic toxic substances that disturbs its normal functions, which can result in various health problems.¹⁵ In addition, processed food products or foods due to physical, chemical and biological characteristics undergo food spoilage/loss of nutrition. Proteins or peptides from food have been found to be physiologically active or bioactive either directly from the food or by in vitro or in vivo hydrolysis.¹⁶ Protein hydrolysates exhibit potent biological activities such as antihypertensive, antioxidant, antimicrobial, immunomodulatory and anticancer effects. From a nutritional point of view, as compared to other dietary sources, it has been shown that marine sources provide a favorable fatty acid composition of DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), which have been proven to exhibit health benefits.¹⁷

Aquatic organisms have numerous bioactive compounds, which can protect them from predators as well as provide health benefits to humans. Protein is among one of the major biological macromolecules, which is physiologically involved in metabolism and also present in diet.¹⁸ Protein hydrolysis is carried out in the intestine of mammalian digestive system in the presence of numerous proteolytic enzymes. Digested protein is absorbed by the body and it elucidates functionality. Marine protein hydrolysates are prepared by enzymatic, simulated gastrointestinal digestion, solvent extraction and fermentation processes. Therefore, it can be suggested that marine-derived hydrolysates or bioactive peptides are alternative sources of synthetic ingredients.¹⁹ Significant research efforts have been focused on marine bioactive peptides and their potential biological activities. In the relationship between food and health, bioactive peptides have been shown to develop functional foods, which are defined as food with specific health benefits.²⁰ Recently, researchers have focused on improving the bioavailability and bioaccessibility of these marine protein hydrolysates for validating functional ingredients for healthy foods. The objective of this review is to provide an overview of the chemistry of marine protein hydrolysates, their production, purification, characterization and perspectives in food chemistry.

Methods used for the preparation of marine protein hydrolysates

The word peptide comes from the Greek word “πεπτíδια”, which is translated as “small digestible”. Proteins are known as the source of various physico-chemical processes and sensory properties in foods and they can also act as functional as well as health promotional ingredients.⁴ The preparation of protein hydrolysates from different marine sources and the adopted methods are shown in Table 1. Marine bioactive peptides have been prepared by enzymatic hydrolysis, solvent extraction and microbial fermentation from the proteins present.¹⁴ Protein hydrolysis, the cleavage of peptide bonds, can be carried out enzymatically or by chemical processes. The chemical processes that include alkaline or acid hydrolysis are often difficult to control and tend to give modified amino acids.¹⁰ In recent years, extraordinary research evidence has shown that food-derived bioactive peptides and proteins have beneficial effects on human health. These food proteins are easily digested, and they release soluble peptides, which have greater resistant to gastric acid, heat, and proteolytic enzymes. These peptides consist of 3–20 amino acids from the digested protein, although some have been reported to contain >20 amino acids.²¹ The essential proteins found in the muscles of vertebrates and invertebrates are myosin, actin, and collagen. Myosin is present in thick filamentous muscle and its action in thin filamentous muscle is responsible for contraction along with the regulatory proteins troponin and tropomyosin.⁷ Marine protein hydrolysates have a broad range of ionic strength, good solubility and they can tolerate steady heat without precipitating.⁶ Proteins in our foods can act as health promoters in two ways, first by acting as indigestible substances in the digestive tract; thus, they can trap and expel toxins. Second, they can reduce the re-absorption of cholesterol in the large intestine.²²

Table 1 The techniques used to recover biologically active peptides from marine protein hydrolysates^a

Protein source	Techniques used		References
a MWCO – molecular weight cut-off; RP-HPLC – reverse phase-high performance liquid chromatography; FPLC – fast protein liquid chromatography; MALDI-TOF – matrix assisted laser desorption and ionization-time of flight; ESI-MS – electrospray ionization-mass spectroscopy; Q-TOF MS – quadrupole-time of flight-mass spectroscopy and DH – degree of hydrolysis.
Marine fish by-products	Separation	MWCO – fractionation 1, 5 and 10 kDa.	Ahn^68
		Ion exchange chromatography, RP-HPLC and Q-TOF MS	Lee^99
		Size exclusion, MALDI-TOF, auto amino-acid analyzer	Ahn^9
	Identification	Ultrafiltration, RP-HPLC and analytical HPLC	Girgih^7
		Gel filtration, RP-HPLC and Q-TOF with ESI	Lee^67
		Gel filtration, RP-HPLC and ESI-MS(MS/MS)	Bougatef^100
		FPLC, RP-HPLC and Q-TOF MS	Himaya^30
Marine fish	Separation	Pico-Taq HPLC, ultrafiltration MWCO – 1, 3 and 10 kDA and HPLC	Samaranayaka^101
		SDS-PAGE and HPLC	Salampessy^79
		Precipitation, sequential ultrafiltration and FAST-AAA MS	Taheri^66
		Ultrafiltration and nanofiltration	Vandanjon^33
		FPLC and DH	Slizyte^6
	Identification	Automatic amino-acid analyzer, gel-permeation, RP-HPLC and Q-TOF MS	Gu^65
		DH, gel filtration, HPLC and Q-TOF MS	Hsu^102
Shrimp and shrimp by-products	Separation	DH, amino acid analyzer	Sila^103
	Identification	Ion exchange, gel filtration, RP-HPLC and ESI-MS (MS/MS)	Huang^104
Mollusks – oyster, mussel	Identification	MWCO – 1, 3 and 10 kDa, size exclusion, amino-acid analyzer, RP-HPLC, off gel fractionation and MS/MS	Aleman^105
		Size exclusion, RP-HPLC, nano ESI-MS/MS	Wang^73
		MWCO – 1, 3 and 10 kDa, SE-HPLC, SDS-PAGE, ESI-Q-TOF MS/MS and SPR	Jung^70
		MWCO – 1, 3 and 10 kDa, gel filtration, RP-HPLC and ESI-Q-TOF MS/MS	Wang^34
		MWCO 3 kDA, RP-HPLC	Wang^72
Echinoderms – sea cucumber	Separation	MWCO – <3 kDa and >3 kDa	Vega^74
Cartilaginous skeleton – fish	Separation	DH, gel filtration, GC-MS	Bougatef^93
Seaweed	Separation	SDS-PAGE, GPC-HPLC	Harnedy^69

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Numerous methods have been utilized to release bioactive peptides from meat and marine food protein. However, the enzymatic hydrolysis of whole protein is used in most of the techniques. Several researchers have succeeded to produce bioactive peptides from milk protein followed by lactobacilli fermentation.^23,24 However, lactobacilli fermentation is less successful in meat and marine food protein due to its lower proteolytic activity. To the best of our knowledge, no microbial fermentation process has been carried out to produce protein hydrolysates in muscle proteins.²⁵ Enzymatic hydrolysis is one of the best methods to prepare marine protein hydrolysates, and can facilitate the production of short sequence peptides that can be obtained by the in vitro hydrolysis of protein substrates using valid proteolytic enzymes. Proteolytic enzymes sources can be microbes, plants, and animals, which can be used to develop bioactive peptides.²⁶ Usually, enzymatic reactions do not involve side reactions and do not reduce the nutritional value of the protein source. Native proteins are well-packed structures with secondary and tertiary structures due to their amino acid linking sequence. These interactions are based on the catalytic cleft of protein sites.¹⁰ However, enzymatic hydrolysis method is preferred in food and pharmaceutical industries because other methods lead to the release of organic solvents and toxic substances in the hydrolysates. Hydrolysis reactions should be carefully controlled to maintain and deliver an equal quality of the end products. The physico-chemical conditions of the reaction media should be optimized for the activity of the enzymes. The choice of proteolytic enzyme for the hydrolysis plays a vital role because it provides the cleavage patterns of the peptide bonds.²⁷

The degree of hydrolysis (DH), defined as a percentage of cleaved peptide bonds, is used to describe the hydrolysis of food proteins and it serves as a monitoring parameter for the reaction.²⁸ Quantification of the degree of hydrolysis is achieved using different methods, including spectrophotometric and micro-Kjeldahl apparatus for determining the percentage of cleaved peptides. Enzymatic reactions are measured using DH, which can be used by researchers to understand the further process of bioactive peptides. Many side chains and reactive functional groups of amino acids can react with reagents by intra and intermolecular cross-linking or covalent reactions.²⁹ In recent studies, the simulation of the gastrointestinal digestion of protein in vitro has been used to hydrolyse complex proteins into bioactive peptides. The simulated human gastrointestinal digestion was carried out using pepsin (gastric digestion) at pH 2 (acidic condition), followed by the addition of trypsin and α-chymotrypsin (duodenal absorption) with pH 6.5–7 for the neutralization of the peptides.³⁰ Newer technologies have been developed to improve the process of enzymatic hydrolysis such as the immobilization of enzymes. Immobilized enzymes allow the reaction conditions to be controlled more easily, preventing the generation of secondary metabolites from the autolysis of the enzymes, and also allow the recovery and re-use of the used enzymes.³¹

Purification and characterization of bioactive peptides

The isolation and purification of bioactive peptides are crucial for determining their in vitro and in vivo bioactivity. The traditional methods to purify a mixture of peptides from the hydrolysates include different types of chromatography and membrane based separation techniques.⁴ The purification of peptides is mainly based on their ionic charges, size, and hydrophobicity. Electrophoresis can separate the migration of charged particles according to their size and molecular weight. SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is used as a preliminary analysis method for protein molecules to confirm the mode of the protein molecule. Membrane ultrafiltration and size exclusion chromatography would be the best methods to concentrate peptides into different molecular weight ranges, and they can be used to obtain fractions that may contain low-molecular-weight peptides.¹⁵ Membrane processes based on the type of cut-off membrane and filtration methods are used to produce bioactive peptides. A novel membrane technology known as electrodialysis–ultrafiltration (EDUF) has been proved useful to separate cationic, anionic, and neutral peptides with defined molecular sizes.³² Refined peptides of biological interest of white fish hydrolysates were obtained by ultrafiltration and nanofiltration. A combination of these filtration methods improved the purification and diafiltration mode of the most active fractions from the hydrolysates.³³ Bioactive low-molecular weight peptides can be obtained using two different cut-off membranes, namely, 3 kDa and 10 kDa, from blue mussel protein hydrolysates. The peptides were proven to show good radical scavenging activity and inhibited auto-oxidation.³⁴ Many researchers have found that ultrafiltration using membranes with a low-molecular cut-off can be used to obtain enriched ACE (angiotensin converting enzyme) inhibitor peptides.^35,36

HPLC is one of the standard methods for peptide separation and it is easy to use because packed and commercially available reverse-phase columns are used to reduce human error. HPLC is usually combined with quantitative/qualitative equipment such as mass spectrometry.⁴ Liquid chromatography followed by tandem mass spectroscopy is the standard method for the characterization of peptide sequences. Matrix assisted laser desorption/ionization and time of flight (MALDI-TOF) is the backbone analysis for generating the peptide profiles of protein hydrolysates or semi-purified fractions.²⁷ A combination of size exclusion, reverse phase-HPLC, and Q-TOF-MS of purified peptides from flounder fish has shown stronger antioxidant activity. In particular, amino-acid residues in the sequence of Pro, Ala, Val, and Cys were claimed to contribute to the antioxidant properties using to these methods.³⁷ The fractionation process often yields peptides depending on the amino acid residues. Furthermore, purification steps based on bio-assays are used to in functional and structural studies.³⁸

Nowadays, consumers demand health benefits from foods beyond basic nutrition. The high complexity and various ranges of biological peptides challenge the capability of analytical methodologies. In silico and in vitro approaches aim to discover bioactive peptides from a food matrix. Recent “omic” approaches consist of cell biology, immunology, biochemistry, synthetic chemistry and a combination of a library of mass spectrometry, to identify and formulate the bioactivity of the peptides in a food sample.³⁹ In the field of proteins and molecular biology, 2-DGE (2-dimensional gel electrophoresis) plays a leading role for measuring the mass of the peptides obtained from the enzymatic hydrolysis of proteins and the identification of the proteins separated by 2-DGE after tryptic gel digestion. Due to the higher resolution and separating power of 2D gels, the identification of protein patterns can be carried out using simple and easy MS instrumentation.⁴⁰ The availability of genome sequences and high throughput technology allows foods to be analyzed at various levels. Recently, the power of proteomic technology has been combined with another technology called nanotechnology. Food proteomics is an emerging field that can act in the multidisciplinary areas of authentication, safety and response of individual diet molecules from a nutritional aspect.⁴¹

Recently, high-performance liquid chromatography (HPLC)-chromatin immunoprecipitation (ChiP)-tandem mass spectrometry (MS/MS) was applied to characterize storage proteins.⁴² Biomarker discovery is another area in food proteomics for major chronic diseases caused by protein inventions. For accuracy and to address the questions of bioavailability and bioefficacy, the proteins must be quantified and qualified both systemically (i.e., blood) and locally (in the gut) in the food matrix. The development of nanoproteomics can offer significant advantages over proteomics, which include high sensitivity, selectivity, and high dynamic range of protein analysis for low volume samples. Novel polypeptides can bind specifically to selected inorganic nanomaterials, which can be genetically engineered using phage-display technologies providing new field molecular biomimetics. Replacing the organic matrix used for the analysis of traditional MALDI-TOF-MS, functionalized nanoparticle probes are employed matrix free direct laser desorption ionization (DLDI-MS).⁴³

Conventional proteomic techniques, such as immunoassays and protein microarrays, are reliant for biomarker analysis. 2-DGE, mass spectrometry (peptide mass fingerprinting) and coupled liquid chromatography can be used for label free proteome and biomarker analysis.⁴¹ Quantitative structure–activity relationship (QSAR) method describes the relationship between bioactivity and structure. QSAR modeling principle allows the activity or function of the particular chemical to be studied by its molecular physico-chemical descriptors, electronic attributes, hydrophobicity and steric properties. The discovery of bioactive peptides from food proteins has greatly advanced by understanding the structure and activity relationships of peptides. The freely available bioinformatics tool peptide cutter (http://www.expasy.ch/tools/peptidecutter/) can carry out the in silico digestion of proteins. The server can use enzymes, such as, trypsin, thermolysin, pepsin, and chymotrypsin either individually or as various combinations, to retrieve the bioactive peptides.⁴⁴

Foodomics has been defined as a new discipline that studies the food and nutrition domains through the application of advanced omics technologies to improve the well-being, health, and confidence of the consumers.^45,46 Foodomics covers the development of new functional foods, health supplements and understanding of molecules through molecular tools. Approaches, such as genomics, transcriptomics, proteomics and metabolomics, have been used significantly to study foods/ingredients for the profiling of molecules and biomarker investigation related to the food quality and bioactivity of molecules.⁴⁷ Human health effects are followed by nutrigenomics and nutrigenetics approaches. Proteomes are different in each individuals, which is based on type of the cells, cells activity and its state. Proteomics is a challenging task because of the extensive concentration of most of the least abundant proteins. Sample preparation includes reducing the proteome complexity via fractionation and depletion, leading to the formation of low-abundant proteins. Proteomic studies include “bottom-up”, “shot-gun” and “top-down” approaches. MS is the last step in the analytical technique of proteomics, which helps to identify the peptides.⁴⁸ Improved mass spectrometers with better sensitivity and high accuracy in mass and resolution are used to identify and quantify the complex protein mixtures in a single experiment. The major mass analyzers utilized for the proteomic studies are, TOF (time-of-flight), Q (quadrupole), FT-ICR (Fourier transform ion cyclotron resonance) and IT (ion-trap). Some of the mass analyzers are combined in one mass spectrometer, e.g. QqQ (triple quadrupole), Q-IT, Q-TOF, TOF-TOF, and IT-FTIMS.

Metabolome is a mixture of endogenous or exogenous low molecular weight entities approximately <1000 Da, which is present in a biological system. Metabolites are downstream products of the operated biological system. Metabolic pattern analysis is critical and considerably interesting to understand from the nutritional point of view because of the variations in the metabolic pathways that arise due to diet.⁴⁹ Metabolome are diverse in the nature is respect to physical and chemical properties (sugars, amino acids, amines, peptides, organic acids, nucleic acid or steroids). Sample preparation entirely depends on the compounds to be analyzed. Two analytical platforms are used in metabolomics, MS, and NMR-based systems. These techniques are either applicable alone or combined with other techniques such as LC-NMR, GC-MS, LC-MS and CE-MS. On the other hand, MS/MS or MSⁿ experiments can be analyzed for ions at high resolution using Q-TOF, TOF-TOF or LTQ-Orbitrap, which can provide additional structural information and aid in the identification of the metabolites.^50,51

The biological potential of bioactive peptides from marine protein hydrolysates

Numerous bioactive peptides have been derived from dietary proteins using enzymatic hydrolysis. Specific peptides have individual or multifunctional activities suitable for functional foods or pharmaceutical products.⁵² The particular bioactivity of a marine peptide for various molecular disease targets is based on the structural conformation arising from the physico-chemical characteristics of the amino acid residues, chain length, molecular charge and bulkiness of the chain.^15,53 Numerous bioactivities are exhibited by bioactive peptides or protein hydrolysates derived from enzymatic hydrolysis, which are shown in Table 2. Aquatic species and their by-products have been extensively investigated in food science and nutrition, including antioxidant, immunomodulatory, anticancer, antimicrobial and anti-inflammatory peptides.⁵⁴ In Asian countries, such as Japan, China, and the Philippines, marine organisms have been part of their diet and also used in traditional medicine for curing major chronic diseases.⁵⁵

Table 2 Marine protein hydrolysates and their biological potential as functional ingredients^a

Marine sources	Mode of hydrolysates	Bioactivities	Reference
a ACE – angiotensin-converting-enzyme.
Tuna frame protein	Cocktail enzymes	Antihypertensive effect	Lee^99
Oyster–mollusc	Pepsin	ACE inhibitory	Wang^83
Oyster–mollusc	Subtilisin	Antioxidant peptide	Wang^72
Blue mussel–mollusc	Cocktail enzymes	Antioxidant peptide	Wang^34
Common smooth-hound–shark	Crude enzyme	Antioxidants	Bougatef^100
Pacific hake-fish	Gastrointestinal digestion	Antioxidants and ACE inhibitory effects	Samaranayaka^101
Pacific oyster–mollusc	Crude enzyme	Antitumor and immunostimulants	Chen^106
Shrimp waste	Alcalase	Antioxidants	Dey^107
White fish	Crude enzyme and ultra filtration	—	Vandanjon^33
Pacific whiting fish	Dried hydrolysate powder	Intestinal protective effect	Marchbank^108
Atlantic salmon skin	Alcalase and papain	ACE inhibitor peptide	Gu^65
Cod backbone waste	Protamax	Antioxidant and radio immune assay	Slizyte^6
Alaska pollock frame	Trypsin	Immunomodulating peptides	Hou^75
Fish waste from different fish muscle	Pepsin, pancreatin and thermolysin from B. thermoproteolyticus	ACE inhibitory and radical scavenging effects	Nakajima^109
Leather jacket-fish	Papain, bromeliin and flavourzyme	Antimicrobial effects	Salampessy^79
Squid gelatin	Cocktail enzymes (protamax, trypsin, neutrase, alcalase)	Antihypertensive, anticancer and antioxidant effects	Aleman^110
Squid skin gelatin	Pepsin	ACE inhibitor and antihypertensive	Lin^35
Squid skin collagen	Esperase, pepsin and pancreatin	ACE inhibitor	Aleman^105
Salmon by product	Cocktail proteases	Antioxidants and anti-inflammatory	Ahn^9
Chum salmon	Complex protease	Immuno modulatroy effect	Yang^111
Chum salmon skin	Complex protease	Neuroprotective effect	Yang^77
Sardinella by-products	Crude protease	Antioxidant effect	Bougatef^93
Seaweed – P. palmata	Alcalase and flavourzyme	Cardioprotective, anti-diabetic & antioxidants	Harnedy^69
Seaweed – P. columbina	Pepsin and pancreatin enzymes	ACE inhibitors and antioxidants	Cian^97
Salmon by-products	Cocktail enzymes	Antioxidant-octa peptide	Ahn^112
Salmon flesh	Pepsin, trypsin and chymotrypsin	Antioxidants	Girigh^7
Surimi by-products	Protamax and alcalase	Functional properties	Liu^113
Shrimp by-products	Alcalase	Caroteno proteins-antioxidant	Sila^103
Pacific cod skin gelatin	Gastrointestinal enzyme	ACE inhibitor and cellular oxidative stress	Himaya^30
Sea cucumber	Gastrointestinal enzyme	Multifunctional peptides	Vega^74
Sphyrna lewini muscle – shark	Ethanol soluble proteins	Antioxidant peptide	Wang^114
Blue mussel	CCl4 treatment and ultrafiltration	Anticoagulant peptide	Jung^70

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Bioactive peptides and depsipeptides with potential anticancer properties have been isolated from marine animals such as tunicates, sponges, soft corals, sea hares, nudibranchs, bryozoans, and sea slugs.⁵⁶ More than 10 000 species of sponges have been diversified in nature and most of them are of marine origin. Three genera (namely, Haliclona, Petrosia, and Discodermia) display anticancer and anti-inflammatory activities. In sponges, most of the studies have been conducted on cyclodepsipeptides, which are secondary metabolites with unusual amino acids and non-amino acid moieties.⁵⁷ Jaspamide is a cyclic depsipeptide identified from genera Jaspis and Hemiastrella. Its molecular structure contains 15-carbon macrocyclic rings, containing three amino acids (Fig. 4A). Homophymine A, cyclic 4-amino-6-carbamoyl-2,3-dihydroxyhexaenoic acid (Fig. 4D), possesses potent anticancer activity. Geodiamolide H (Fig. 4B), isolated from a Brazilian sponge Geodia corticostylifera, has been proven to have anti-proliferative activity against breast cancer cells by affecting the cytoskeleton of the tumor cells. Phakellistatins (Fig. 4C) were identified from a western Indian ocean sponge Phalkellia carteri and investigated for their use towards the treatment of leukemia. Their cyclic depsipeptides inhibited the growth of leukemia cells. The isolated cyclodepsipeptides bioactivities were reported in vitro. Didemnin (Fig. 4E) was obtained from the Caribbean tunicate Trididemnum solidum, and the bioactive peptide showed potential anti-tumor activity and antiproliferative activity against human prostate cancer cell lines. Another bioactive peptide ziconotide (Fig. 4F) was isolated from the mollusc Conus magus, which is a 25 amino acid peptide with three sulphur bonds with proven analgesic activity. A 60 kDa protein obtained from the purple ink of the hare Bursatella leachii, named as Bursatellanin-P, shows anti-HIV activity. Cyclic depsipeptides and bioactive peptides isolated from marine animals need to be investigated for their further detailed mechanism and human intervention studies.^58–62

Fig. 2 A schematic diagram of the recent applications of marine protein hydrolysates in food science and nutrition.

Fig. 3 A schematic representation of the present and future perspectives of marine protein hydrolysates in food science and nutrition.

Fig. 4 The chemical structures of bioactive marine peptides and depsipeptides from marine animal sources; sponges, tunicates, mollusks: (A) jaspamide; (B) geodiamolide H; (C) phakellistatin; (D) homophymine A; (E) didemin; and (F) ziconotide.

Innovations in nutraceuticals are growing enormously because of the awareness of modern consumers about their health. Hydrolyzing protein from marine sources is not only an innovation, but it is also claiming necessary nutritional availability, intervention against human diseases, and promoting food industries to produce functional foods. Cardiovascular disease is a major health disorder responsible for 30% of the world's population deaths,⁶³ and it is estimated that in 2020, heart disease and stroke will be a major cause of death. Oxidative stress is a common factor for the chronic diseases. At present, there is increasing interest in the utilization of food derived biologically active peptides as nutritional supplements or nutraceuticals.^5,30 The peptides generated via in vitro gastrointestinal hydrolysis from seafood waste and Pacific cod skin show effective ACE inhibition and antioxidant properties. The properties of these peptides are directly related to their structural amino acid composition and highly hydrophobic amino acids. Protein rich salmon muscle was analyzed in a computer-aided and experimental approach to identify ACE-inhibitory peptides. The peptides derived from salmon are often consumed in the human diet.⁶⁴ Hypertension is another worldwide problem and affects 15–20% of all adults. Salmon skin collagen peptide powder consists of purified low-molecular-weight peptides and have been shown to have in vitro ACE-inhibitory bioactivity.⁶⁵ The squid gelatin hydrolysates of fractionated HSSG-III were investigated for their antihypertensive effects on oral renal hypertensive rats (RHR) after long-term oral administration. The HSSG-III of squid gelatin hydrolysates possess in vitro ACE-inhibitory activity IC50 value of 0.33 mg ml⁻¹. Oral administration in rats decreased systolic blood pressure and diastolic blood pressure of RHR. The effect of blood pressure reduction was demonstrated in vivo.³⁶ The different peptide fractions of salted herring brine protein hydrolysates obtained by ultrafiltration exhibited antioxidant properties and functional properties. The isolation of peptides from the hydrolysates using ultrafiltration removed the salt content of the fractions. Fractions between 50 kDa and 10 kDa showed good in vitro antioxidant activity. Moreover, the functional properties exhibited by the isolated fractions were lower than sodium caseinate and BSA (bovine serum albumin).⁶⁶ The pectoral fin of salmon by-products, which are rich in proteins, were enzymatically hydrolysed, and their hydrolysates were investigated for antioxidant and anti-inflammatory effects to verify the possibility of their future applications. The isolated highly active SPHF1 (salmon protein hydrolysates fraction 1; 1000–2000 Da) showed a great potential to inhibit the generation of intracellular ROS (reactive oxygen species). It also inhibited lipid peroxidation and increased the level of GSH (glutathione) in Chang liver cells. SPHF1 also exhibits anti-inflammatory effects by inhibiting nitric oxide and proinflammatory cytokine production, and it includes TNF-α, IL-6 and IL-1β in in vitro LPS induced RAW264.7 macrophage cells.⁸

Simulated gastrointestinal digested salmon protein hydrolysates obtained by RP-HPLC fractions were investigated for in vitro antioxidant properties. The peptides reduce and chelate the metal cations that are used for the production of harmful free radicals such as the iron-catalyzed conversion of hydrogen peroxide to hydroxyl radicals.⁷ Skate is popular seafood in South Korea due to its unique taste and flavor. The by-products of skate skin protein hydrolysates were investigated for the first time for ACE-inhibitory activity.⁶⁷ Tuna liver by-products are procured when processing of tuna canned products. The tuna liver protein hydrolysates are prepared using commercially available enzymes and fractionated with different pore sizes of ultrafiltration membrane. The hydrolysates show dual in vitro bioactivity: AchE (acetylcholinesterase) inhibition and antioxidant activity. The fractions above 10 kDa exhibit high AchE inhibitory activity as compared to the low-molecular weight fractions.⁶⁸ Macroalgae are one of the popular seafoods in many oriental countries. Biofunctional ingredients with cardioprotective, antidiabetic and antioxidant properties have been investigated from red algae (Palmaria palmata). Aqueous protein hydrolysates generated by alcalase and corolase PP in in vitro studies showed higher inhibitory effects for Type-2 diabetes, ACE inhibition and antioxidant properties.⁶⁹ An anticoagulant peptide (MEAP) was isolated and investigated from the soluble extracts of the edible parts of a mussel (Mytilus edulis). MEAP prolongs the normal clotting time to 321 ± 2.1 s for APTT (activated partial thromboplastin time) and 81.3 ± 0.8 s for TT (thrombin time) in a dose-dependent manner. MEAP can prolong the time of clotting by inhibiting the activation of FX in the intrinsic tenase complex and the conversion of FII (prothrombin) to FIIa (thrombin) in the prothrombinase complex.⁷⁰ Calcium deficiency is widespread due to the insufficient intake and diminished solubility of calcium in the constituents of food and anti-nutritional factors. Nile tilapia (Oreochromis niloticus) is distributed worldwide and the dumping of processed tilapia scale by-products is also increasing. The calcium binding peptide (DGDDGEAGKIG; Mw 1033.0 Da) has been purified from tilapia scale protein hydrolysates. Asp and Glu residues in the peptide contribute to the substantial calcium binding capacity of the peptide. The physical and biochemical properties of femurs in Ca-deficient rats were significantly improved by the improved calcium bioavailability.⁷¹ Oyster is a good source of quality nutrition in North East China and other parts of the World. The TCA soluble fractions were hydrolyzed from an oyster (Crassotrea talienwhanensis) by using subtilisin enzyme. An attempt was also made to isolate two antioxidant peptides using nano-ESI/MS/MS. The hydrolysates passed through a 3 kDa membrane and exhibited hydroxyl and radical scavenging activities. Two purified peptides, namely, PVMGD (Mw 518 Da) and QGHV (Mw 440 Da), did not have a significant homology as compared to the other antioxidative peptides.⁷² In another study, oyster (Crassostrea gigas) hydrolysates were derived from a protease (Bacillus sp. SM98011), and their production was conducted on pilot to plant scales. The antitumor and immunomodulating effects of the hydrolysates on S-180 bearing BALB/c mice were investigated. The weight coefficient of thymus and spleen, NK cell activity, and spleen lymphocyte proliferation of the phagocytic rate of macrophage cells in S-180 bearing BALB/c mice proved to be significantly different upon the oral administration of the hydrolysates.⁷³ Sea cucumber is another benthic marine organism distributed in the majority of the ocean and highest diversity of shallow tropical waters. It is also used as food in Asian countries such as the Philippines, Malaysia, Japan, Korea, and China. Extensive research on sea cucumber extracts for their potential multiple biological activities has been carried out. Simulated gastrointestinal digested peptides from a sea cucumber (Isostichopus badionotus) were analyzed for antioxidant, antiproliferative and ACE inhibitory activities. Fractions >3 kDa and <3 kDa showed ACE inhibitory and cytotoxic effects against colorectal cancer cells. Released multifunctional peptides are capable of resisting gastrointestinal enzymes and found to have higher concentrations of amino acids (Gly, Arg, and Ala). They play a significant role in physiological effects and reduce serum cholesterol levels.⁷⁴ Pollock is a commercial fish and has enough meat and backbone after processing to obtain by-products utilized in animal feed. Immune functions play a significant role in modulating the immune system and counter-attack chronic diseases. Purified and identified peptides from Pollock frame protein hydrolysates were used for splenocyte lymphocyte proliferation and amino acid sequencing. Three peptides with high lymphocyte proliferation activities were separated, and their amino acid sequences were NGMTY, NGLAP and WT. The proliferation rates were above 30% using 20 μg ml⁻¹ of the peptides.⁷⁵ The hydrolysates obtained from shrimp waste were used for their functional properties and product applications. Using enzymes, approximately 40–50% of hydrolysates could be isolated from certain species of shrimps, possibly by the binding of protein or carbohydrate complex in the shrimp shells. Fractions <10 kDa and 10–30 kDa significantly inhibited the growth of both colon cancer and liver cancer cells by 60% after 72 h.⁷⁶ Marine oligopeptide prepared from chum salmon (Oncorhynchus keta) by enzymatic hydrolysis showed an enhancement of innate and adaptive immunities through the production of cytokines in mice. Gamma radiation-induced immunosuppressed female mice fed with marine oligopeptide exhibited the augmentation of the relative numbers of radioresistant CD4+ T-cells. They also showed an enhancement of IL-12 levels in splenocytes, a reduction in the level of NF-^κB through the induction of I^κB in the spleen and an apoptosis inhibition of splenocytes. Therefore, marine oligopeptide can be used as a supplementary therapy, and its shows a protective effect in cancer.⁷⁷ Baked products are widely consumed foods in the world and a suitable vehicle for delivering bioactive ingredients.⁷⁸ The identification of antimicrobial peptides from marine origins is lower than that found from terrestrial origins. The enzymatic hydrolysis of fish muscle leather jacket (Meuschenia sp.) purified fractions 9 and 12 were carried out for an antimicrobial MIC (minimum inhibition concentration) assay. Fraction 12 exhibited MIC against Bacillus cereus and Staphylococcus aureus pathogenic bacteria.⁷⁹ The protein hydrolysates of red seaweed (Palmaria palmata) were used for next level studies to discover functional foods or health supplements. The renin inhibition assay showed that the bioactive properties of the hydrolysates were retained during the baking process. Furthermore, the developed seaweed hydrolysates bread did not affect the sensory quality of the product.⁸⁰

Commercial marine-derived protein hydrolysates and peptides have been approved as functional ingredients in Japan. They are labeled as FOSHU (several foods for specific health use) products. Lapis Support™ (Tokiwa Yakuhin Co. Ltd.) and Valtyron® (Senmi Ekisu Co. Ltd.) are examples of two such products sold in Japan.⁷⁶ Lapis Support™ is available in beverage format and Valtyron® is incorporated in 33 other products including soft drinks, jelly and dietary supplements. The production of Valtyron® is via the hydrolysis of sardine muscle with commercially available food grade alkaline protease from Bacillus licheniformis. Another, FOSHU approved functional product is ‘Peptide soup’ made from katsuobushi (bonito) hydrolysate generated using thermolysin.^81,82 The active peptide (LKPNM) in the product shows a significant reduction in systolic blood pressure in mildly hypertensive subjects. In addition to beverages (soup and tea), bonito peptide has been sold as a powdered ingredient and also in tablet form called ‘Peptide ACE 3000’ in Japan (Nippon Supplement Inc.). Apart from this, other marine-derived protein hydrolysates without approved health claims are sold as food supplements in Europe and North America. The products are Stabilium® 200, Protizen®, AntiStress 24, Nutripeptin™ and Seacure®. Nutripeptin™ (Nutrimarine Life Science AS, Norway) is a product of cod protein hydrolysate exhibiting a postprandial blood glucose lowering activity. Seacure® (Proper Nutrition, US) is a product of Pacific whiting hydrolysate marketed as a supplement for gastrointestinal health improvement. Furthermore, Fortidium liqumen® (Biothalassol, France) is a product from white fish (Molva molva) autolysate, which is commercially available and has multifunctional effects; it is an antioxidant, anti-stress and glycemic index reducing agent. Based on these evidences of potential health benefits of marine protein hydrolysates or peptides, it can be inferred that they have a promising role in functional ingredients or nutraceuticals. Marine protein derived hydrolysates products were commercially listed, which can be an alternative to health supplements. Although a number of studies exist for proving the biological effects using in vitro or animal models, it is now important to use human intervention trials to study the biological effects and their mechanisms in more detail. Ultimately, regulatory approvals from various standard agencies, such as FDA, EFSA and FOSHU, are required to commercialize marine protein-derived products.^81–83

Recent approaches to bioactive peptides and functional delivery systems

Microencapsulation is the entrapment of tiny molecules, liquid droplets and gases in a suitable coating. Microencapsulation can allow the protection of a broad range of materials of biological interest that can be applied to biomedicine and biopharmaceuticals. Recently, this technology has been utilized in the applications of food industry for providing high-value products or nutraceuticals. Bioactive peptides, as added products, can undergo processing, storage, and transport. To protect the bioactivity, an encapsulated form is a suitable delivery system. The applications of marine protein hydrolysates and their bioactive peptides and recent approaches are shown as a schematic representation in Fig. 2. The encapsulation of proteins depends on the type of proteins and envisioned health effects served by the vehicle of the bioactive peptide.^84,85 Nanotechnology is another field that can utilize, create and manipulate the materials in devices or systems on a nanometer scale. The entrapment of bioactive peptides using nanotechnology is a promising method for supplying active functional ingredients in the food industry.⁸⁶ Nanoemulsions, functional hydrogels, and nanoparticles deliver bioactives to target organs. To retain the bioactivities and improve the stability of bioactives in digestive system as well as their bioavailability, the abovementioned technologies will be suitable and helpful for the development of functional foods, nutraceuticals or health supplements.^87,88

Nutritionally enriched marine based processed food products

Marine animal foods are rich in protein content on an edible fresh weight basis than most terrestrial meats. Marine animals such as fishes, crustaceans and mollusks are the widely consumed seafoods amongst others. The food proteins of marine animals are highly digestible and have a biological value of releasing essential amino acids (EAA), which is recommended for human diet. Because plants and other terrestrial proteins consumed by humans lack EAA, aquatic food products should be added to a plant-based diet consumed by humans.⁸⁹ Humans are counter-attacked by free radicals from both, inside the body and their surrounding environment, exclusively by reactive oxygen species (ROS) during the metabolic process. In addition to oxidative stress that leads to the attack of macromolecules, DNA, proteins, carbohydrates and lipids to cause health disorders, the oxidation of foods is a major problem that causes the deterioration of food quality, leading to rancidity and reduction of the shelf life of the products. To retard these issues, many synthetic antioxidants have been made by pharmaceutical and food industries. However, these synthetic antioxidants must be used under strict regulations due to their potential health hazards. To overcome these issues, natural antioxidants from foods that are based on biological substances have been addressed recently. The present and future directions of marine protein hydrolysates in food science and nutrition are diagrammatically represented in Fig. 3. Due to their safety, nutritional and therapeutic properties, the level of interest in natural antioxidants has increased significantly. Marine organisms are believed to be a potential source of biologically active peptides for the development of pharmaceuticals, functional ingredients and human nutrition. The development of bioactive peptides from the seafood protein depends on two factors, the primary sequence of the protein substrate and the specificity of the enzymes used. Structure–activity relationships of thee generated peptides have not been fully established, but the few reported examples have been used to identify their influence on biological actions. For example, the binding action of angiotensin converting enzyme (ACE (EC. 3.4.15.1)) inhibitory peptides is a consequence of the presence of amino acids such as tyrosine, phenylalanine, tryptophan, proline, lysine, isoleucine, leucine, valine and arginine. The lipid reducing and antioxidant activity of peptides is also totally dependent on the configuration of the hydrophobic or hydrophilic residues of the amino acids.⁹⁰

In addition, marine food processing by-products, such as standard muscles, viscera, skins, trimmings, and shells, can be used efficiently to produce nutraceuticals and functional food ingredients with biofunctional activity.⁹¹ Marine species and processing by-products containing plenty of proteins may have undiscovered novel sequences encrypted within their primary structures with potential biofunctional activity. However, growing scientific evidence shows that many marine-derived species, including molluscs, crustaceans and processing by-products, protein hydrolysates and peptides can improve health in addition to the treatment of chronic diseases.⁸³ Recently, the addition of the protein hydrolysates of a seaweed (Palmaria palmata) to the bakery food (bread) has been validated to be beneficial for the human heart. These breads incorporated with hydrolysates are not affected by organoleptic characteristics and they also improved the overall product quality with beneficial effects.⁸⁰

Peptides, consisting of 2–6 amino acids, are compared to complex protein; proteins display less absorbance across the gastrointestinal tract. Their limitations may be based on intrinsic factors such as physico-chemical and biological properties. The reason is a poor permeation of biological membranes because of their molecular size, physical and chemical instability, degradation by intrinsic proteolytic enzymes and aggregation. Transcription factors and signaling molecules adsorption, as well as immunogenicity, are thought to play roles in the process. Therefore, foods with incorporated marine-derived bioactive proteins play a critical role to assess their in vivo biological potential.⁸³

Humans began to consume microalgae in their diets, either in the form of capsule, powder, tablet, and pastille in early 1950. Most of the marine-derived microalgae species consumed are Spirulina, Chlorella, Dunaliella, and Aphanizomenon. They have rich proteins and essential phytochemicals that can contribute more physiological effects to humans. Microalgae can easily be incorporated into food products such as pastas, biscuits, breads, candies, yogurts and soft drinks. It is reported that the consumption of Spirulina incorporated foods can stimulate gastrointestinal tract Lactobacillus sp. Moreover, microalgae also act as animal nutrition to stimulate physiological functions. The cost of animal feed price is double the amount of a human diet. Animal feed industries are looking for functional and low cost food supplements, which can give more potential to animal as well as the animal farm owners. Microalgae (Schizochytrium sp.) were incorporated into ruminants feed and has been proven to enrich the products of polyunsaturated fatty acids in milk fat, while saturated fat was reduced. Another study in rabbits showed that the incorporation of microalgae (Spirulina platensis) in their feed was proven to reduce serum cholesterol levels and increased high-density lipoprotein cholesterol levels. Poultry feed is another growing research area in the world. The addition of 10% microalgae (Chlorella sp.) powder showed an increase in linoleic acid and DHA in egg yolk and reduced docosatetraenoic acid levels. Aquaculture industries are also benefiting from these tiny microalgae because phytoplankton communities are the primary feed for macro level organisms. Powdered or pellet forms of microalgae can be used as feed or pigments for carp, salmon, and shrimp. Being simple aquatic, photosynthetic organisms they are promising sources for novel products and applications.^92,93

Meat oxidation in stored or processed products is a significant concern in the food industry. Meat oxidation leads to the production of off-flavor, reduced shelf life, dark colors, and potentially toxic products. Due to these problems, the food industry sector cannot deliver a fresh product to the consumers once processed the meat is processed or chopped. To handle this matter, the inhibition of meat oxidation can be carried out using antioxidant peptides. Recently, antioxidant peptides from Goby muscle protein hydrolysates (GPH) have been obtained by treatment with various fish crude alkaline protease, and their activity was determined against lipid peroxidation in turkey meat sausage during a 25 day storage period. Malondialdehyde (MDA) is a widely studied marker of oxidative stress and the lipid peroxidation index in food products. When MDA reacts with TBA (thiobarbituric acid) it gives TBA reactive substances, which are detectable by spectrophotometry at 532 nm. The decrease in TBARS was probably due to the interactions of the peptides, which inhibited the oxidation of the turkey meat sausage up to 12 days.⁹⁴

One of the most relevant and significant food processing technologies is extrusion cooking, which has been used since the 1930s for the production of breakfast cereals, ready to eat snacks and other textured products.⁹⁴ Edible seaweed is an ingredient aimed to develop food-based applications in extrusion products to make them attractive and also reach non-seaweed eaters. Two Indian seaweeds (Sargassum marginatum and Undaria pinnatifida) based semolina extruded pasta products were developed and their biofunctional and nutritional qualities were analyzed.^95,96 However, to the best of our knowledge, very few research articles exist reporting such incorporated food products. Recently, maize-based extruded products of seaweed (Porphyra columbina) were investigated and their retention of the properties of bioactive compounds was studied. Maize (control) and maize-seaweed extruded products were digested with gastrointestinal enzymes and in vitro studies on the potential ACE inhibitory properties of their bioactive peptides as well as on their antioxidant properties were performed.⁹⁷ Another recent study utilizing a marine mussel (Perna canaliculus) as an ingredient for product quality and biofunctional evaluation was reported in gluten-free pasta products.⁹⁸ Gluten is the backbone of the food industry; however, it can cause allergy to genetically susceptible consumers. A gluten-free diet is the only solution to handle this problem for the consumers. Marine based protein can act as a replacement of other protein sources and helps to develop a network of other molecules for developing gluten-free products. These situations may help food technologists to understand and carry out research on the utilization of marine sources for nutritional retention and enriched functional ingredients.

Conclusions and recommendations for future research

The production of marine protein hydrolysates and their potential biological activity studies have been reported and are evolving in the direction towards the development of functional foods, nutraceuticals and functional food ingredients. Currently, very few studies on the development of protein hydrolysates or bioactive peptides enriched food products or coated products have been reported. In the modern world, due to the time demands and lower availability of terrestrial food products, we have to look at other food sources, which have a huge biodiversity and less utilization in consumption. Health related disorders are another trend nowadays, and to combat and treat these disorders, we have to utilize these natural sources and deliver it to the population in need. Future research and studies should be in multidisciplinary areas to produce functionally enriched food products with improved bioavailability and stability and with the retention of their potential biological activity.

Abbreviations

PUFA	Poly unsaturated fatty acid
DHA	Docosahexaenoic acid
EPA	Eicosapentaenoic acid
SDS-PAGE	Sodium dodecyl sulphate-poly acrylamide gel electrophoresis
kDa	Kilo Daltons
EDUF	Electro dialysis ultrafiltration
ChiP	Chromatin immunoprecipitation
QSAR	Quantitative structure–activity relationship
MWCO	Molecular weight cut-off
RP-HPLC	Reverse phase-high performance liquid chromatography
FPLC	Fast protein liquid chromatography
MALDI-TOF	Matrix assisted laser desorption and ionization-time of flight
ESI-MS	Electrospray ionization-mass spectrometry
Q-TOF MS	Quadrupole time of flight mass spectrometry
NMR	Nuclear magnetic resonance
DH	Degree of hydrolysis
TNF-α	Tumor necrosis factor-alpha
IL-6	Interleukin-6
IL-1β	Interleukin-1beta
LPS	Lipo polysaccharide
ROS	Reactive oxygen species
NF-kB	Nuclear factor kappa-light chain enhancer of B cells
FOSHU	Several food for specific health use
FDA	Food and drug administration
EFSA	European food safety authority
ACE	Angiotensin converting enzyme

Acknowledgements

The author Vijaykrishnaraj M. gratefully acknowledges the funding agency, (Department of Science & Technology, Govt. of India) for providing a DST-INSPIRE-Fellowship to conduct this research and he is also thankful to the CSIR-CFTRI for allowing access to its facilities.

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