Peptides from Fish By-product Protein Hydrolysates and Its Functional Properties: an Overview

The inadequate management of fish processing waste or by-products is one of the major problems that fish industry has to face nowadays. The mismanagement of this raw material leads to economic loss and environmental problems. The demand for the use of these by-products has led to the development of several processes in order to recover biomolecules from fish by-products. An efficient way to add value to fish waste protein is protein hydrolysis. Protein hydrolysates improve the functional properties and allow the release of peptides of different sizes with several bioactivities such as antioxidant, antimicrobial, antihypertensive, anti-inflammatory, or antihyperglycemic among others. This paper reviews different methods for the production of protein hydrolysates as well as current research about several fish by-products protein hydrolysates bioactive properties, aiming the dual objective: adding value to these underutilized by-products and minimizing their negative impact on the environment.     

General review of fish protein hydrolysates

Fish protein hydrolysates are generally considered to be the liquefied products obtained from fish by the action of proteolytic enzymes under accelerated conditions of digestion. The proteolytic enzymes which are used for the digestion of fish proteins must be active either above the survival temperature of spoilage bacteria or outside the range of pH which would support their growth. By suitable selection of proteolytic enzymes and conditions for hydrolysis some control over the digestion can be achieved to give a range of products. This paper reviews the work done here and elsewhere on fish protein hydrolysates and discusses their potential for making more effective use of the fish resources.     

Hydrolysates from Atlantic cod (Gadus morhua L.) viscera as components of microbial growth media

Three hydrolysates made from cod viscera by different enzymatic hydrolysis procedures were tested as a combined source for nitrogen, amino acids and vitamins in microbial growth media. Using a panel of five different microbes: Escherichia coli, Bacillus subtilis, Lactobacillus sakei, Saccharomyces cerevisiae and Aspergillus niger, the performance of these viscera hydrolysates was compared to the performance of common commercial peptones in an automated growth analyzer (Bioscreen C). The results show that the fish hydrolysates in general are promising alternatives to currently available commercial nitrogen sources of other origins. In the case of the food-grade and nutritionally fastidious L. sakei, two of the fish hydrolysates were clearly superior to all tested commercial peptones. For several microbes, the choice of the proteolytic enzymes used to produce the fish hydrolysate had considerable impact on performance of the resulting hydrolysate, both in terms of maximum growth rate and biomass production. In terms of hydrolysate performance, the generally best enzyme for production of a fish peptone from cod viscera was found to be Alcalase.     

Preparation and testing of Sardinella protein hydrolysates as nitrogen source for extracellular lipase production by Rhizopus oryzae

Various fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were used as nitrogen sources for the production of extracellular lipase by the filamentous fungus Rhizopus oryzae. The best results were obtained with defatted meat–fish protein hydrolysates (DMFPH), indicating the presence in the lipid fraction of some constituents which may repress lipase synthesis. Furthermore, it was found that the extensive hydrolysis of fish proteins resulted in a higher lipase production. The use of 40 g DMFPH l^–1 for the growth of Rhizopus oryzae in medium R1 resulted in a lipase production of 394 U ml^–1, higher than the yield obtained with standard soy peptone as nitrogen source (373 U ml^–1). The most appropriate medium for the growth and the production of lipase is composed only of 24 g DMFPH l^–1 and 10 g glucose l^–1, indicating that the strain can obtain its nitrogen and salts requirements directly from fish substrate.     

Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis

To utilize fish processing waste, tuna backbone protein was hydrolyzed using different proteases (alcalase, α-chymotrypsin, neutrase, papain, pepsin and trypsin) for production of antioxidant peptide. Antioxidant activities of hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, peptic hydrolysate exhibited the highest antioxidant activity compared to other hydrolysates. To identify antioxidant peptide, peptic hydrolysate was purified using consecutive chromatographic methods, and the antioxidant peptide was identified to be VKAGFAWTANQQLS (1519 Da) by Q-TOF ESI mass spectroscopy. The antioxidant activities of antioxidant peptide from tuna backbone protein (APTBP) was evaluated, and the results show that APTBP significantly inhibited lipid peroxidation in linoleic acid emulsion system and also quenched free radicals (DPPH, hydroxyl and superoxide) in a dose-dependent manner. Moreover, APTBP did not show any cytotoxic effect against MRC-5 and ECV304 cell lines.     

抗鱼肉蛋白冷冻变性机理的研究进展

简述了鱼肉蛋白冷冻变性机理的研究现状,综述了糖类、盐类、乳蛋白、不同水解物等添加物的抗冷冻变性机理,介绍了鱼肉蛋白冷冻变性的评价指标及其测定方法,展望了抗鱼肉蛋白冷冻变性的新途径及应用前景。论文内容对于深入研究抗肉类蛋白冷冻变性具有较大的参考价值。     

Functional,bioactive and antioxidative properties of hydrolysates obtained from cod (Gadus morhua) backbones

Fish protein hydrolysates (FPH) have good and well documented functional properties. Peptides obtained from various fish protein hydrolysates have also shown bioactive and antioxidative activities.The aim of this study was to evaluate how storage and preparation of cod (Gadus morhua) backbones influence the yield, functionality, bioactivity (CGRP and gastrin/CCK related molecules) and antioxidative properties of fish protein hydrolysates. A series of hydrolysis trials have been carried out using backbones from cod that were initially fresh or frozen and further hydrolysed for different times (10, 25, 45 and 60 min). Use of fresh raw material significantly increased yield of dry FPH, gave lighter and less yellow powders with better emulsification properties. Longer time of hydrolysis gave higher FPH yield, increased degree of hydrolysis and decreased water holding capacity of the powders. Among the hydrolysis times tested, 25 and 45 min hydrolysis demonstrated the best emulsification properties.FPH have potential to enhance product stability by preventing oxidative deterioration. The DPPH scavenging activity showed that antioxidative activity of hydrolysates could be due to the ability to scavenge lipid radicals. The ability of hydrolysates to inhibit iron induced lipid oxidation was not influenced by time of hydrolysis.This work also shows that it is possible to obtain bioactive molecules from cod backbones by protein hydrolysis. The content of bioactive peptides (gastrin/CCK- and CGRP-like peptides) could make the cod hydrolysates useful for incorporation in functional foods.     

Biosynthesis of protease by Pseudomonas aeruginosa MN7 grown on fish substrate

Fish powders and fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were prepared and tested as growth media for alkaline protease production by Pseudomonas aeruginosa MN7. Cultivated in fish substrate as carbon source, the strain exhibited a slightly greater protease production (about 7800 U ml^–1) than that obtained with commercial peptones (about 7222 U ml^–1). Furthermore, P. aeruginosa MN7 produced the same amount of protease when cultivated in medium containing only fish substrate or that containing all ingredients, indicating that the strain can obtain its carbon and nitrogen requirements directly from whole fish proteins. Moreover, it was found that extensive hydrolysis of fish proteins did not increase protease formation. Protease production in media containing only FPH prepared by Alcalase was about 70% of those obtained with MN7 protease digest of fish protein or with meat-fish powder. These results indicate that sardinella substrates are an excellent carbon and nitrogen source for the growth of P. aeruginosa MN7 and the production of protease.     

微波水解制备鱼蛋白的研究

采用微波水解的方法制备鱼蛋白水解液,结果表明:微波可以明显增加蛋白质回收率,正交实验得到微波酸解的最适条件,即HCl浓度4 mol·L-1、微波功率450w、作用时间为30min,其水解液的蛋白质回收率可达到91.02％,相当于酶解的效果,且腥苦昧较小。     

Proteolysis in vitro as the Way of Modification of the Protein Trophic Component

The protein hydrolysates of two types, fodder and for microbiological medium, have been obtained in vitro, using protein containing wastes of Iceland scallop fishery (WSF) and enzyme preparation from the red king crab hepatopancreas. The degree of protein degradation and composition of obtained hydrolysates were analyzed. Intensive protein hydrolysis was necessary to obtain the microbiological diagnostic cultural media that fit trophic requirements for 12 microbial test-cultures. Addition of the fodder protein hydrolysate to diet of juvenile salmons Salmo salar during mixed feeding produces a positive effect on their survival; the 5 and 20% replacement of fish flour by the fodder hydrolysate results in a reduction of fish mortality by 21 and 57%, respectively. The living weight of chickens increased by 15% after substitution of the 10% fish flour by the fodder WSF hydrolysate in their diet. The results obtained can be explained by a more effective assimilation of partly hydrolyzed proteins in comparison with native ones.     

Functional,antioxidant and antibacterial properties of protein hydrolysates prepared from fish meat fermented by Bacillus subtilis A26

Composition, functional properties and in vitro antioxidant and antibacterial activities of protein hydrolysates prepared with a proteolytic bacterium, Bacillus subtilis A26, through fermentation of fish proteins were investigated. Fermented fish meat protein hydrolysates (FPHs) were prepared from sardinelle (SPH), zebra blenny (ZPH) goby (GPH) and ray (RPH). The protein content of freeze-dried FPHs ranged from 74.3% to 81%. All fermented hydrolysates had an excellent solubility and possessed interfacial properties. The antioxidant activities of FPHs were evaluated by different methods, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power assay, β-carotene bleaching and DNA nicking assay. All hydrolysates showed dose-dependent antioxidant activities. Further, FPHs exhibited antibacterial activity and SPH was the most effective, particularly against Gram positive bacteria.     

Hydrolysis of Soybean Proteins with Kamchatka Crab Hepatopancreas Enzyme Complex

To perform hydrolysis with the enzyme complex from the hepatopancreas of the Kamchatka crab, a protein mixture was isolated from soybean meal by extraction at alkaline pH values. Extractable low-molecular impurities were removed by ultrafiltration and precipitation of proteins with alcohol. The amino acid composition of the obtained protein extract turned out to be similar to the composition of the fish meal traditionally used in the production of fish feeds. Analysis of the products of fermentolysis by DDS-electrophoresis, HPLC, and mass spectrometry showed a high degree of hydrolysis of soybean proteins. Depending on the time of fermentolysis, the hydrolysates contained up to 60% (18 h of hydrolysis) of free amino acids (the fraction of the weight of the hydrolyzed protein mixture) and short peptides (2–20 amino acid residues).     

鱼蛋白粉的营养评价

对采用酶解法和酸水解法制备的白鲢鱼蛋白粉的营养成分进行了分析,从氨基酸组成上表明鱼蛋白粉具有较高的营养价值,在食品行业有较大应用前景。     

Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture

To investigate the effect of size-excluded fraction of non-animal protein hydrolysate on growth, viability and longevity of Chinese hamster ovary (CHO) cells, several commercially available protein hydrolysates were evaluated as a feed supplement to chemically-defined protein-free suspension culture. Soy protein hydrolysates showed better supporting capability for cell growth and viability than the other types of hydrolysates. Maximal cell growth was not affected greatly by size exclusion of some soy hydrolysates such as bacto soytone and soy hydrolysates. CHO cells supplemented with size-excluded fractions of the two hydrolysates showed viable cell density and viability almost equal to those with their crude hydrolysates, although soy hydrolysates showed a little better performance. This suggested that the size-excluded hydrolysate fractions of some soy hydrolysate might be a potential culture medium additive to achieve better downstream operation in a large-scale production as well as enhanced productivity.     

Preparation of fish protein hydrolysates

A process for the preparation of fish protein hydrolysates using commercially available proteolytic enzymes is described. Both non-fatty and fatty species of fish have been used as the raw material and the hydrolysates obtained have been assessed in nutritional studies on neonatal animals. Data on the release of nitrogenous substances during the digestion of cod with the enzyme papain are presented, together with amino acid analyses of the various fractions produced. As far as hydrolysates from non-fatty species are concerned there are no technical difficulties in preparing a spray-dried or concentrated product suitable for animal feeding. For hydrolysates from fatty species, however, it is necessary either to remove the oil mechanically or to stabilise it with suitable antioxidants. For the present exercise, hydrolysates were frozen and stored at ?30°C until required for the nutritional studies. Before any industrial development is considered, further studies on the drying and storing of hydrolysates from fatty species are necessary.     

Chemometric analysis of soy protein hydrolysates used in animal cell culture for IgG production – An untargeted metabolomics approach

Soy protein hydrolysates are used as the most cost effective medium supplement to enhance cell growth and recombinant protein productivity in cell cultures. Such hydrolysates contain diverse classes of compounds, such as peptides, carbohydrates and phenolic compounds. To identify if specific compounds dominate the functionality of hydrolysates in cell cultures, thirty samples of hydrolysates with different cell culture performances were analyzed for chemical composition using an untargeted metabolomics approach. Out of 410 detected compounds, 157 were annotated. Most of the remaining 253 compounds were identified as peptides, but could not be annotated exactly. All compounds were quantified relatively, based on their average signal intensities. The cell growth and total immunoglobulin (IgG) production, relative to the CD medium (100%), ranged from 148 to 438% and 117 to 283%, respectively. Using bootstrapped stepwise regression (BSR), the compounds with the highest inclusion frequency were identified. The most important compound, i.e. phenyllactate and ferulate explained 29% and 30% of the variance for cell growth and total IgG production, respectively. Surprisingly, all compounds identified in the BSR showed a positive correlation with cell growth and total IgG production. This knowledge can be applied to monitor the production and accumulation of these compounds during the production process of hydrolysates. Consequently, the processing conditions can be modulated to produce soy protein hydrolysates with enhanced and consistent cell culture performance.     

Development of an effective process for utilization of collagen from livestock and fish waste

A procedure for the extraction of protein and production of peptides by enzymic hydrolysis from bone and skin wastes containing collagen was developed. Fat and inorganic components were first removed in a pretreatment step and a high molecular weight protein extracted under acidic conditions (pH 3) using a 1 h reaction time at 60 °C. The molecular weight of extract from pig skin was greater than 100 kDa. The extract had a high water retention capacity, was beneficial for repair of rough skin, had no odor problem and was demonstrated to be safe in skin patch tests. It was thus considered acceptable for use in cosmetic materials. Pretreated fish bone and pig skin were hydrolyzed with a commercial enzyme. The hydrolysates had a high anti-radical activity (IPOX₅₀, 0.18 and 0.45 mg ml⁻¹) and a high potential for decreasing blood pressure (IC₅₀, 0.16 and 0.41 mg ml⁻¹), suggesting the hydrolysates could be a useful additive in food materials.     

Use of sugarcane bagasse and grass hydrolysates as carbon sources for xylanase production by Bacillus circulans D1 in submerged fermentation

The use of sugarcane bagasse and grass as low cost raw material for xylanase production by Bacillus circulans D1 in submerged fermentation was investigated. The microorganism was cultivated in a mineral medium containing hydrolysate of bagasse or grass as carbon source. High production of enzyme was obtained during growth in media with bagasse hydrolysates (8.4 U/mL) and in media with grass hydrolysates (7.5 U/mL). Xylanase production in media with hydrolysates was very close to that obtained in xylan containing media (7.0 U/mL) and this fact confirm the feasibility of using this agro-industrial byproducts by B. circulans D1 as an alternative to save costs on the enzyme production process.     

High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensin-I-converting enzyme inhibitory activity by capillary electrophoresis

Twelve kinds of marine protein materials, including fish, shrimp, seashell, algae and seafood wastes were selected for the hydrolysis using four different proteases. The IC(50) values for angiotensin-converting enzyme (ACE) inhibitory activity of 48 hydrolysates were rapidly determined by capillary electrophoresis (CE). The values ranged from 0.17 to 501.7mg/ml, and were affected by both the marine protein resources and the selected proteases. Hydrolysates of the lowest IC(50) values were from shrimp (Acetes chinensis), shark meat, mackerel bone, Polysiphonia urceolata and Spirulina platensis, indicating these five kinds of marine food proteins contained beneficial materials for the production of ACE inhibitory peptides by proteolysis. The hydrolysates obtained using proteases Protamex and SM98011 had lower IC(50) values, showing these two proteases were superior to others. The CE method achieved the same sensitivity as the high performance liquid chromatography (HPLC) method. However, the CE method was faster and, as a result, more economical. Therefore, CE had potential for rapid screening of marine protein hydrolysates enriched in ACE inhibitory peptides.     

Synthesis and in vitro antioxidant functions of protein hydrolysate from backbones of Rastrelliger kanagurta by proteolytic enzymes

Every year, a huge quantity of fishery wastes and by-products are generated by fish processing industries. These wastes are either underutilized to produce low market value products or dumped leading to environmental issues. Complete utilization of fishery wastes for recovering value added products would be beneficial to the society and individual. The fish protein hydrolysates and derived peptides of fishery resources are widely used as nutritional supplements, functional ingredients, and flavor enhancers in food, beverage and pharmaceutical industries. Antioxidants from fishery resources have attracted the attention of researchers as they are cheaper in cost, easy to derive, and do not have side effects. Thus the present investigation was designed to produce protein hydrolysate by pepsin and papain digestion from the backbones of Rastrelliger kanagurta (Indian mackerel) and evaluate its antioxidant properties through various in vitro assays. The results reveal that both hydrolysates are potent antioxidants, capable of scavenging 46% and 36% of DPPH (1,1-diphenyl-2 picrylhydrazyl) and 58.5% and 37.54% of superoxide radicals respectively. The hydrolysates exhibit significant (p < 0.05) reducing power and lipid peroxidation inhibition. Among the two hydrolysates produced, pepsin derived fraction is superior than papain derived fraction in terms of yield, DH (Degree of hydrolysis), and antioxidant activity.