Valorization of fish processing by-products for protein hydrolysate recovery: Opportunities, challenges and regulatory issues.

Protein-rich fish processing by-products, often called rest raw materials (RRM), account for approximately 60% of the total fish biomass. However, a considerable amount of these RRM is utilized for low-value products such as fish meal and silage. A promising and valuable approach for maximizing the utilization of RRM involves the extraction of bioactive fish protein hydrolysate (FPH). This review assesses and compares different hydrolyzation methods to produce FPH. Furthermore, the review highlights the purification strategy, nutritional compositions, and bioactive properties of FPH. Finally, it concludes by outlining the application of FPH in food products together with various safety and regulatory issues related to the commercialization of FPH as a protein ingredient in food. This review paves the way for future applications by highlighting efficient biotechnological methods for valorizing RRM into FPH and addressing safety concerns, enabling the widespread utilization of FPH as a valuable and sustainable source of protein.

Valorization of Saithe (Pollachius virens) Residuals into Protein Hydrolysates-Silaging as Preservation Technology.

Silaging can be used as preservation technology to valorize currently discarded raw material into protein hydrolysate on board deep-sea vessels. The aim of this study was to investigate the effect of sorting and raw material freshness on the quality and yield of protein hydrolysates obtained through silaging of saithe (Pollachius virens) viscera. Additionally, the effect of using acid-containing antioxidants was tested. Out sorting of the liver prior to silaging resulted in slightly higher hydrolysate yields. The hydrolysates with the highest protein contents were obtained from silages made from fresh raw materials (day 0), and the content decreased significantly after longer storage of the raw material (2-3 days at 4 °C). Storage of the raw material for 1 day did not affect the quality. However, a significantly higher degree of hydrolysis (DH), content of free amino acids (FAA), and total volatile basic nitrogen (TVB-N) were obtained when raw materials were stored for 3 days. The FAA composition was influenced by the raw material's freshness, with increases in free glutamic acid and lysine and a decrease in free glutamine after longer storage. None of the studied parameters were significantly affected by out sorting of liver or the addition of antioxidants. Overall, the results indicate that the whole fraction of the viscera can be utilized without reducing the quality of the hydrolysate and that the raw material should be stored for a maximum of 1 day prior to preservation to optimize the quality.

Exploring the Potential of Atlantic Mesopelagic Species Processed on Board Commercial Fishing Vessels as a Source of Dietary Lipids.

This study investigates the use of untapped mesopelagic species as a source of long-chain polyunsaturated omega-3 fatty acids (LC n-3 PUFAs) to meet the growing demand. The challenges faced by commercial fishing vessels, such as varying catch rates and species distribution affecting lipid levels, are addressed. Marine oils were produced post-catch using thermal separation and enzymatic hydrolysis during four commercial cruises, screening approximately 20,000 kg of mixed mesopelagic species. Maurolicus muelleri and Benthosema glaciale were the dominant species in the catch, while krill was the primary bycatch. The lipid composition varied, with B. glaciale having a higher prevalence of wax esters, while triacylglycerols and phospholipids were more predominant in the other species. LC n-3 PUFAs ranged from 19% to 44% of lipids, with an average EPA + DHA content of 202 mg/g of oil. Both processing methods achieved oil recoveries of over 90%. Estimates indicate that the mesopelagic biomass in the Northeast Atlantic could supply annual recommended levels of EPA + DHA to 1.5 million people, promoting healthy heart and brain functions. These findings offer valuable insights for considering mesopelagic species as a potential source of dietary marine lipids, laying the groundwork for further research and innovation in processing and obtaining valuable compounds from such species.

Optimization of single-step gelatin extraction from pink perch (Nemipterus japonicus) skin and bone obtained from surimi industry using a green solvent.

Surimi industry produces a large quantity of byproducts that are currently being utilized to produce low-value commodities. This study aims to extract gelatin from pink perch skin and bone obtained from the surimi industry using a green single-step extraction method. In addition to using a green solvent, that is, acetic acid, the new method combines the multiple steps of pre-treatment and hydrolysis into one single-step extraction process. Response surface methodology was used to optimize extraction parameters (pH, temperature, and time) to maximize yield and l-hydroxyproline (l-hyp) content. The optimum condition for gelatin extraction was obtained at pH 3, 75°C, and 30 min. At optimum conditions, gelatin yield and l-hyp content were observed to be 16.07% and 41.26 mg g-1 , respectively. The gelatin obtained at optimized condition was further compared with commercial bovine gelatin (BG) in terms of chemical composition and textural, functional, and rheological properties. The results suggested that the optimized pink perch gelatin had higher protein content (92.06%), better gel strength (251.08 g), higher imino acid (18.01%), and improved textural and functional properties than the commercially available BG. The optimized single-step gelatin extraction method from pink perch skin and bones is a promising, rapid, and efficient method for the production of good-quality gelatin, which can be further used for the development of high-value products such as food formulations. PRACTICAL APPLICATION: Fish gelatin is widely used in food product development. Most of the existing methods of the development of high-value product such as gelatin, use multi-step process and harsh mineral acid, therefore, are time-consuming and harmful to the environment. This study provides a green single-step gelatin extraction method that provides an efficient, rapid, and convenient method of gelatin extraction and a sustainable solution for fish industry byproduct utilization. The data obtained with this laboratory-scale study provides a strong basis for scale-up studies.

Silage for upcycling oil from saithe (Pollachius virens) viscera - Effect of raw material freshness on the oil quality.

The main objective of this study was to investigate how the freshness of saithe (Pollachius virens) viscera affected the quality, composition and yield of oil obtained by silaging. Minced viscera with and without liver were stored separately for up to 3 days at 4 °C before silaging at pH 3.8 for 6 days at 10 °C. An antioxidant mixture was added to evaluate the effect on the lipid oxidation. Oil was extracted thermally from untreated raw material during storage (day 0-3) and after silaging. For oil obtained after silaging of viscera with liver, the oil yields increased significantly when the raw material was stored for more than one day prior to the treatment. Use of fresh raw material (collected at day 0) led to significantly lower oxidation compared to longer raw material storage. After one day of storage, the oxidation was less dependent on the freshness. Silaging with antioxidants resulted in significantly lower formation of oxidation products compared to acid without antioxidants and the most significant differences were observed after one day of storage. Contents of docosahexaenoic acid (DHA) and total omega-3 fatty acids decreased significantly when the raw material was stored for 1-3 days prior to silaging compared to fresh raw material. Results obtained by high resolution nuclear magnetic resonance (NMR) spectroscopy indicated that oxidation of esterified DHA might explain the DHA decrease. The free fatty acid content was highest when fresh raw material was used and was most likely affected by the formation of cholesteryl esters observed in NMR spectra after longer storage. The study shows that although the oil quality is reduced during silaging, processing shortly after catch and use of antioxidants can optimize the quality resulting in less oxidized oil richer in omega-3 fatty acids.

Effect of Pink Perch Gelatin on Physiochemical, Textural, Sensory, and Storage Characteristics of Ready-to-Cook Low-Fat Chicken Meatballs.

In recent years consumer demand for low-fat convenience food is increasing rapidly. This study was designed to develop low-fat ready-to-cook (RTC) chicken meatballs using pink perch gelatin. Meatballs were prepared using different concentrations of fish gelatin (3%, 4%, 5%, and 6%). The effect of fish gelatin content on the physico-chemical, textural, cooking, and sensory properties of meatballs was studied. Further, the shelf-life of meatballs was also studied at 4 °C for 15 days and -18 °C for 60 days. The addition of fish gelatin to meatballs decreased the fat content by 67.2% and 79.7% and increased the protein content by 20.1% and 66.4% in comparison to control and Branded Meatballs, respectively. As compared to the Control Meatballs, the addition of fish gelatin also reduced hardness by 26.4% and increased yield and moisture retention in the RTC meatballs by 15.4% and 20.9%, respectively. Sensory analysis suggested that a 5% fish gelatin addition in meatballs has the best acceptability among all tested treatments. Storage study indicated that the addition of fish gelatin to RTC meatballs delayed lipid oxidation during both refrigerated and frozen storage. The results suggested that pink perch gelatin can be used as a fat replacer in chicken meatballs and can potentially increase their shelf-life.

Optimization of a Calcium-Based Treatment Method for Jellyfish to Design Food for the Future.

Edible jellyfish are a traditional Southeast Asian food, usually prepared as a rehydrated product using a salt and alum mixture, whereas they are uncommon in Western Countries and considered as a novel food in Europe. Here, a recently developed, new approach for jellyfish processing and stabilization with calcium salt brining was upgraded by modifying the pre-treatment step of freshly caught jellyfish and successfully applied to several edible species. Treated jellyfish obtained by the application of the optimized version of this method respected both quality and safety parameters set by EU law, including no pathogenic microorganisms, absence or negligible levels of histamine and of total volatile basic nitrogen, no heavy metals; and the total bacterial, yeast, and mold counts were either negligible or undetectable. Jellyfish treated by the presented method exhibited unique protein content, amino acid and fatty acid profiles, antioxidant activity, and texture. The optimized method, initially set up on Rhiszostoma pulmo, was also successfully applied to other edible jellyfish species (such as Cotylorhiza tuberculata, Phyllorhiza punctata, and Rhopilema nomadica) present in the Mediterranean Sea. This study discloses an innovative process for the preparation of jellyfish-based food products for potential future distribution in Europe.

Biochemical Characterization of Cassiopea andromeda (Forsskål, 1775), Another Red Sea Jellyfish in the Western Mediterranean Sea.

Increasing frequency of native jellyfish proliferations and massive appearance of non-indigenous jellyfish species recently concur to impact Mediterranean coastal ecosystems and human activities at sea. Nonetheless, jellyfish biomass may represent an exploitable novel resource to coastal communities, with reference to its potential use in the pharmaceutical, nutritional, and nutraceutical Blue Growth sectors. The zooxanthellate jellyfish Cassiopea andromeda, Forsskål, 1775 (Cnidaria, Rhizostomeae) entered the Levant Sea through the Suez Canal and spread towards the Western Mediterranean to reach Malta, Tunisia, and recently also the Italian coasts. Here we report on the biochemical characterization and antioxidant activity of C. andromeda specimens with a discussion on their relative biological activities. The biochemical characterization of the aqueous (PBS) and hydroalcoholic (80% ethanol) soluble components of C. andromeda were performed for whole jellyfish, as well as separately for umbrella and oral arms. The insoluble components were hydrolyzed by sequential enzymatic digestion with pepsin and collagenase. The composition and antioxidant activity of the insoluble and enzymatically digestible fractions were not affected by the pre-extraction types, resulting into collagen- and non-collagen-derived peptides with antioxidant activity. Both soluble compounds and hydrolyzed fractions were characterized for the content of proteins, phenolic compounds, and lipids. The presence of compounds coming from the endosymbiont zooxanthellae was also detected. The notable yield and the considerable antioxidant activity detected make this species worthy of further study for its potential biotechnological sustainable exploitation.

Sustainable resource production for manufacturing bioactives from micro- and macroalgae: Examples from harvesting and cultivation in the Nordic region.

Micro- and macroalgae are a great and important source of raw material for manufacturing of bioactives and ingredients for food, feed, cosmetics, or pharmaceuticals. Macroalgae (or seaweeds) have been harvested locally from wild stocks in smaller volumes for a long time, and a production chain based on cultivated seaweed for the harvest of considerably larger amounts is in progress for several species. Microalgae and cyanobacteria such as Spirulina have been produced in "backyard ponds" for use in food and feed also for a long time, and now we see the establishment of large production plants to control the cultivation process and increase the production yields. There is also a shift from harvesting or cultivation centered in warmer, sunnier areas to increasing exploitation of natural resources in temperate to boreal regions. In locations with strong seasonal variations in solar irradiance and temperatures, we need to develop procedures to maximize the biomass production in the productive seasons and ensure efficient stabilization of the biomass for year-round processing and product manufacturing. Industrialized biomass production and large-scale manufacturing of bioactives also mean that we must employ sustainable, cost-effective, and environmentally friendly processing methods, including stabilization and extraction methods such as ensiling and subcritical water extraction (SWE) and advanced analytic tools to characterize the products. These topics are focus areas of the Nordic Centre of Excellence (NCoE) NordAqua, and here we present a review of current activities in the field of micro- and macroalgae biomass production sectors illustrated with some of our experiences from the NordAqua consortium.

Energetic and Economic Evaluation of Zero-Waste Fish Co-Stream Processing.

This study evaluates the possibility of recovery of high-quality valuable fish oil and proteins from fish co-streams by traditional means or a combination of several technologies. A techno-economically feasible and sustainable zero-waste process is needed for full utilisation of this co-stream's potential. This study aims to determine the energy efficiency and economic feasibility of four different zero-waste bio-refineries based on salmon filleting co-streams. The study covers four concepts: (I) biogas and fertiliser production from salmon co-streams, (II) fish silage production, (III) thermal processing of salmon co-streams for producing oil, protein concentrate, and meal, and (IV) novel two-stage thermal and enzymatic process for producing high-quality oil and protein hydrolysate, while the solid residue is converted to biogas and fertilisers. Monte Carlo simulation is used to evaluate uncertainties in economic evaluation. The results show that the two-stage processing of fish co-streams leads to recovery of both high-quality marine oil and proteins, showing the largest profitability and return on investment during the economic analysis. It is a more tempting option than the currently used thermal treatment or traditional silage processes. The possibility of producing food-grade fish protein hydrolysate is the biggest benefit here. Concepts studied are examples of zero-waste processing of bioproducts and illustrate the possibilities and benefits of fully utilising the different fractions of fish as fillets, oil, protein, fertilisers, and energy production.

A chicken protein hydrolysate exerts anti-atherosclerotic effect beyond plasma cholesterol-lowering activity in Apoe-/- mice.

Chicken protein hydrolysates (CPHs) generated from rest raw materials through enzymatic hydrolysis using Corolase PP or Alcalase were shown to reduce inflammation and stimulate hepatic mitochondrial fatty acid oxidation in high-fat-fed mice. This study investigates the effect of CPH diets in atherosclerosis-prone apolipoprotein E-deficient (Apoe-/-) mice. Apoe-/- mice were divided into three groups of 12 animals and fed high-fat diets with casein (control), Alcalase CPH, or Corolase PP CPH. After 12 weeks, mice were sacrificed, blood samples were collected, and aorta was dissected for subsequent én face analysis. Mice fed Corolase PP CPH but not Alcalase CPH had significantly lower % atherosclerotic plaque area in the aortic arch compared to controls (p = .015 and p = .077, respectively). Plasma and liver cholesterol and triacylglycerol remained constant, but levels of the fatty acid C20:5n-3 were increased, accompanied by an elevated delta-5 desaturase index in both CPHs groups. Moreover, a significant reduction of plasma MCP-1 was detected in Corolase PP CPH compared to control. Overall, our data show that protein hydrolysates from chicken reduced atherosclerosis and attenuated systemic risk factors related to atherosclerotic disorders, not related to changes in the level of plasma cholesterol.

Utilization of egg-laying hens (Gallus Gallus domesticus) for production of ingredients for human consumption and animal feed.

BACKGROUND: In Norway, 3 million discarded egg-laying hens are destructed annually, which equals 1500 tons pure hen meat. Due to the slaughter methods used, this raw material is handled as a high-risk waste, while in reality it constitutes a source of valuable components like proteins and lipids. METHODS: This study assess different processing methods (thermal treatment, enzymatic hydrolysis and silaging) for utilization of discarded egg-laying hens for the production of ingredients for human consumption and animal feed. The processing methods were evaluated on the basis of quantity and quality of the obtained products. RESULTS: Thermal treatment and enzymatic hydrolysis resulted in extraction of good quality lipids from the raw material. The separated oil (50.1-82.3% of the total lipid content in the raw material) was of high quality based on the content of free fatty acids (≤ 1.0%) and total oxidation value (≤ 3.9). Enzymatic hydrolysis also enabled separation of protein in the form of protein hydrolysate. Addition of Protamex and Papain+Bromelain significantly (p ≤ 0.05) increased the protein content (85.1-94.6%) and decreased the lipid content (0.3-1.1%) in the hydrolysate compared to autolysis (protein content: 64.8-72.3%, lipid content: 1.0-2.6%). Silaging increased the protein digestibility (63.2-79.7% compared to 57.3-66.2% for untreated raw material), and thus constitutes a good method for utilizing the protein content of the raw material for animal feed. CONCLUSION: The biotechnological processing methods thermal treatment, enzymatic hydrolysis and silaging can be used to increase the utilization of discarded egg-laying hens for production of ingredients for human consumption and animal feed.

Effects of low doses of fish and milk proteins on glucose regulation and markers of insulin sensitivity in overweight adults: a randomised, double blind study.

PURPOSE: To examine whether supplementation with low doses of fish or milk proteins would affect glucose regulation and circulating lipid concentrations in overweight healthy adults. METHODS: Ninety-three overweight adults were assigned to receive 2.5 g protein/day from herring (HER), salmon (SAL), cod (COD) or milk (CAS, a casein-whey mixture as positive control) as tablets for 8 weeks. RESULTS: Seventy-seven participants were included in the analyses. HER and SAL did not affect glucose and insulin concentrations. COD significantly reduced within-group changes in 90 and 120 min postprandial glucose concentrations but changes were not different from HER and SAL groups. CAS supplementation significantly reduced the area under the curve for glucose concentrations (- 7%), especially when compared to SAL group, and reduced postprandial insulin c-peptide concentration (- 23%). Reductions in acetoacetate (- 24%) and ß-hydroxybutyrate (- 29%) serum concentrations in HER group were more prominent compared to SAL and COD groups, with no differences between fish protein groups for α-hydroxybutyrate. Serum concentrations of α-hydroxybutyrate (- 23%), acetoacetate (- 39%) and ß-hydroxybutyrate (- 40%) were significantly reduced within CAS group, and the decreases were significantly more pronounced when compared to SAL group. Serum lipid concentrations were not altered in any of the intervention groups. CONCLUSION: Findings indicate that 2.5 g/day of proteins from fish or milk may be sufficient to improve glucose regulation in overweight adults. The effects were most pronounced after supplementation with proteins from cod, herring and milk, whereas salmon protein did not affect any of the measurements related to glucose regulation. CLINICAL TRAIL REGISTRATION: This trial was registered at clinicaltrials.gov as NCT01641055.

Hydrolyzed proteins from herring and salmon rest raw material contain peptide motifs with angiotensin-I converting enzyme inhibitors and resulted in lower urine concentrations of protein, cystatin C and glucose when fed to obese Zucker fa/fa rats.

The use of angiotensin-I converting enzyme (ACE) inhibitors is a common strategy for treating kidney disease. Several amino acid sequences with ACE inhibiting activity are identified in filet and rest raw material from various species of fish, and fish protein hydrolysates could be of interest for possible treatment or prevention of kidney disease. Therefore, we hypothesized that protein hydrolysates from rest raw material from herring and salmon contained ACE inhibiting motifs, and could beneficially affect typical markers for kidney function in an obesity rat model prone to developing renal failure. We identified 81 and 49 peptide sequences with known ACE inhibiting activity in herring and salmon protein hydrolysates from rest raw material, respectively. To investigate the effects of fish protein hydrolysates on markers of kidney function, obese Zucker fa/fa rats consumed diets with 25% of protein from herring (HER) or salmon (SAL) protein hydrolysate from rest raw material and 75% of protein from casein/whey, or 100% protein from casein/whey (CAS) for 4 weeks. Rats fed HER or SAL diets had lower urine concentrations (relative to creatinine) of protein, cystatin C and glucose when compared to rats fed CAS diets, with no differences between groups for serum concentrations of protein, creatinine and cystatin C. To conclude, protein hydrolysates from herring and salmon rest raw material contained several peptide sequences with known ACE inhibiting activities, and resulted in lower urine concentrations of proteins, cystatin C and glucose when fed to obese Zucker rats.

Chicken Protein Hydrolysates Have Anti-Inflammatory Effects on High-Fat Diet Induced Obesity in Mice.

Background: Studies have shown that dietary source of protein and peptides can affect energy metabolism and influence obesity-associated diseases. This study aimed to investigate the impact of different chicken protein hydrolysates (CPHs) generated from chicken rest raw materials in a mouse obesity model. Methods: Male C57BL/6 mice were fed a high-fat, high-sucrose diet with casein or CPHs generated using Papain + Bromelain, Alcalase, Corolase PP, or Protamex for 12 weeks (n = 12). Body weight, feed intake, and intraperitoneal glucose tolerance was determined, and plasma and liver and adipose tissues were collected at sacrifice. Results: The average feed intake and body weight did not differ between the groups and white adipose tissue depots were unchanged, except for a reduction in the subcutaneous depot in mice fed the Protamex CPH diet. Moreover, the CPH diets did not prevent increased fasting glucose and insulin levels. Interestingly, the hepatic mitochondrial fatty acid ß-oxidation was increased in mice fed Alcalase and Corolase PP CPHs. All CPH diets reduced plasma interleukine (IL)-1ß, interferon-γ, tumor necrosis factor α, and monocyte chemotactic protein 1 compared to control, indicating anti-inflammatory effects. In addition, Corolase PP and Protamex CPHs significantly reduced plasma levels of IL-1α, IL-2, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor. Conclusions: CPH diets were not able to counteract obesity and glucose intolerance in a mouse obesity model, but strongly reduced inflammatory parameters associated with obesity. Alcalase and Corolase PP CPHs also stimulated mitochondrial fatty acid ß-oxidation. The possibility that hydrolysates from chicken rest raw materials could alleviate obesity-associated metabolic disease should be investigated further.

Dietary fish protein hydrolysates containing bioactive motifs affect serum and adipose tissue fatty acid compositions, serum lipids, postprandial glucose regulation and growth in obese Zucker fa/fa rats.

The world's fisheries and aquaculture industries produce vast amounts of protein-containing by-products that can be enzymatically hydrolysed to smaller peptides and possibly be used as additives to functional foods and nutraceuticals targeted for patients with obesity-related metabolic disorders. To investigate the effects of fish protein hydrolysates on markers of metabolic disorders, obese Zucker fa/fa rats consumed diets with 75 % of protein from casein/whey (CAS) and 25 % from herring (HER) or salmon (SAL) protein hydrolysate from rest raw material, or 100 % protein from CAS for 4 weeks. The fatty acid compositions were similar in the experimental diets, and none of them contained any long-chain n-3 PUFA. Ratios of lysine:arginine and methionine:glycine were lower in HER and SAL diets when compared with CAS, and taurine was detected only in fish protein hydrolysate diets. Motifs with reported hypocholesterolemic or antidiabetic activities were identified in both fish protein hydrolysates. Rats fed HER diet had lower serum HDL-cholesterol and LDL-cholesterol, and higher serum TAG, MUFA and n-3:n-6 PUFA ratio compared with CAS-fed rats. SAL rats gained more weight and had better postprandial glucose regulation compared with CAS rats. Serum lipids and fatty acids were only marginally affected by SAL, but adipose tissue contained less total SFA and more total n-3 PUFA when compared with CAS. To conclude, diets containing hydrolysed rest raw material from herring or salmon proteins may affect growth, lipid metabolism, postprandial glucose regulation and fatty acid composition in serum and adipose tissue in obese Zucker rats.

Quality changes of salmon by-products during storage: Assessment and quantification by NMR.

Safe utilization of fish by-products is an important task due to increasing fish consumption. It can provide new valuable food/feed and will increase the economical profit and sustainability of the fishery industry. NMR spectroscopy is a reliable tool able to monitor qualitative and quantitative changes in by-products. In this work the trichloroacetic acid extracts of salmon backbones, heads and viscera stored at industrially relevant temperatures (4 and 10°C) were studied using NMR. Twenty-five metabolites were detected and the possibility of salmon by-products utilization as a source of anserine, phosphocreatine and taurine was discussed. Statistical data elaboration allowed determining the main processes occurring during by-products storage: formation of trimethylamine and biogenic amines, proteolysis and different types of fermentations. By-products freshness was evaluated using a multi-parameter approach: the trimethylamine and biogenic amines concentration changes were compared with Ki and H-values and safe temperatures and times for storage of salmon by-products were proposed.

Bioactivities of fish protein hydrolysates from defatted salmon backbones.

Bioactivities of bulk fish protein hydrolysates (FPH) from defatted salmon backbones obtained with eight different commercial enzymes and their combinations were tested. All FPH showed antioxidative activity in vitro. DPPH scavenging activity increased, while iron chelating ability decreased with increasing time of hydrolysis. All FPH showed ACE inhibiting effect which depended on type of enzyme and increased with time of hydrolysis. The highest effect was found for FPH produced with Trypsin. Bromelain + Papain hydrolysates reduced the uptake of radiolabelled glucose into CaCo-2 cells, a model of human enterocytes, indicating a potential antidiabetic effect of FPH. FPH obtained by Trypsin, Bromelain + Papain and Protamex showed the highest ACE inhibitory, cellular glucose transporter (GLUT/SGLT) inhibitory and in vitro antioxidative activities, respectively. Correlation was observed between the measured bioactivities, degree of hydrolysis and molecular weight profiles, supporting prolonged hydrolysis to obtain high bioactivities.