Production of Valuable Compounds and Bioactive Metabolites from By-Products of Fish Discards Using Chemical Processing, Enzymatic Hydrolysis, and Bacterial Fermentation.

The objective of this report was to investigate the isolation and recovery of different biocompounds and bioproducts from wastes (skins and heads) that were obtained from five species discarded by fishing fleets (megrim, hake, boarfish, grenadier, and Atlantic horse mackerel). Based on chemical treatments, enzymatic hydrolysis, and bacterial fermentation, we have isolated and produced gelatinous solutions, oils that are rich in omega-3, fish protein hydrolysates (FPHs) with antioxidant and antihypertensive activities, and peptones. FPHs showed degrees of hydrolysis higher than 13%, with soluble protein concentrations greater than 27 g/L and in vitro digestibilities superior to 90%. Additionally, amino acids compositions were always valuable and bioactivities were, in some cases, remarkable. Peptones that were obtained from FPHs of skin and the heads were demonstrated to be a viable alternative to expensive commercial ones indicated for the production of biomass, lactic acid, and pediocin SA-1 from Pediococcus acidilactici.

Rice bran protein hydrolysates attenuate diabetic nephropathy in diabetic animal model.

INTRODUCTION: Diabetic nephropathy (DN) is an important microvascular complication of uncontrolled diabetes. The features of DN include albuminuria, extracellular matrix alterations, and progressive renal insufficiency. Rice bran protein hydrolysates (RBPs) have been reported to have antihyperglycemic, lipid-lowering, and anti-inflammatory effects in diabetic rats. Our study was to investigate the renoprotective effects of RBP in diabetic animals and mesangial cultured cells. METHODS: Eight-week-old male db/m and db/db mice were orally treated with tap water or RBP (100 or 500 mg/kg/day) for 8 weeks. At the end of the experiment, diabetic nephropathy in kidney tissues was investigated for histological, ultrastructural, and clinical chemistry changes, and biomarkers of angiogenesis, fibrosis, inflammation, and antioxidant in kidney were analyzed by Western blotting. Protection against proangiogenic proteins and induction of cytoprotection by RBP in cultured mesangial cells was evaluated. RESULTS: RBP treatment improved insulin sensitivity, decreased elevated fasting serum glucose levels, and improved serum lipid levels and urinary albumin/creatinine ratios in diabetic mice. RBP ameliorated the decreases in podocyte slit pore numbers, thickening of glomerular basement membranes, and mesangial matrix expansion and suppressed elevation of MCP-1, ICAM-1, HIF-1α, VEGF, TGF-ß, p-Smad2/3, and type IV collagen expression. Moreover, RBP restored suppressed antioxidant Nrf2 and HO-1 expression. In cultured mesangial cells, RBP inhibited high glucose-induced angiogenic protein expression and induced the expression of Nrf2 and HO-1. CONCLUSION: RBP attenuates the progression of diabetic nephropathy and restored renal function by suppressing the expression of proangiogenic and profibrotic proteins, inhibiting proinflammatory mediators, and restoring the antioxidant and cytoprotective system.

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.

Modelling of the production of ACE inhibitory hydrolysates of horse mackerel using proteases mixtures.

Fish protein hydrolysates from Mediterranean horse mackerel were produced by using a mixture of two commercial endoproteases (i.e. subtilisin and trypsin) at different levels of substrate concentration (2.5 g L(-1), 5 g L(-1), and 7.5 g L(-1) of protein), temperature (40 °C, 47.5 °C, and 55 °C) and percentage of subtilisin in the enzyme mixture (0%, 25%, 50%, 75% and 100%). A crossed mixture process model was employed to predict the degree of hydrolysis (DH) and the ACE inhibitory activity of the final hydrolysates as a function of the experimental factors. Both models were optimized for a maximum DH and ACE inhibition. A maximum DH (17.1%) was predicted at 2.54 g L(-1) of substrate concentration, 40 °C and an enzyme mixture comprising 38.3% of subtilisin and 61.7% of trypsin. Although its proteolytic activity is limited, the presence of trypsin in the enzyme mixture allowed obtaining higher degrees of hydrolysis at low temperatures, which is desirable to minimize thermal deactivation of the proteins. Similarly, a percentage of ACE inhibition above 48% was attained at 2.5 g L(-1) of protein, 40 °C and a 1 : 1 mixture of both proteases. Higher values of ACE inhibition could be attained by increasing both the temperature and the amount of trypsin in the enzyme mixture (e.g. 50% ACE inhibition at 55 °C and 81.5% of trypsin). Finally, those hydrolysates exhibiting the highest levels of ACE inhibition were subjected to simulated gastrointestinal digestion. These assays confirmed the resistance of active fractions against their degradation by digestive enzymes.

Production of the natural iron chelator deferriferrichrysin from Aspergillus oryzae and evaluation as a novel food-grade antioxidant.

BACKGROUND: Deferriferrichrysin (Dfcy) is a siderophore found in foods fermented by Aspergillus oryzae and is a promising candidate for an antioxidant food additive because of its high binding constant toward iron. However, the Dfcy concentration is typically low in foods and cultures. RESULTS: We optimised culture conditions to improve Dfcy production to 2800 mg L(-1) from 22.5 mg L(-1) under typical conditions. Then, we evaluated the potential of Dfcy as a food additive by measuring its safety, stability, and antioxidant activity. Dfcy was sufficiently stable that over 90% remained after pasteurisation at 63 °C for 30 min at pH 3-11, or after sterilisation at 120 °C for 4 min at pH 4-6. Dfcy showed high antioxidant activity in an oil-in-water model, where inhibition of lipid oxidation was measured by peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) assays. Dfcy decreased PV and TBARS by 83% and 75%, respectively. Antioxidant activity of Dfcy was equal to or higher than that of the synthetic chelator EDTA. CONCLUSION: Our study provides the first practical method for production of Dfcy. Dfcy can be a novel food-grade antioxidant and the first natural alternative to the synthesised iron chelator EDTA. © 2015 Society of Chemical Industry.

The Maillard reaction of a shrimp by-product protein hydrolysate: chemical changes and inhibiting effects of reactive oxygen species in human HepG2 cells.

Recently, much attention has been given to improving the antioxidant activity of protein hydrolysates via the Maillard reaction, but little is known about the cellular antioxidant activity of Maillard reaction products (MRPs) from protein hydrolysates. We first investigated chemical characterization and the cellular antioxidant activity of MRPs in a shrimp (Litopenaeus vannamei) by-product protein hydrolysate (SBH)-glucose system at 110 °C for up to 10 h of heating. Solutions of SBH and glucose were also heated alone as controls. The Maillard reaction greatly resulted in the increase of hydroxymethylfurfural (HMF) and browning intensity, high molecular weight fraction, and reduction of the total amino acid in SBH with the heating time, which correlated well with the free radical scavenging activity of MRPs. MRPs had stronger inhibiting effects on oxidative stress of human HepG2 cells than the original SBH, and its cellular antioxidant activity strongly correlated with free radical scavenging activity, but less affected by the browning intensity and HMF level. The caramelization of glucose partially affected the HMF level and free radical scavenging activity of MRPs, but it was not related to the cellular antioxidant activity. The cellular antioxidant activity of MRPs for 5 h of heating time appeared to reach a maximum level, which was mainly due to carbonyl ammonia condensation reaction. In conclusion, the Maillard reaction is a potential method to increase the cellular antioxidant activity of a shrimp by-product protein hydrolysate, but the higher HMF levels and the lower amino acid content in MRPs should also be considered.

Improvement of glycemic control in streptozotocin-induced diabetic rats by Atlantic salmon skin gelatin hydrolysate as the dipeptidyl-peptidase IV inhibitor.

In our previous study, Atlantic salmon skin gelatin hydrolysed with flavourzyme possessed 42.5% dipeptidyl-peptidase (DPP)-IV inhibitory activity at a concentration of 5 mg mL(-1). The oral administration of the hydrolysate (FSGH) at a single dose of 300 mg per day in streptozotocin (STZ)-induced diabetic rats for 5 weeks was evaluated for its antidiabetic effect. During the 5-week experiment, body weight increased, and the food and water intake was reduced by FSGH in diabetic rats. The daily administration of FSGH for 5 weeks was effective for lowering the blood glucose levels of diabetic rats during an oral glucose tolerance test (OGTT). After the 5-week treatment, plasma DPP-IV activity was inhibited; the plasma activity of glucagon-like peptide-1 (GLP-1), insulin, and the insulin-to-glucagon ratio were increased by FSGH in diabetic rats. The results indicate that FSGH has the function of inhibiting GLP-1 degradation by DPP-IV, resulting in the enhancement of insulin secretion and improvement of glycemic control in STZ-induced diabetic rats.

Purification and identification of angiotensin I-converting enzyme-inhibitory peptides from apalbumin 2 during simulated gastrointestinal digestion.

BACKGROUND: Bee larvae are considered to be an important reservoir for proteins. However, little attention has been paid to the release of potential bioactive peptides from bee larva proteins. In this study the major protein in bee larvae was hydrolyzed in vitro by gastrointestinal enzymes. The peptide profile of the hydrolysis was characterized by gel filtration chromatography and tricine-SDS-PAGE. Furthermore, the bioactive peptide was isolated and identified by Q-TOF-MS/MS. RESULTS: The major bee larva protein was identified as apalbumin 2 and was more digestible into peptides with molecular weights lower than 3 kDa. The hydrolysate obtained after 3 h of digestion exhibited angiotensin I-converting enzyme (ACE)-inhibitory activity and was purified sequentially by gel filtration and RP-HPLC. The molecular weights of peptide fractions with ACE-inhibitory activity were distributed between 0.5 and 1.5 kDa. A novel peptide with highest ACE-inhibitory activity (IC50 54.9 µmol L(-1) ) was purified by further RP-HPLC. The amino acid sequence of this peptide was identified as LLKPY (632.40 Da). CONCLUSION: ACE-inhibitory peptides could be formed from bee larvae through gastrointestinal digestion. The most active peptide (LLKPY) is potentially useful as a therapeutic agent in treating hypertension.

Application of taste sensing system for characterisation of enzymatic hydrolysates from shrimp processing by-products.

The objective of this study was to investigate the potential of an instrumental taste-sensing system to distinguish between shrimp processing by-products hydrolysates produced using different proteases and hydrolysis conditions, and the possible association of taste sensor outputs with human gustatory assessment, salt content, and bioactivity. Principal component analysis of taste sensor output data categorised samples according to the proteases used for hydrolysis. High umami sensor outputs were characteristic of bromelain- and Flavourzyme-produced hydrolysates, compared to low saltiness and high bitterness outputs of Alcalase-produced hydrolysates, and high saltiness and low umami outputs of Protamex-produced hydrolysates. Extensively hydrolysed samples showed higher sourness outputs. Saltiness sensor outputs were correlated with conductivity and sodium content, while umami sensor responses were related to gustatory sweetness, bitterness and umami, as well as angiotensin-I converting enzyme inhibitory activity. Further research should explore the dose dependence and sensitivity of each taste sensor to specific amino acids and peptides.

Is prevention of atopic eczema with hydrolyzed formulas cost-effective? A health economic evaluation from Germany.

OBJECTIVE: The German Infant Nutritional Intervention (GINI) trial, a prospective, randomized, double-blind intervention, enrolled children with a hereditary risk for atopy. When fed with certain hydrolyzed formulas for the first 4 months of life, the risk was reduced by 26-45% in PP and 8-29% in intention-to-treat (ITT) analyses compared with children fed with regular cow's milk at age 6. The objective was to assess the cost-effectiveness of feeding hydrolyzed formulas. PATIENTS AND METHODS: Cost-effectiveness was assessed with a decision tree model programmed in TreeAge. Costs and effects over a 6-yr period were analyzed from the perspective of the German statutory health insurance (SHI) and a societal perspective at a 3% effective discount rate followed by sensitivity analyses. RESULTS: The extensively hydrolyzed casein formula would be the most cost-saving strategy with savings of 478 € per child treated in the ITT analysis (CI95%: 12 €; 852 €) and 979 € in the PP analysis (95%CI: 355 €; 1455 €) from a societal perspective. If prevented cases are considered, the partially whey hydrolyzed formula is cost-saving (ITT -5404 €, PP -6358 €). From an SHI perspective, the partially whey hydrolyzed formula is cost-effective, but may also be cost-saving depending on the scenario. An extensively hydrolyzed whey formula also included into the analysis was dominated in all analyses. CONCLUSIONS: For the prevention of AE, two formulas can be cost-effective or even cost-saving. We recommend that SHI should reimburse formula feeding or at least the difference between costs for cow's milk formula and the most cost-effective formula.

Cost-effectiveness of using an extensively hydrolysed formula compared to an amino acid formula as first-line treatment for cow milk allergy in the UK.

OBJECTIVE: To estimate the cost-effectiveness of using an extensively hydrolysed formula (eHF; Nutramigen) compared with an amino acid formula (AAF; Neocate) as first-line treatment for cow milk allergy (CMA) in the UK, from the perspective of the National Health Service (NHS). METHOD: A decision model was constructed depicting the treatment paths and associated resource use attributable to first-line management of CMA with the two formulae. The model was based on the case records of 145 AAF-treated infants and 150 matched eHF-treated patients from The Health Improvement Network (THIN) database [a nationally representative database of patients registered with general practitioners (GPs) in the UK]. The model estimated the costs and consequences of patient management over 12 months following their initial GP visit for CMA. RESULTS: Patients presenting with a combination of gastrointestinal symptoms and eczema accounted for 44% of all patients in both groups. Those with gastrointestinal symptoms alone and eczema alone accounted for up to a further 39% and 13%, respectively. Those with urticaria and faltering growth accounted for <5% and ≤6% of all patients, respectively. Patients' age and weight at presentation was a mean 2.6-2.8 months and 4.4 kg, respectively. It took a mean 2.2 months to start a formula after initially seeing a GP. Time to symptom resolution following the start of treatment was 1.2 months in both groups; hence, the mean number of symptom-free months during the 12 months following the initial GP visit was estimated to be 8.6 months in both groups. Patients treated with an eHF had a mean 13.1 GP visits over the 12 months compared to 17.5 visits made by AAF-treated patients (p < 0.001). The NHS cost of managing a CMA infant over the first 12 months following initial presentation to a GP was estimated to be £1853 and £3161 for an eHF-treated and AAF-treated patient, respectively. CONCLUSION: Starting treatment for CMA with an eHF was the cost-effective option, as there were no significant differences in clinical outcomes between the two groups. A prospective, randomized controlled trial would allow a definitive confirmation of these findings.

Hydrolysis of whey protein isolate using subcritical water.

Hydrolyzed whey protein isolate (WPI) is used in the food industry for protein enrichment and modification of functional properties. The purpose of the study was to determine the feasibility of subcritical water hydrolysis (SWH) on WPI and to determine the temperature and reaction time effects on the degree of hydrolysis (DH) and the production of peptides and free amino acids (AAs). Effects of temperature (150 to 320 °C) and time (0 to 20 min) were initially studied with a central composite rotatable design followed by a completely randomized factorial design with temperature (250 and 300 °C) and time (0 to 50 min) as factors. SWH was conducted in an electrically heated, 100-mL batch, high pressure vessel. The DH was determined by a spectrophotometric method after derivatization. The peptide molecular weights (MWs) were analyzed by gel electrophoresis and mass spectrometry, and AAs were quantified by high-performance liquid chromotography. An interaction of temperature and time significantly affected the DH and AA concentration. As the DH increased, the accumulation of lower MW peptides also increased following SWH (and above 10% DH, the majority of peptides were <1000 Da). Hydrolysis at 300 °C for 40 min generated the highest total AA concentration, especially of lysine (8.894 mg/g WPI). Therefore, WPI was successfully hydrolyzed by subcritical water, and with adjustment of treatment parameters there is reasonable control of the end-products.

The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties.

The tryptic meat digest Primatone RL is a low-cost medium supplement of a complex nature which serves as a source of amino acids, oligopeptides, iron salts, some lipids and other trace low molecular weight substances. Its addition to mammalian and insect cell culture media significantly improves the cell growth properties of many cell lines. In this work the growth promoting effects of Primatone RL are described in more detail using different mouse hybridomas, a mouse myeloma cell line, and human promyelocytic leukemia HL-60 cells. The positive effects on cell growth induced by Primatone were observed in the presence of serum but were even more pronounced in serum-free culture. In addition the adaptation time from high serum to low (1%) or serum-free growth in the presence of Primatone is also significantly reduced. Primatone RL, when added to HL and DHI medium, improves cell growth under low serum or serum-free conditions by increasing the maximum cell numbers and in particular the viability of the culture. The observed decrease in cell death (apoptosis) induction leads to a significant improvement in antibody (recombinant protein) production by increasing the volumetric yields during long-term batch culture. The so-called anti-apoptotic effects of Primatone RL for mouse hybridomas, which is concentration dependent, is not fully understood.