Chelating peptides from rapeseed meal protein hydrolysates: identification and evaluation of their capacity to inhibit lipid oxidation.

An optimized proteolysis process was applied to rapeseed meal proteins (RP) and the hydrolysate was separated by membrane filtration allowing the production of highly metal-chelating peptides in the permeate. In order to identify the chemical structure of the most active obtained metal-chelating peptides, immobilized metal affinity chromatography (IMAC) was applied. The RP-IMAC peptide fraction was mainly composed of small peptides from 2 to 20 amino acids. Using the Ferrozine assay, RP-IMAC peptides showed a significant chelating efficiency higher than sodium citrate and close to that of EDTA. The peptide sequences were identified by UHPLC-MS and several possible iron binding sites were found. ß-carotene oxidation assay and lipid oxidation in bulk oils or emulsion were carried out to evaluate the potential of such peptides as efficient antioxidants to protect lipids from oxidation. While chelating peptides showed a limited efficiency in bulk oil, they performed more efficiently in emulsion.

Improving the in vitro digestibility of rapeseed albumins resistant to gastrointestinal proteolysis while preserving the functional properties using enzymatic hydrolysis.

Study of in vitro digestibility of high-quality isolate of rapeseed albumins (RA) was carried out in this work, using Size-Exclusion Chromatography. A poor in vitro digestibility of the RA isolate was highlighted (15%). The aim of this study was therefore to improve the RA in vitro digestibility by enzymatic hydrolysis while preserving its attractive functional properties. Alcalase, Flavourzyme and Prolyve were used to obtain 12 hydrolysates with various degrees of hydrolysis (DH) and compositions. All hydrolysates showed improved digestibility and those with the highest DH showed the best improvements. Techno-functional properties of these hydrolysates were also characterized. The poor emulsion capacity of initial RA was improved and results showed that extent proteolysis can be a good way to improve both digestibility and functional properties. Moreover, optimal conditions for RA proteolysis were identified to produce with Flavourzyme a partial hydrolysate (still containing 50% intact RA) that is both digestible and functional.

Optimization of Selective Hydrolysis of Cruciferins for Production of Potent Mineral Chelating Peptides and Napins Purification to Valorize Total Rapeseed Meal Proteins.

Preventing oxidation and microbial spoilage are both major concerns in food industries. In this context, this study aimed to valorize the total rapeseed meal proteins with controlled enzymatic proteolysis to generate potent mineral-chelating peptides from cruciferins while keeping intact the antimicrobial napins. Implementation of proteolysis of total rapeseed protein isolate with the Prolyve® enzyme highlighted an interesting selective hydrolysis of the cruciferins. Hence, the mechanism of this particular hydrolysis was investigated through a Design of Experiments method to obtain a model for the prediction of kinetics (cruciferin degradation and napin purity) according to the operating conditions applied. Then, multicriteria optimization was implemented to maximize the napin purity and yield while minimizing both enzymatic cost and reaction time. Antioxidant assays of the peptide fraction obtained under the optimal conditions proved the high metal-chelating activity preservation (EC50 = 247 ± 27 µg) for more than three times faster production. This fraction might counteract lipid oxidation or serve as preventing agents for micronutrient deficiencies, and the resulting purified napins may have applications in food safety against microbial contamination. These results can greatly help the development of rapeseed meal applications in food industries.

Using the dual isotope method to assess cecal amino acid absorption of goat whey protein in rats, a pilot study.

Measurement of ileal amino acids (AA) bioavailability is recommended to evaluate protein quality. A dual isotope tracer method, based on plasma isotopic enrichment ratios, has been proposed to determine true digestibility in humans. In a pilot study, we aimed to evaluate whether this method could be implemented in rats to determine AA bioavailability based on isotopic enrichment ratios measured in cecal digesta or plasma samples. Goat milk proteins were intrinsically labeled with 15N and 2H. Wistar rats were fed a meal containing the doubly labeled goat whey proteins and a tracer dose of 13C-spirulina. Blood samples were collected 0, 1 h and 3 h after meal ingestion from the tail vein. The rats were euthanized 4 h (n = 6) or 6 h (n = 6) after meal to collect plasma and intestinal contents. True orocecal protein digestibility and AA bioavailability were assessed by means of 15N and 2H enrichment in cecum content and compared with absorption indexes determined at the plasma or cecum level using isotopic ratios. Plasma kinetics of isotopic enrichment could not be completed due to the limited quantity of plasma obtained with sequential blood collection. However, the absorption indexes determined from cecal 15N or 2H/13C ratios gave coherent values with true orocecal AA bioavailability. This dual isotope approach with measurements of isotopic ratios in digestive content could be an interesting strategy to determine true AA bioavailability in ileal digesta of rats.

Production and antioxidant capacity of bioactive peptides from plant biomass to counteract lipid oxidation.

Preventing lipid oxidation, especially with the polyunsaturated fat-based products, is a major concern in sectors as agri-food and cosmetic. Even though the efficiency of synthetic antioxidants has been recognized, both consumers and manufacturers are looking for more innovative, healthy and quality products while rejecting synthetic additives due to their concern about safety, along with their environmental impact issues. In this context, plant biomass, which have shown to be rich in compounds, have raised interest for the isolation of novel naturally occurring antioxidants. Among their myriad of molecules, bioactive peptides, which are biologically active sequence of amino acid residues of proteins, seem to be of a great interest. Therefore, the number of identified amino acids sequences of bioactive peptides from plant biomass with potential antioxidant action is progressively increasing. Thus, this review provides a description of 129 works that have been made to produce bioactive peptides (hydrolysate, fraction and/or isolate peptide) from 55 plant biomass, along with the procedure to examine their antioxidant capacity (until 2019 included). The protein name, the process, and the method to concentrate or isolate antioxidant bioactive peptides, along with their identification and/or specificity were described. Considering the complex, dynamic and multifactorial physico-chemical mechanisms of the lipid oxidation, an appropriate in-vitro methodology should be better performed to efficiently probe the antioxidant potential of bioactive peptides. Therefore, the results were discussed, and perspective for antioxidant applications of bioactive peptides from plant biomass was argued.

A Rational Approach for the Production of Highly Soluble and Functional Sunflower Protein Hydrolysates.

Exploitation of plant proteins as an alternative to animal proteins currently presents an important challenge for food industries. In this contribution, total sunflower protein isolate from cold press meal was used as a starting material for the generation of highly soluble and functional hydrolysates that could be used in various food formulations. To do this, a rational and complete approach of controlled hydrolysis was implemented using the individual Alcalase and Prolyve enzymes. The method of stopping the hydrolysis reaction was also evaluated. The influence of operating conditions on hydrolysis kinetics and enzymatic mechanism was studied to identify the appropriate hydrolysis conditions. The gain of the solubility was then analyzed and compared to that of the initial proteins. Finally, the emulsifying and foaming properties (capacities and stabilities) of the resulting hydrolysates were also assessed. As a result, controlled enzymatic proteolysis significantly improved the sunflower protein solubility at neutral pH (twofold increase) and generated highly soluble hydrolysates. The limited proteolysis also maintained the good foam capacities and allowed an improvement in the initial foam stabilities and emulsifying capacities and stabilities of sunflower proteins. This contribution can greatly increase the value of sunflower meal and help in the development of sunflower protein products in the future.

Ultrafiltration Fractionation of Bovine Hemoglobin Hydrolysates: Prediction of Separation Performances for Optimal Enrichment in Antimicrobial Peptide.

Hydrolysis of bovine hemoglobin (bHb), the main constituent of bovine cruor by-product, releases a natural antimicrobial peptide (NKT) which could present a major interest for food safety. To enrich this, tangential ultrafiltration can be implemented, but ultrafiltration conditions are mainly empirically established. In this context, the application of a simulation method for predicting the NKT yield and enrichment was investigated. Ultrafiltration performances were studied for decolored bHb hydrolysates at different degrees of hydrolysis (DH; 3%, 5%, 10% and 18%) and colored hydrolysates (3% and 5% DH) with 1 and 3 kg·mol-1 regenerated cellulose membranes. The simulation method helped to identify the most promising hydrolysate (in terms of NKT enrichment, yield and productivity) as the 3% DH colored hydrolysate, and UF conditions (volumetric reduction factor of 5 and 3 with 1 and 3 kg·mol-1 membrane, respectively) for higher antimicrobial recovery. A maximal enrichment factor of about 29 and NKT purity of 70% in permeate were observed. The results showed that the antimicrobial activity was in relation with the process selectivity and NKT purity. Finally, this reliable method, applied for predicting the ultrafiltration performances to enrich peptides of interest, is part of a global approach to rationally valorize protein resources from various by-products.

Simultaneous quantification of the degree of hydrolysis, protein conversion rate and mean molar weight of peptides released in the course of enzymatic proteolysis.

This paper describes an original analytical methodology for a simultaneous measurement of the protein conversion rate, the mean molar weight of peptide and the degree of hydrolysis in the course of proteolysis by Size-Exclusion High-Performance Liquid Chromatography. Peak area of dead volume eluents reflects the non-converted protein. The protein conversion rate is thus determined by comparing the area at a given time to the initial area. The peptide signal allows determining the peptide molar weight distribution and degree of hydrolysis of hydrolysates. As a first step, the approach was tested on the hydrolysis of bovine serum albumin, lysozyme and rapeseed albumin by Alcalase 2.4L. Values of degree of hydrolysis were also determined by TNBS and pH-stat methods. Most of the hydrolysate obtained showed relative differences < 20% with the reference methods. The method was also adapted to fit the TNBS assay. 39 experimental validation tests were analyzed by size-exclusion chromatography, TNBS and pH stat methods. 90% of the validation data show non-significant differences between the degree of hydrolysis predicted and the degree of hydrolysis measured by TNBS method. Hence, the proposed methodology can be efficient to study the process of enzymatic proteolysis while minimizing time and quantity of sample assay required.