Identification and Capture of Phenolic Compounds from a Rapeseed Meal Protein Isolate Production Process By-Product by Macroporous Resin and Valorization Their Antioxidant Properties.

In this study, phenolic compounds from an aqueous protein by-product from rapeseed meal (RSM) were identified by HPLC-DAD and HPLC-ESI-MS, including sinapine, sinapic acid, sinapoyl glucose, and 1,2-di-sinapoyl gentibiose. The main phenolic compound in this by-product was sinapine. We also performed acid hydrolysis to convert sinapine, and sinapic acid derivatives present in the permeate, to sinapic acid. The adsorption of phenolic compounds was investigated using five macroporous resins, including XAD4, XAD7, XAD16, XAD1180, and HP20. Among them, XAD16 showed the highest total phenolic contents adsorption capacities. The adsorption behavior of phenolic compounds was described by pseudo-second-order and Langmuir models. Moreover, thermodynamics tests demonstrated that the adsorption process of phenolic compounds was exothermic and spontaneous. The highest desorption ratio was obtained with 30% (v/v) and 70% (v/v) ethanol for sinapine and sinapic acid, respectively, with a desorption ratio of 63.19 ± 0.03% and 94.68 ± 0.013%. DPPH and ABTS tests revealed that the antioxidant activity of the hydrolyzed fraction was higher than the non-hydrolyzed fraction and higher than the one of vitamin C. Antioxidant tests demonstrated that these phenolic compounds could be used as natural antioxidants, which can be applied in the food industry.

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.

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.

Steam explosion of Aucoumea klaineana sapwood: Membrane separation of acetylated hemicelluloses.

The fractionation of the aqueous effluent of Aucoumea klaineana Pierre (Okoumé) sapwood steam explosion was examined by a sequential-dilution type membrane diafiltration. The permeate and retentate fractions were characterized by HPLC-SEC, HSQC-NMR, FTIR, UV-visible and HPAE-PAD ion chromatography. Diafiltration with 10 kDa regenerated cellulose membrane has been shown to provide efficient fractionation without fouling. O2 and/or O3 acetylated xylans with a lower proportion of O2 and/or O3 acetylated glucomannans were isolated in the retentate (≈35% w/w and 1.08 w/w% based on initial effluent solid content and on initial dry wood respectively, including 65% w/w in the range 9-22 kDa). The molecular weights of the polysaccharides were significantly higher than those obtained by ethanolic precipitation. The permeate concentrated low molecular mass oligomers (90% w/w < 2.3 kDa, 1.88 w/w% based on initial dry wood) composed of pectic sugars, highly acetylated xylans (DS ≈ 0.9) and relatively high proportion of soluble lignin (≈40% w/w) including Lignin-Carbohydrate Complexes (LCCs).

Multicriteria Optimization of Phenolic Compounds Capture from a Sunflower Protein Isolate Production Process by-Product by Adsorption Column and Assessment of Their Antioxidant and Anti-Inflammatory Effects.

The aim of this study was to valorize liquid effluent from the sunflower protein isolate process by extracting phenolic compounds it contains. To do so, XAD7 resin was used. A multicriteria optimization methodology based on design of experiments showed the optimal conditions were adsorption flow rate of 15 BV/h at pH 2.7, a desorption flow rate at 120 BV/h with ethanol/water 50% (v/v). The best trade-off between purity and recovery yields resulted in the production of a fraction containing 76.05% of chlorogenic acid (CGA) whose biological properties were evaluated. DPPH and ABTS tests showed that this fraction had a higher radical scavenging capacity than vitamin C. In vitro assays have shown that this fraction, when used at a concentration corresponding to 50 or 100 µM of CGA, does not present any cytotoxicity on human THP-1 cells differentiated into macrophages. In addition, this fraction when added prior to the inflammatory stimulus (LPS) can reduce tumor necrosis factor-alpha (TNF-α) production by 22%, thereby highlighting its protective properties against future inflammation.

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.

A new size-exclusion chromatography method for fast rapeseed albumin and globulin quantification.

The method described in the article aims at the quantification of both main storage proteins, globulins and albumins, in aqueous extract from rapeseed, as an alternative to the current reference methods, Kjeldahl and SDS-PAGE electrophoresis. The new method lies on the analytical separation of extracted compounds by Size-Exclusion High Performance Liquid Chromatography (SE-HPLC) (Biosep-SEC-s2000, 5 µm). The elution of rapeseed extracts with water/acetonitrile/trifluoroacetic acid (45/55/0.1% v/v) during 30 min yields two distinct peaks for the main proteins of rapeseed. Based on the protein extinction coefficients, a calibrationless methodology was developed for their quantification on the basis of the UV signal. The SE-HPLC method was successfully compared to references: Kjeldahl and SDS-PAGE densitometry for the determination of the proportion of each protein. Then, it was successfully applied on two other oleoproteagineous plants, linseed and sunflower.

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α-position and peptides on N-terminal position.

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N-α or ε-acetyl-L-lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε-position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N-acylation on the α-position of the lysine or of the amino-acid in N-terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N-α-acetyl-L-lysine could be attributed to the product of SAM23877_1734 gene.

Recombinant pediocin in Lactococcus lactis: increased production by propeptide fusion and improved potency by co-production with PedC.

We describe the impact of two propeptides and PedC on the production yield and the potency of recombinant pediocins produced in Lactococcus lactis. On the one hand, the sequences encoding the propeptides SD or LEISSTCDA were inserted between the sequence encoding the signal peptide of Usp45 and the structural gene of the mature pediocin PA-1. On the other hand, the putative thiol-disulfide oxidoreductase PedC was coexpressed with pediocin. The concentration of recombinant pediocins produced in supernatants was determined by enzyme-linked immunosorbent assay. The potency of recombinant pediocins was investigated by measuring the minimal inhibitory concentration by agar well diffusion assay. The results show that propeptides SD or LEISSTCDA lead to an improved secretion of recombinant pediocins with apparently no effect on the antibacterial potency and that PedC increases the potency of recombinant pediocin. To our knowledge, this study reveals for the first time that pediocin tolerates fusions at the N-terminal end. Furthermore, it reveals that only expressing the pediocin structural gene in a heterologous host is not sufficient to get an optimal potency and requires the accessory protein PedC. In addition, it can be speculated that PedC catalyses the correct formation of disulfide bonds in pediocin.

Determination of reducing power and metal chelating ability of antioxidant peptides: revisited methods.

The purpose of this study was to improve two common tests used for antioxidant capacity measurements, i.e. the reducing power and chelating ability measurements, for appropriate comparisons between the molecules tested and chosen references, as the usual methods are often performed in a qualitative way rather than a quantitative way. After revision, it was then possible to determine an AERC indice (Ascorbate Equivalent Reducing Capacity) and a CECC (Carnosine Equivalent Chelating Capacity) or EECC (EDTA Equivalent Chelating Capacity) indice according to the standard chosen, by analogy to the TEAC indice (Trolox Equivalent Antioxidant Capacity) already used in many reported works to determine the free radical scavenging activity. Thus, the determination of these relative indices enables the comparison of antioxidative capacities obtained in various studies. The adaptation of these two tests to micro-scales and the calculation of AERC, EECC and CECC were performed on model peptides.