Hydrolysis of rapeseed protein isolates: Kinetics,characterization and functional properties of hydrolysates

Two rapeseed protein isolates corresponding to albumins and globulins, respectively, were produced from an industrial defatted rapeseed meal. A pilot-scale process of protein extraction has been developed to remove major anti-nutritional compounds, easy to scale-up and using recyclable solvents. The kinetics of the hydrolyses of these two protein substrates using Alcalase 2.4L^® were compared by the measurement of the degree of hydrolysis (DH) when varying the initial proteins and enzyme concentrations. The globulins isolate was hydrolysed more efficiently than the albumins isolate mainly due to the compact and globular conformation of albumin (napin). Kinetic parameters have been determined for both substrates and a log-linear relation has been established between the DH values at a definite time and the initial enzyme/substrate ratio. Such relationships allow an effective monitoring of hydrolysis process since the hydrolysates analysis using reverse-phase chromatography coupled with mass spectrometry revealed that peptide maps corresponding to peptides of molecular weight inferior to 1 kDa are similar at a specific DH, independently of the reaction temperature and initial concentrations of substrate and enzyme. Thus, it is demonstrated that the DH is the sole parameter needed to control the physico-chemical properties and consequently the functionalities (solubility, foaming and emulsifying properties) which depend on the nature of peptides present in the hydrolysates.     

Optimization of Enzymatic Hydrolysis Conditions of Caspian kutum (Rutilus frisii kutum)ˮ By-product for Production of Bioactive Peptides with Antioxidative Properties

The enzymatic hydrolysis was performed by Alcalase to recover the fish protein hydrolysate from Caspian kutum by-product (CB). The degree of hydrolysis (DH) was applied for monitoring the hydrolysis reaction of CB. The response surface methodology was applied based on a D-optimal design to perform the optimization process for obtaining the high yield of CB protein hydrolysate. The effect of four independent variables including pH (7.5–8.5), temperature (45–55 °C), time (1–3 h), and enzyme concentration (0.5–1.5% w/w) on DH was studied. The results indicated that the predicted and actual values of the optimum condition had no significant difference. The optimum enzymatic hydrolysis conditions were achieved at pH 8.5, temperature of 55 °C, enzyme concentration of 1.5% w/w, and time of 3 h, which resulted in the maximum value of DH (19.08%). Antioxidant assays including DPPH scavenging and metal chelating activities showed that Caspian kutum protein hydrolysates had antioxidant properties.

     

Opposite Contributions of Glycinin- and β-Conglycinin-Derived Peptides to the Aggregation Behavior of Soy Protein Isolate Hydrolysates

The aggregation behavior as a function of pH was studied for hydrolysates obtained by hydrolysis of soy protein isolate (SPI) and glycinin- and β-conglycinin-rich protein fractions with subtilisin Carlsberg. The substrates were hydrolyzed up to degrees of hydrolysis (DH) of 2.2% and 6.5%. Compared with nonhydrolyzed SPI, a decrease in solubility was observed for the hydrolysates of SPI [0.8% (w/v) protein, I = 0.03 M] around neutral pH. At pH 8.0, glycinin hydrolysates had a much lower solubility (∼43% and 60%, respectively, for DH 2.2% and 6.5%) than SPI and β-conglycinin-derived hydrolysates, which were almost completely soluble. Peptides that aggregated were all larger than 5 kDa, and as estimated by size-exclusion chromatography their composition was almost independent of the aggregation pH. The solubility of hydrolysates of SPIs with a varying glycinin and β-conglycinin composition showed that glycinin-derived peptides are the driving force for the lower solubility of SPI hydrolysates. The solubility of SPI hydrolysates at pH 8.0 was shown not to be the sum of that of glycinin and β-conglycinin hydrolysates. Assuming that the separate hydrolysis of glycinin and β-conglycinin did not differ from that in the mixture (SPI), this indicates that β-conglycinin-derived peptides have the ability to inhibit glycinin-derived peptide aggregation.     

大豆分离蛋白不同酶解方式水解度与乳化性和起泡性关系

研究了大豆分离蛋白酶解产物水解度与乳化和起泡功能特性的关系。试验结果表明:大豆分离蛋白酶解产物起泡性随着水解度的增加而增加,不同酶处理液的起泡性以AS1.398酶解液最好,水解度为20%时起泡性达到了360±2.46%。研究还发现,乳化性随着水解度增加而逐渐下降,以双酶复合酶解液最差(DH=25%时乳化性最差,17.60±0.80m l.g-1)。     

Protein hydrolysate from organic fraction of municipal solid waste compost as nitrogen source to produce lactic acid by Lactobacillus fermentum ATCC 9338 and Lactobacillus plantarum NCIMB 8826

In this work a strategy for obtaining free amino-acids concentrate from an organic fraction of municipal solid waste compost and its use as a nitrogen source for lactic acid production, a compound widely used in different industries, using L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 strains is described. Enzymatic digestion is based on the combined action of endoprotease Alcalase 1.5 MG and exoprotease Flavourzyme 500 MG. The highest degree of hydrolysis obtained under the optimal conditions was 41%. The use of glucanase Viscozyme L prior to protein hydrolysis helped to reduce the viscosity of the solution and promote the action of proteases, increasing its hydrolysis degree by 76%. The hydrolysate contained all 21 amino-acids, making it ideal for lactic acid bacteria growth. During shake flask cultivations the culture media was complemented with glucose as carbon source. Finally, with the hydrolysate, a maximum lactic acid concentration of 9.0 ± 0.2 g·L⁻¹ and 11.1 ± 0.1 g·L⁻¹ for L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 respectively was obtained after 27 h. The innovation of the approach lies in exploiting the overproduction of compost for the production of lactic acid.     

In vitro assessment of the cardioprotective, anti-diabetic and antioxidant potential of Palmaria palmata protein hydrolysates

The contribution of protein fraction and proteolytic enzyme preparation to the in vitro cardioprotective, anti-diabetic and antioxidant activity of Palmaria palmata protein hydrolysates was investigated. Aqueous, alkaline and combined aqueous and alkaline P. palmata protein fractions were hydrolysed with the food-grade proteolytic preparations, Alcalase 2.4 L, Flavourzyme 500 L and Corolase PP. The hydrolysates had angiotensin converting enzyme (ACE) and dipeptidyl peptidase (DPP) IV inhibitory activity with IC₅₀ values in the range 0.19–0.78 and 1.65–4.60 mg mL^?1, respectively. The oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) values ranged from 45.17 to 467.54 and from 1.06 to 21.59 μmol trolox equivalents/g, respectively. Furthermore, hydrolysates (1 mg mL^?1) were show to inhibit renin within the range 0–50 %. In general, Alcalase 2.4 L and Corolase PP hydrolysates of aqueous protein displayed the highest in vitro activity. The results indicate that protein fraction and enzyme preparation used have significant effects on in vitro biofunctional activity of the hydrolysates. This study demonstrates the potential of P. palmata protein hydrolysates as multifunctional functional food ingredients for the prevention/control of hypertension and type II diabetes.     

Structural characteristics of hydrolysates of proteins from extracted sunflower flour at the air-water interface

The structural and topographical characteristics of a sunflower protein isolate (SPI) and its hydrolysates at different degrees of hydrolysis (DH = 5.62%, 23.5%, and 46.3%) spread at the air-water interface at pH 7 and 20 degrees C were determined from pi-A isotherms coupled with Brewster angle microscopy (BAM). The structural characteristics of SP hydrolysate spread monolayers depend on the degree of hydrolysis. We observed a significant shift of the pi-A(APPARENT) isotherms toward lower molecular areas as the degree of hydrolysis (DH) increased. This phenomenon was attributed to spreading of the protein at the interface, especially at DH 46.3%. A change in the monolayer structure was observed at a surface pressure of 12-15 mN/m. At a microscopic level, the heterogeneous monolayer structures visualized near the monolayer collapse and during the monolayer expansion proved the existence of large regions of protein aggregates. Reflectivity increased with surface pressure and was a maximum at the monolayer collapse. The monolayer thickness decreased as the degree of hydrolysis increased. These phenomena explain the poor functional properties for the formation and stabilization of a dispersion (emulsion or foam) of protein hydrolysates at high degrees of hydrolysis.     

Evaluation of iron-binding capacity,amino acid composition,functional properties of Acetes japonicus proteolysate and identification of iron-binding peptides

Recovery of functional iron-binding protein hydrolysate from Acetes japonicus employing enzymatic hydrolysis and iron-chelating peptide identification were conducted in this study. The result showed that under the optimal hydrolysis condition including Flavourzyme, pH 5, 50 °C, E:S ratio of 27.4 U/g protein and hydrolysis time of 4.8 h, the obtained proteolysate displayed the maximal iron-binding capacity (IBC) of 177.7 μgFe²⁺/g protein and comprehended 38,77 % of essential amino acids. Functional features of the Acetes proteolysate encompassing solubility, heat stability, foaming and emulsifying property, oil and water holding capacity were also noteworthy. Peptide fractionation was performed using ultrafiltration and the 1−3 kDa fraction expressed the highest IBC of 120.43 ± 0.15 μgFe²⁺/g protein, 13.7 times lower than that of disodium ethylenediaminetetraacetate (Na₂EDTA). From this fraction, two iron-binding peptides of DSVNFPVHGL (1083.53 Da) and FKVGQENTPILK (1372.77 Da) were identified utilizing nano-UHPLC-MS/MS as well as their de novo spatial structures and interaction with ferrous ion were simulated by PEP-FOLD 3. As a whole, the proteolysate/peptides could be filled as an iron chelator which could shield human body from iron deficient-related disorders or as a functional proteolysate preparation to upgrade food properties.     

Enzymatic preparation of immunomodulating hydrolysates from soy proteins

Soy protein hydrolysates with lower molecular weight were enzymatically prepared by several commercially available proteases (Alcalase 2.4L, Flavourzyme, Trypsin, Papain, Protease A and Peptidase R) with protein recovery varied from 42.59% to 79.87%. Relative content of positively charged peptides was determined on SP Sephadex C-25 using gradient sodium chloride solution as eluents. Immunomodulating properties were evaluated by measuring their effect on in vitro proliferation of murine spleen lymphocytes and phagocytic activity of peritoneal macrophages. The results showed that soy protein hydrolysates (SPHs) prepared with Alcalase and insoluble soy protein (InSP), preferable to other enzymes and soy proteins, have the highest immunomodulating activity and the optimum conditions were determined as follows: E/S=2% (Alcalase), 60 degrees C, pH 8.0, InSP concentration 6% and 225min. Positive correlations were obtained between the immunomodulating activity and content of positively charged peptides. The results suggested that lower molecular weight and positively charged peptides released from soy protein were effective in stimulating immunomodulating activity, thus provided insights into the preparation of potent immunomodulating products.     

Enzymatic hydrolysis of the Eisenia andrei earthworm: Characterization and evaluation of its properties

Some studies have carried out in order to retrieve proteins from the by-product of animal-processing industries. Earthworms are rich in protein and usually are used in animal feed. Thus, this study aimed to optimize the hydrolysis process of Eisenia andrei earthworms by employing Alcalase enzyme. Using the response surface methodology, we evaluated the following conditions: temperature, hydrolysis time, stirring speed, and enzyme/substrate ratio. The optimal conditions for the experimental design were determined through the analysis of the foaming and emulsifying properties, in vitro starch digestibility, and antioxidant activity. The results demonstrate that the highest degree of hydrolysis (i.e., 92%) was obtained under the following conditions: pH, 9.5; temperature, 25?°C; hydrolysis time, 2.25?h; stirring speed, 200?rpm; and enzyme/substrate ratio, 1.77%, using Alcalase enzyme. Evaluation of the amino acid composition under these conditions revealed higher concentrations of aspartic acid, glutamic acid, and leucine. The in vitro protein digestibility of the hydrolysate was approximately 73%. There were no significant improvements in either foam stability or emulsification after enzymatic hydrolysis. Additional studies on the antioxidant activity are required. This bioproduct could potentially serve as a promising supplementary food product.     

Influence of the rapeseed protein hydrolysis process on CHO cell growth

Different protein hydrolysates were prepared from enzymatic hydrolyses of a rapeseed isolate (>90% protein content) using different commercial enzymes of non-animal origin. The extent of hydrolysis was controlled to produce hydrolysates corresponding to various degrees of hydrolysis (DH) from 5 to 30. These hydrolysates were characterized according to their solubility and size peptide pattern. Different growth behaviours of Chinese Hamster Ovary cells were observed when these various hydrolysates were added in serum-free medium containing transferrin, albumin and insulin. Hydrolysates from low degree of hydrolysis generally did not exhibit significant positive effect on cell growth; conversely hydrolysates from extensive hydrolysis, corresponding to a major low molecular size peptides content, usually allowed an increase of the maximal cell density. However, depending on the enzyme used, the supplementation with hydrolysates corresponding to a high degree of hydrolysis and composed of at least 70% peptides with a molecular size under 1kDa, led to different maximal cell density values, indicating the importance of enzyme specificity and consequently the nature of the released peptides. This result showed that the positive influence of the rapeseed hydrolysates on cell growth was not only due to a nutritional support tied to the addition of small peptides but may be related to the presence of peptides exhibiting growth or survival factor effects. Furthermore, total substitution of proteins (transferrin, albumin and insulin) in the cell culture medium by some rapeseed hydrolysates appeared to be a promising alternative to improve the cell growth in protein-free media.     

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.     

Enzymatic generation of peptides from potato proteins by selected proteases and characterization of their structural properties

The use of low grade starting material for the generation of peptides with bioactivity properties is of interest. The proteins from the potato starch industry byproduct is a promising source, as several health benefits may be associated with their hydrolysates. The efficiency of selected proteases (Novo Pro‐D, Alcalase, Flavourzyme, and Papain), exhibiting different substrate specificities and cleavage action modes, to hydrolyze potato protein isolate (patatin and protease inhibitors) was investigated. Novo Pro‐D resulted in the lowest degree of hydrolysis, whereas Alcalase, Flavourzyme, and Papain exhibited a high catalytic efficiency for the hydrolysis of potato proteins. The degree of hydrolysis behaved in a concentration dependent manner. However, the end‐product profile (peptides and free amino acids) was dependent not only on the protease specificity, its cleavage action mode (endo/exo) and the availability of the intermediate substrates but also on the contribution of the protease inhibitors to the reaction kinetics through their inhibitory effects. Indeed, the dependence of the exo‐activity on the catalytic efficiency of the endo‐action of protease was shown to be significant. Papain generated more unique peptide sequences with homology assessment matching several potato proteins when compared with Flavourzyme. This can be attributed to the high exo‐peptidase activity of Flavourzyme resulting in the generation of shorter peptides which were difficult to match. Flavourzyme produced more peptides originated from patatin fraction, whereas Papain resulted in the release of more peptides corresponding to the protease inhibitor fractions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:420–429, 2016     

Production of hydrolysate with antioxidative activity by enzymatic hydrolysis of extruded corn gluten

Hydrolysate of extruded corn gluten with higher solubility and antioxidative property was prepared. Extrusion and starch removal of corn gluten were applied as pretreatment before enzymatic hydrolysis by Alcalase. The amylase hydrolysis of starch at 70°C for 3 h resulted in the removal of the starch from the extruded corn gluten. The best hydrolysis results can be obtained by conducting the hydrolysis at 60°C with water addition 20 g/g protein, enzyme addition 0.048 Ansen units/g protein, pH 8.5, and 120 min. Degree of hydrolysis of extruded and nonextruded corn gluten reached 39.54 and 31.16%, respectively, under the optimal condition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the optimal hydrolysate revealed that proteolysis of extruded corn gluten was more extensive than proteolysis of its counterpart which was not subjected to extrusion. The molecular weight of the peptides in the optimal hydrolysate was mainly over 3,710–660 Da as determined by gel filtration chromatography. The hydrolysates displayed good solubility and antioxidative activity. The separation profile of the hydrolysate on an ion exchange chromatography of Q-Sepharose Fast Flow showed that many kinds of peptides had antioxidative effect. A new peptide with antioxidative activity was purified, and its amino acid sequence was Phe-Pro-Leu-Glu-Met-Met-Pro-Phe, which was identified by Q-TOF2 mass spectrometry.     

Trypsin Hydrolysed Protein Fractions as Radical Scavengers and Anti-bacterial Agents from <Emphasis Type="Italic">Ficus deltoidea</Emphasis>

Different molecular sizes of protein hydrolysates were prepared from the crude protein extract of Ficus deltoidea using the technique of membrane ultrafiltration after trypsin hydrolysis. Gel electrophoretic images shows the presence of 12, 8, 7 and 7 protein bands for the protein fractions prepared from the molecular weight cut-off of 3, 10, 30 and 100 kDa, respectively. The protein hydrolysates were found to have higher radical scavenging activity than those unhydrolysed fractions at the similar molecular size. They exhibited significant differences in the radical scavenging activities based on one-way analysis of variance, except for the protein hydrolysates of 30 and 100 kDa. The smallest protein hydrolysates, 3 kDa appeared to have the comparable activity (30%) with bovine serum albumin as a positive control in this study. Similarly, the 3 kDa protein hydrolysates achieved the highest inhibitory activity (87.5%) against Pseudomonas aeruginosa at the concentration of 128 µg/mL. The protein hydrolysates were found to be more effective against gram negative bacteria (P. aeruginosa and Escherichia coli) because of lower minimum inhibitory concentration (MIC) and effective inhibitory concentration at 50% (EC₅₀) than gram positive bacterium (Staphylococcus aureus). Trypsin catalysed hydrolysis seemed to improve the anti-bacterial activity of protein hydrolysates in a bacterial strain dependent manner. The MIC could achieve 1–55 µg/mL at different molecular sizes of protein fractions. Mass spectra matching revealed that 26% of 226 identified proteins belonged to the category of plant defensive proteins in stress management and metal handling.     

Production of angiotensin I-converting enzyme inhibitory peptides from defatted canola meal

To simplify the method of ACE-inhibitory peptide production, defatted canola meal was subjected to enzymatic proteolysis. Alcalase 2.4L and protease M “Amano” were found to be the most efficient enzymes in releasing ACE-inhibitory peptides from canola proteins among 13 tested enzymes. The IC₅₀ values of canola protein hydrolysates ranged from 18.1 to 82.5 μg protein/mL. Differences in ACE-inhibitory activities of various protein hydrolysates reflected varied enzyme specificities. A positive correlation was determined between ACE-inhibitory activity and the degree of hydrolysis (r = 0.5916, p < 0.001). Ion-exchange chromatography of canola protein hydrolysate increased the protein content greater than 95% without loss of ACE-inhibitory activity. This fraction was resistant to the degradation of gastrointestinal enzyme and ACE during in vitro incubation and may be a useful ingredient in the formulation of hypotensive functional food products.     

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.     

木瓜蛋白酶水解马鹿茸血制备免疫活性肽的研究

选用木瓜蛋白酶对天山马鹿茸血进行水解制备免疫活性肽,确定了最佳水解工艺条件,并测定了三种不同水解度水解物的抗氧化活性和免疫活性。水解度(DH)为6.65%时的水解物具有最高DPPH.清除率,DH12.2%时的水解物具有最高OH.清除率。免疫活性体外检测实验显示,DH12.2%时的水解物促进细胞增值率随着浓度的增大而增大,呈现出良好的线性关系(P<0.05)。结果还显示,OH.清除率与免疫活性之间存在相关性,r=0.87(P<0.05).