Enzyme membrane reactor in isolation of antioxidative peptides from oil industry waste: A comparison with non-peptidic antioxidants

Edible oil industries suffer from the problem of seed meal utilization, which is recognized as a byproduct of edible oil. Present work has investigated production of peptide antioxidants, from oil seed meals to meet the increasing crave for natural antioxidants in food and pharmaceutical industries. Metallo-endopeptidase ‘Protease A Amano 2G’ (Aspergillus oryzae) was used to hydrolyze seed protein isolate in enzyme membrane reactor (EMR) and protein hydrolysate was sequentially fractionated by ultrafiltration to obtain potential peptide fraction. Degree of hydrolysis was varied within enzyme to substrate ratio 0.1–2 g/100 g and hydrolysis time 10–60 min to maximize peptide yield and antioxidant activity of peptides in vitro. Controlled hydrolysis with enzyme dose of 2 g/100 g, exhibited peptide yield of 4.60 ± 0.08 mg/100 mg meal protein in membrane reactor (DH 30.7%) and 4.23 ± 0.22 mg/100 mg meal protein in batch mode of hydrolysis (DH 29.3%). Antioxidant potential of peptide fractions were compared with commercial non-peptidic antioxidants and major findings confirm superior activity for protein fragments (IC₅₀-0.062 mg/mL) as compared to protein isolate (IC₅₀-0.038 mg/mL), with enhanced product stability by preventing oxidative deterioration. Results substantiate the prospect of EMR in obtaining peptides from edible oil industry waste, having comparable antioxidative potential with commercial non-peptidic antioxidants.     

Continuous hydrolysis of modified wheat gluten in an enzymatic membrane reactor

BACKGROUND: The low solubility of wheat gluten is one of the major limitations to its use in food processing, and enzymatic hydrolysis has been found to be an effective way to prepare more soluble bioactive peptides from gluten. The aim of this study was to prepare bioactive peptides from modified wheat gluten (MWG) in a continuous enzymatic membrane reactor (EMR) that allowed rapid separation of low‐molecular‐weight peptides from hydrolysates, thus avoiding the disadvantages of batch reaction such as inefficient use of enzymes, inconsistent products due to batch‐to‐batch variation, substrate–product inhibition, low productivity and excessive hydrolysis. RESULTS: Wheat gluten was modified to decrease its lipid and starch contents in order to prevent membrane fouling. The optimal working conditions for Alcalase to hydrolyse MWG in the EMR were a substrate concentration of 20 g L^?1, an enzyme/substrate ratio of 0.03, an operating pressure of 0.04 MPa, a temperature of 40 °C and a pH of 9. The operating stability of the EMR (including residual enzyme activity, productivity and capacity) was high. The permeate fractions showed antioxidant activities that were mostly due to low‐molecular‐weight peptides. A simple theoretical kinetic model was successfully applied to the enzymatic hydrolysis of MWG in the EMR. CONCLUSION: Modification of wheat gluten made the continuous enzymatic membrane reaction more efficient and the EMR proved to be an effective means of producing peptides with particular properties and bioactivities. The permeate fractions (mainly < 1000 Da) were homogeneous and stable and also showed strong antioxidant activities. Copyright © 2011 Society of Chemical Industry     

Experimental study on the continuous production of velvet bean-based bioactive peptides in a membrane reactor and bioactivity mapping

This study investigates the continuous production of bioactive peptides from fermented velvet bean (Mucuna pruriens) using a cost-effective enzymatic membrane reactor. The optimal operating conditions for continuous hydrolysis were residence time of 9 h, enzyme-to-substrate ratio of 10%, and the use of a 5-kDa PES membrane for separation. The resulting permeate exhibited notable antioxidant activity with DPPH and FRAP assay values of 0.28 and 0.12 mg AEAC/mL, respectively, and an angiotensin-converting enzyme (ACE) inhibition of 83.28%. Further fractionation of the permeate with a 2-kDa membrane led to increased antioxidant activity and ACE inhibition, with resulting half maximal inhibitory concentration (IC₅₀) values of 7.6 and 0.6 μg protein/mL, respectively. Liquid chromatography-mass spectrometry was used to identify peptide sequences from the <2-kDa fraction, which were matched with the BIOPEP-UWM™ database. The results revealed that the fermented velvet bean peptides were predominantly dipeptidyl peptidase (DPP)-4 inhibitors and ACE inhibitors.Industrial relevanceThe combination of a stirred tank reactor and membrane separation has the potential to enable the continuous production of bioactive peptides from velvet bean through the use of a freely suspended enzyme system. This reactor design offers an alternative solution to the existing delay in commercializing bioactive peptides, by (i) simplifying the industrial process development for continuous bioactive peptide production, (ii) eliminating the need for enzyme immobilization, and (iii) eliminating batch-to-batch variability in product quality and unproductive time associated with batch-wise production.     

Process Characteristics of Hydrolysis of Chitosan in a Continuous Enzymatic Membrane Reactor

ABSTRACT: Crude enzyme from Bacillus cereus NTU-FC-4 was used to hydrolyze chitosan of 66% deacetylation in a membrane reactor, operated at 45 °C and pH 5, to continuously produce chitooligosaccharides. Major oligomers in the product from the reactor were chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose. When the membrane reactor was operated at an enzyme/substrate ratio of 0.2 (unit/mg) and residence time of 100 min, it reached steady state in 2.5 h. The system could be operated for 15 h and still maintained a stable product composition. When the volume replacement exceeded 2.5, the productivity of the membrane reactor became higher than that of the batch reactor, and the difference between them became even greater when the volume replacement was further increased. The apparent Michaelis constant (K_m) for the enzyme in the membrane reactor was 18.8 mg/mL, but the apparent K_m was 5.4 mg/mL for the batch reactor, suggesting that the affinity of the enzyme for chitosan was lower in the membrane reactor compared with the enzyme in the batch reactor. The estimated values of apparent V_max were 0.18 and 0.20 mg reducing sugar/mL/min for the enzyme in the membrane reactor and in the batch reactor, respectively, indicating that the enzyme activity was not greatly altered when used in the membrane reactor.     

不同蛋白酶对小麦蛋白酶解物抗氧化活性的影响

小麦蛋白是小麦淀粉加工的副产物,酶解是提高小麦蛋白溶解性和功能性的有效方式,而酶解用酶种类可能对酶解产物的功能性如抗氧化活性有一定影响。采用碱性蛋白酶、中性蛋白酶、胃蛋白酶、风味蛋白酶、胰蛋白酶、木瓜蛋白酶6种常用的蛋白酶分别对小麦蛋白进行酶解,并对酶解4 h后酶解物的多肽得率、分子质量分布、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率、超氧阴离子自由基(O_2~-·)清除率、羟自由基(·OH)清除率等反映水解程度和抗氧化能力的主要指标进行评价。结果表明,风味蛋白酶酶解物中多肽得率最高,达91.44%,且分子质量小于3 000 D的多肽含量达76.9%;酶解物质量浓度为3 mg/m L时,木瓜蛋白酶酶解物对DPPH自由基清除作用最好,清除率为65.12%(P0.01),其次是风味蛋白酶(58.43%)和碱性蛋白酶(55.29%);碱性蛋白酶酶解物对O_2~-·清除率效果最好,清除率为58.68%(P0.01),其次是风味蛋白酶(49.25%);碱性蛋白酶和木瓜蛋白酶酶解物对·OH清除效果最佳,清除率分别为59.23%和58.16%。结果说明,蛋白酶种类对小麦蛋白酶解物抗氧化活性影响显著,风味蛋白酶对提高蛋白水解程度和生成小分子质量多肽的作用明显,而碱性蛋白酶、木瓜蛋白酶和风味蛋白酶对提高酶解产物抗氧化活性效果较好。     

Continuous production of tempe-based bioactive peptides using an automated enzymatic membrane reactor

The utilization of bioactive peptides from food-grade raw materials has received increasing interest, especially for the production of functional foods. Within this study, a combination of fermentation and in vitro papain hydrolysis was devised to produce bioactive peptides exhibiting antioxidant property and angiotensin I-converting enzyme (AICE) inhibitory activity. The utilization of an automated membrane reactor system developed in this study could facilitate the continuous hydrolysis of tempe flour-rich in protein under constant flux, thus constant residence time operation. The optimum operating conditions were: [E]/[S] = 10% (w/v) and τ = 9 h. With a 10-kDa membrane filtration, the resulted antioxidant capacity and AICE inhibition were 0.033 mg AEAC/mL and 50.9%, respectively. Further separation of permeate with a 2-kDa membrane cut-off leveled off the antioxidant capacity but increased the AICE inhibitory activity (i.e., Reduction of IC₅₀ of ~40%). By this, the obtained IC₅₀ for AICE was 0.08 mg protein/mL, and this value was comparable with the IC₅₀ values reported in the literature.Industrial relevanceThe production of bioactive peptides from soybean through a combination of fermentation and in vitro enzymatic hydrolysis is limitedly reported. In this work, fermented soybean (tempe) was further hydrolyzed continuously using an automated membrane reactor. The hydrolysis under optimum operating conditions could increase the antioxidant activity and AICE inhibition of permeate. This work is expected to increase the commercialization of tempe-based food products by using safer technological approaches (i.e., fermentation and membrane technology).     

Conjugation of gluten hydrolysates with glucosamine at mild temperatures enhances antioxidant and antimicrobial properties

Gluten represents one of the principal by-products of the wheat starch industry. Peptides obtained by wheat hydrolysis can be used for specific functional and biological activities, albeit at relatively low yields. Although the Maillard reaction (glycation) is widely used to increase functionality of proteins, its main disadvantage is the production of undesirable compounds due to high processing temperature. In this research, functional and biologically active glycopeptides were obtained from gluten. Alcalase or Flavourzyme proteases were used to hydrolyse gluten protein, and the resulting peptides were conjugated with glucosamine by enzymatic glycosylation, using transglutaminase, or through glycation. Both reactions were performed at mild temperatures (25 or 37 °C). The formation of glycopeptides depended mostly on the glycation process, as demonstrated by MALDI-TOF-MS. The bioactivities of the conjugated hydrolysates were compared to the native hydrolysates. Although a reduction in the anti-ACE activity was detected, improved DPPH scavenging activity and enhanced antimicrobial activity against Escherichia coli were observed in the glycated Alcalase-derived hydrolysates and in the glycated Flavourzyme-derived hydrolysates, respectively. This study showed that mild conditions are an alternate approach to the traditional Maillard process conducted at elevated temperatures in creating conjugated gluten hydrolysates with enhanced bioactivities.     

Production of high degree polymerized chitooligosaccharides in a membrane reactor using purified chitosanase from Bacillus cereus

Crude chitosanase from Bacillus cereus NTU-FC-4 was separated by a cation exchanger to three fractions named CBCI, CBCII, and CBCIII. The CBCI hydrolyzed chitosan to yield dimers. The primary hydrolytic products of CBCII were low degree polymerized (DP) chitooligosaccharides. The CBCIII had the fastest reaction rate and yielded high DP chitooligosaccharides (heptamer and higher DP oligomers). When CBCIII was used in the ultrafiltration membrane reactor with enzyme/substrate ratio 0.06 unit/mg and 100 min of residence time (RT), the concentration of high DP oligomers was 9.78 mg/mL which occupied ca. 48% of total oligomers in the final product as compared to ca. 29% resulted from the crude enzyme. Decrease of RT to 50 min and 33 min, the high DP oligomers in the products were ca. 61% and 69%, respectively. This system could be operated for at least 24 h and kept a constant permeate flux and product output rate.     

Enzymatic hydrolysis of hard‐to‐cook bean (Phaseolus vulgaris L.) protein concentrates and its effects on biological and functional properties

Inadequate postharvest handling and storage under high temperature and relative humidity conditions produce the hard‐to‐cook (HTC) defect in beans. However, these can be raw material to produce hydrolysates with functional activities. Angiotensin I‐converting enzyme (ACE) inhibitory and antioxidant capacities were determined for extensively hydrolysed proteins of HTC bean produced with sequential systems Alcalase‐Flavourzyme (AF) and pepsin–pancreatin (Pep‐Pan) at 90 min ACE inhibition expressed as IC₅₀ values were 4.5 and 6.5 mg protein per mL with AF and Pep‐Pan, respectively. Antioxidant activity as Trolox equivalent antioxidant capacity (TEAC) was 8.1 mm  mg^?1 sample with AF and 6.4 mm  mg^?1 sample with Pep‐Pan. The peptides released from the protein during hydrolysis were responsible for the observed ACE inhibition and antioxidant activities. Nitrogen solubility, emulsifying capacity, emulsion stability, foaming capacity and foam stability were measured for limited hydrolysis produced with Flavourzyme and pancreatin at 15 min. The hydrolysates exhibited better functional properties than the protein concentrate.     

Structure–mechanism relationship of antioxidant and ACE I inhibitory peptides from wheat gluten hydrolysate fractionated by pH

The aims of this study were to assess bioactive properties (ACE inhibition and antioxidant capacity) from wheat gluten hydrolysate peptides fractionated by pH (4.0, 6.0 and 9.0), to determine peptide action mechanism, and to relate it to the secondary structure and functional groups of peptides. Gluten hydrolysate extracts (GHE) were enriched in peptides with medium hydrophobicity and molecular weight (≈ 60% MH and 5.5 kDa, respectively). Gluten peptides inhibited ACE I by uncompetitive mechanism and a direct relationship between α-helix structure and IC50% value was obtained (r = 0.9127). TEAC and cooper chelating activity from GHE 6.5 were the highest and directly correlated with MH peptides. GHE 9.0 had high carotene bleaching inhibition (47.5 ± 0.3%) and reducing power activity (163.1 ± 2.9 mg S₂O₃^2 − equivalent g^− 1 protein), which were directly related to disulfide bonds content of peptides (r = 0.9982 and 0.9216, respectively). pH was a good alternative to select bioactive peptides from wheat gluten hydrolysate.     

USE OF IMMOBILIZED LACTASE IN PROCESSING CHEESE WHEY ULTRAFILTRATE

Enzymatic hydrolysis of lactose in cottage cheese whey ultrafiltrate was investigated. Lactase of A. niger was immobilized on an alumina-silica catalyst support by the linking agent tolylene-2, 4-diisocyanate. The resulting immobilized enzyme preparation had an activity of 3 standard international units of lactase per gram at pH 4 and 37 °C. The optimum pH and temperature for hydrolysis of lactose by immobilized lactase were 3.5 and 50°C, respectively. Immobilization of the enzyme resulted in reductions of 1.1 pH units and 15°C in optimum pH and temperature, respectively. The two constants of the simple Michaelis-Menten rate expression were obtained from Lineweaver-Burk plots of the initial reaction rate data obtained at 37°C. Estimated values for V_max and the apparent K_m were 7.8 (μmoles/min-g) and 0.26 (M), respectively. Inhibition by the product galactose was measured by studying the hydrolysis reaction in a batch reactor. The inhibition constant K_i was estimated from batch reactor data to be 0.005 and 0.053 (M) at 35 and 50°C, respectively. Activation energies of 8.1 and 6.4 (kcal/gmole) were obtained for the immobilized and soluble enzyme reactions, respectively. The behavior of the batch reactor as measured in terms of a plot of conversion versus time was essentially the same for both conventional and deionized whey ultrafiltrate.     

A study on malt modification,used as a tool to reduce levels of beer hordeins

Storage proteins from barley, wheat and rye are toxic to gluten sensitive consumers. These consumers include those suffering from coeliac disease, which account for up to 1% of the global population, and non‐coeliac gluten sensitivity that may affect even greater numbers of the population. Codex Alimentarius has published guidelines and limits of gluten in gluten‐free foods, which are applied in Europe, and similar guidelines apply in the rest of the world. The storage proteins present in barley are hordeins. These proteins are broken down and used by the plant as a source of amino acids during germination and growth of the barley embryo. The objective of this study was to extend the germination stage of the malting process and look at the effect on beer hordeins. Standard MEBAK methods were used to develop an extended malting process and produce three different malts, germinated for 3, 5 or 7 days. The quality of malt was assessed and model beers were produced from each malt to test the effect of modification on levels of beer hordeins. Malt germinated for 7 days produced beer 18 mg/kg hordeins corresponding to a reduction of 44% compared with the beer made from malt germinated for 3 days characterized by a hordein content equal to 32 mg/kg. The malting loss was increased during the 7 days of germination but otherwise all malts were of high quality. The results showed that malting conditions have a significant impact on beer hordeins. Copyright © 2018 The Institute of Brewing & Distilling     

Simultaneous rice bran oil stabilization and extraction using sub-critical water medium

Sub-critical water technique was used for simultaneous inactivation of lipase enzyme existing in rice bran and extraction of its oil in order to obtain the stabilized edible rice bran oil. Sub-critical water treatment was carried out in the temperature range between 120 and 240 °C for 10 and/or 20 min residence time in a batch reactor. The quality of the extracted oil was evaluated with respect to its total free fatty acids concentration over a 12 week period, and compared with the oil obtained by conventional extraction methods. Without sub-critical water treatment, the concentration of total free fatty acids in the rice bran significantly increased from 5.6% to 36.0%. In contrast, no increase was observed in the total free fatty acids concentration in the samples treated by sub-critical water. Experimental evidence showed that total free fatty acids concentration increased somewhat in the oils treated by conventional methods. Considering no change was observed in total free fatty acid concentration in the treated oils by sub-critical water, it was found that sub-critical water not only could efficiently extract oil from rice bran in a short residence time but also completely stabilized the extracted oil. Oil extraction yields generally increased with increases in sub-critical water treatment temperature and residence time. The highest extraction yield of oil was 249 (mg/g dry matter) obtained at 240 °C and 10 min residence time. Oil extraction by sub-critical water could be conducted in a very short residence time (10 and/or 20 min). Also, the kinetics of free fatty acids formation in untreated rice bran was investigated and developed which successfully describes the concentration of total free fatty acids in the course of rice bran storage.     

Preparation and Characterization of Hydrolyzed Proteins from Defibrinated Bovine Plasma

ABSTRACT: Proteins from defibrinated bovine blood plasma were enzymatically hydrolyzed with food-grade microbial proteases Alcalase 2.4 L® and Flavourzyme L&TM;, and a substrate consisting of small peptides and free amino acids was obtained. Hydrolysis of the plasma proteins with Flavourzyme resulted in a maximum degree of hydrolysis of 43% at an enzyme concentration of 110 LAPU/g protein after 15.5 h of hydrolysis. Among the free amino acids in the hydrolysate, hydrophobic amino acids were predominant. The major plasma proteins were degraded due to hydrolysis; peptides of less than 1.04 kDa were dominant in the product when a high degree of hydrolysis was employed.     

Is the calculation of the gluten content by multiplying the prolamin content by a factor of 2 valid?

Presently, the only essential therapy of celiac disease (CD) is the permanent strict withdrawal of gluten from the diet. With respect to gluten containing foods from wheat, rye, barley, and oats, CD patients have to consume surrogates that must be gluten-free according to the “Codex Alimentarius Standard for Gluten-Free Foods”. The recent “Draft Revised Standard” proposes a gluten threshold of 20 mg per kg gluten-free product. For gluten quantitation, the alcohol-soluble prolamins should be extracted and analyzed by an immunochemical method; the amount of gluten is calculated by multiplying the prolamin content by the factor of 2. To investigate, whether this calculation is valid in any case of contamination of gluten-free products by wheat, rye, barley, and oats, wholemeal or white flours from common wheat, spelt, durum wheat, kamut, emmer, einkorn, rye, barley and oats were analyzed for the ratio of prolamins to glutenins (PROL/GLUT) by a combination of extraction and reversed-phase HPLC procedures. Additionally, different industrial wheat starches were analyzed for their prolamin and total gluten content using different extraction and concentration steps followed by gel permeation HPLC. The results for the cereal flours revealed that the ratio PROL/GLUT was generally higher than 1.0 as proposed by the “Draft Revised Standard” and strongly influenced by cereal species and variety. Common wheat showed the lowest ratio (1.5–3.1), followed by oats and spelt (1.7–3.3), barley (1.4–5.0), durum wheat and kamut (3.1–3.4), emmer (3.5–7.6), rye (6.3–8.2), and einkorn (4.0–13.9). In any case, the gluten content of gluten-free products contaminated with CD activating cereals was generally overestimated, when the prolamin content was multiplied by the factor of 2. In extreme cases, e.g., contamination with rye, the overestimation amounted to 72–79%. Completely different PROL/GLUT ratios were found in ten commercial wheat starches ranging from 0.2 to 4.9. Obviously, the quality of wheat cultivars used for starch production and/or different process parameters, e.g., washing steps, influenced the composition of gluten proteins adherent to starch granules. For wheat starch, the calculation of the gluten content by 2 × PROL may either lead to underestimation (−71% at most) or overestimation (+66% at most). In conclusion, this calculation is invalid; therefore, a future task will be the development of immunoassays with antibodies against all types of storage proteins from wheat, rye, barley, and oats.     

Process optimisation and physicochemical characterisation of enzymatic hydrolysates of proteins from co‐products of Atlantic Salmon (Salmo salar) and Yellowtail Kingfish (Seriola lalandi)

The aim of this study was to develop an enzymatic hydrolysis process of protein co‐products for two major commercial fish species in Australia: Atlantic salmon (AS) and Yellowtail kingfish (YTK). The outcomes are to produce high protein recovery of fish protein hydrolysates within controlled molecular weight ranges that display enhanced physicochemical properties of oil binding and emulsification. Three enzymes (Flavourzyme, Neutrase and Alcalase) were applied to processing co‐products. Protein recovery and physicochemical properties were evaluated with increasing hydrolysis time from 30 min to 180 min and ratio of enzyme to substrate (E/S) from 0.5% to 3.0%. In order to achieve a product with optimum emulsifying capacity (50 ± 0.6 m² g^?1), an E/S ratio of 0.6–1.3% Flavourzyme was applied for 30–111 min with a protein recovery of 55%; in order to achieve a product with optimum oil‐binding capacity (8.3 ± 0.3 g oil g hydrolysates^?1), an E/S ratio of 2.3–3.0% Flavourzyme was applied for 25–64 min with a protein recovery of 70%. YTK protein hydrolysates were further membrane‐fractionated into five fractions (>100 kDa, 50–100 kDa, 30–50 kDa, 10–30 kDa and <10 kDa), and of these, the 10–30 kDa exhibited the best properties of oil binding (19 ± 0.3 g oil g hydrolysates^?1) and emulsification (57 ± 0.7 m² g^?1). These results demonstrate the importance of enzymatic hydrolysis of seafood co‐products into high‐value ingredients for food products and processing.     

Variation in gluten protein and peptide concentrations in Belgian barley malt beers

Gluten is the main family of storage proteins found in barley. During malting and brewing, some of the barley malt's proteinaceous material is hydrolysed into peptides or to amino acids. Most of the gluten proteins are removed with the spent grains and with hot‐ and cold‐breaks. However, some gluten proteins and especially gluten‐derived peptides can remain throughout the brewing process and will hamper the gluten‐free (≤20 ppm) status of the beer. In this work, three production batches (a, b and c) of 51 Belgian barley malt beers from 24 breweries were analysed with the sandwich (R7001) and competitive (R7021) Ridascreen gliadin R5‐ELISA to quantify gluten proteins and peptides. Although the majority of the beers contained low‐gluten protein concentrations of ≤20 ppm (a/45, b/47, c/48), only a minority were truly gluten‐free with ≤20 ppm gluten peptides (a/18, b/17, c/15). The grain bill had no influence on the measured gluten concentration, but the use of (combined) clarification techniques and presence of wheat malt in the grist was respectively a positive and negative influence. Ten beers, from four breweries, were gluten free in all analysed samples. These included two wheat beers, reflecting the importance of effective clarification in the management of gluten. These results explore the feasibility of the production of gluten‐free barley malt beers. Copyright © 2018 The Institute of Brewing & Distilling     

Angiotensin I-converting enzyme inhibitory properties of lentil protein hydrolysates: Determination of the kinetics of inhibition

ACE inhibitory activity was studied for different hydrolysates obtained from protein concentrates of two lentil varieties by in vitro gastrointestinal simulation, Alcalase/Flavourzyme, papain and bromelain. Protein/peptide profiles studied by electrophoresis and HPLC-SEC showed a rich composition of the hydrolysates in small peptides ranging in size from 0.244 to 1.06 kDa. ACE inhibitory activity was measured using the HPLC Hippuryl-His-Leu (HHL) substrate method. Significantly different (P < 0.05) IC₅₀ values ranging between 0.053 and 0.190 mg/ml were obtained for different hydrolysates. Furthermore, the inhibition mechanism investigated using Lineweaver–Burk plots revealed a non-competitive inhibition of ACE with inhibitor constants (K_i) between 0.16 and 0.46 mg/ml. These results demonstrate that hydrolysates of lentil proteins obtained by different enzymatic digestions may contain bioactive components.     

Production of wheat gluten hydrolyzates by enzymatic process at high pressure

A novel process for producing wheat gluten enzyme hydrolyzates (WGEHs) was developed, using combinations of Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E at the high pressure of 300 MPa, and the resultant hydrolyzates were analyzed for electrophoretic and hydrolytic properties. It was found that multiple-enzyme treatments increased the proportion of the electrophoretic bands less than 5 kDa in the hydrolyzates greatly both at ambient pressure and 300 MPa compared with one-enzyme hydrolysis. The contents of total soluble solids in the WGEHs increased considerably up to 89.75% according to the increase in the number of enzymes used at 300 MPa compared with 79.37% found for the ambient-pressure hydrolysis. These characteristics together with the contents of soluble nitrogen and free amino acids clearly indicated that the high-pressure enzymatic process of this study is an efficient method for obtaining WGEHs with increased degree of hydrolysis.     

Continuous production of wheat gluten peptide with foaming properties using immobilized enzymes

Gluten peptides are prepared using limited hydrolysis of wheat gluten, resulting in improved solubility and physico-chemical properties. In general, the hydrolysis is performed using a batch-wise process. In this study, we developed a bioreactor system that can continuously produce gluten peptides with foaming properties. Two kinds of acid protease, pepsin and rapidase, were immobilized on porous chitosan beads. The partial deamidation of gluten in advance increased initial velocity of hydrolysis by immobilizing protease and also worked to enhance foaming properties. A packed-bed reactor filled with immobilized protease was designed, and operating parameters were optimized. The optimized conditions were as follows: pH, 3.0; temperature, 40 °C; substrate concentration, 40 mg/ml; and space velocity, 2.0 h^–1. Based on these results, a bench-scale reactor was manufactured to determine the stability of continuous operation. The half-life of the reactor was approximately 45 days, and both productivity and quality were stable and excellent.