Preparation and nutritional characteristics of a hydrolysate from pepitona (Arca zebra)

Two soluble products resulting from the hydrolysis of pepitona (Arca zebra) were prepared as flour. Papain at its optimum hydrolysis conditions, previously established, was the enzyme used (40 degrees C for two hours at a pH of 7 in the proportion of 0.3% weight/enzyme/100 g meat). The hydrolysate obtained was then subjected to two different dehydration techniques: drum drying at 121 degrees C and 18 seconds retention, and spray drying at 101 degrees C and 40 psi pressure. The products were then stored for a five-month period at a temperature of 25 degrees C +/- 2 degrees C, time during which chemical determinations were performed in both hydrolysates. Findings showed that the time of storage does exert a significant effect of deterioration on the products. The greater and more significant quality losses occur during the first two months. The dehydration techniques used also affect significantly the soluble nitrogen content, and non-protein nitrogen and soluble solids content, as well as color of pepitona hydrolysates. Spray-drying dehydration technique does not have a significant deteriorating effect. Biological studies undertaken demonstrated that the quality of both hydrolysates is satisfactory from the nutritional and amino acid composition points of view. A protein efficiency ratio (PER) of 2.27 and 2.29 was determined for the hydrolysate dehydrated by drum drier and for the dehydrated by spray drier, respectively. With regard to amino acid composition, both had satisfactory levels of essential amino acids, with a lysine content of 6.9 g/100 g protein for the hydrolysate dehydrated by drum drying, and 8.6 g/100 g protein for the other hydrolysate dehydrated by spray drying.     

Lipase activity and fatty acid typoselectivities of plant extracts in hydrolysis and interesterification

Lipase fatty acid typoselectivities of Euphorbia characias latex and commercially available crude preparation of bromelain were determined in the hydrolysis of homogeneous triacylglycerols (TAG) and natural TAG mixtures. Their activities were compared to a commercially available crude preparation of papain. Under optimal lipolysis conditions at pH 8.0 and 10 min of incubation time, maximal activities were observed at 45, 55, and 50°C, respectively, for E. characias latex, crude bromelain, and crude papain. Commercially available crude preparations of bromelain exhibited very poor hydrolysis activity. Latex from E. characias, which contained 340 mg of dried material per milliliter of fresh latex, exhibited a high lipase activity and a short-chain fatty acid preference in the hydrolysis of homogeneous TAG. For all substrates, it showed a better activity than crude papain. Lipase fatty acid typoselectivities of crude bromelain and crude papain also were studied in interesterification reactions of tributyrin with a series of homogeneous TAG. Experiments showed that crude bromelain [water activity (A _w )∶ 0.21] had no activity in interesterification. Regarding reactions with crude papain (A _w ∶ 0.55), yields of newly formed TAG decreased with increasing chain length of TAG, except for the reaction with trimargarin. For interesterification of tributyrin with unsaturated TAG, triolein reacted faster than polyunsaturated TAG. During these interesterification reactions, the proportion of new TAG with two butyroyl residues was higher than new TAG with only one butyroyl residue. This phenomenon was more pronounced for reactions with long-chain TAG.     

Enzymatic hydrolysis of rawhide using papain and neutrase

Rawhide split was hydrolysed separately by two proteolytic enzymes, papain and neutrase. The effects of enzymatic conditions of the hydrolysis reaction were investigated. During the first 10 min of the enzymatic hydrolysis, the yield of the hydrolysed protein increased sharply, then it slowly increased or became essentially constant due to the limited availability of the substrate. The optimum hydrolysis conditions of papain and neutrase for highest protein yield are at 70 °C, pH 6–7 and 40–50 °C, pH 6–7, respectively. The product from papain hydrolysis is a gelatin with low gel strength and viscosity, while that from neutrase hydrolysis is collagen hydrolysate with viscosity as low as water. This is considered to indicate that longer fragments of protein are obtained from papain hydrolysis than that from neutrase implying different mechanisms of papain and neutrase hydrolysis.     

青霉素菌丝中蛋白质酶法水解工艺

采用碱热法溶解青霉素菌丝,研究酶法催化水解菌丝溶解液中蛋白质转化为氨基酸的过程.考察酶种类、溶解液pH值、酶与蛋白质质量比、反应温度和时间等因素对蛋白质水解度的影响,建立最佳水解工艺.结果 表明,酶种类、酶与蛋白质之比、pH、温度和时间均对蛋白质水解过程产生影响.以单酶为催化剂,碱性蛋白酶催化水解效果最好,蛋白质水解度...     

Hydrolysis of raw hide using proteolytic enzyme extracted from papaya latex

Crude proteolytic enzyme was extracted from papaya latex using two solvents, water and phosphate buffer pH 6. The yield of extracted enzyme using water as a solvent was similar to that using phosphate buffer. Following the solvent extraction, the extracted enzyme was precipitated in 45 wt% saturated ammonium sulfate solution. The yield and activity of precipitated enzyme considerably decreased. Crude proteolytic enzyme extracted using water as an extracting liquid was, therefore, selected to use in gelatin production from raw hide hydrolysis, comparing to the use of commercial papain. The effects of hydrolysis conditions on gelatin recovery and properties of obtained gelatin were investigated. The optimum conditions for the activities of both crude extracted enzyme and commercial papain were at 75 °C and pH 7. At this condition, the highest percentages of gelatin recovery were obtained from raw hide hydrolysis reactions. The gelatin recovery and gel strength of gelatin obtained from crude extracted enzyme and commercial papain hydrolysis were similar. This proved that crude extracted enzyme from papaya latex could be effectively used in gelatin production, instead of the use of commercial papain, with a comparatively low cost.     

Enzymatic modification of sesame seed protein,sourced from waste resource for nutraceutical application

The functional characteristics which include protein solubility at different pH, emulsifying and foaming properties, degree of hydrolysis, molecular weight distribution, antioxidant and ACE inhibitory activity of sesame protein hydrolysates prepared with pepsin, papain and alcalase enzymes were evaluated. The rate of degree of hydrolysis was found to reach maximum (25–30%) within the first time fragment i.e 10 min but 80% of hydrolysis was obtained in 120 min with alcalase. SDS-PAGE of hydrolysates with papain, pepsin and alcalase evinced bands of low molecular weight protein of 14.3 kDa and even lower for alcalase treatment of 120 min. Hydrolysates so formed were of improved functional properties as evident from emulsifying and foaming property. Hydrolysis with different proteases enhanced the protein solubility significantly at pH 7.0. Antioxidative assay revealed radical scavenging activity of the hydrolysates with papain hydrolysates showing maximum antioxidative efficacy. The ultra-filtered peptide fractions which showed comparable ACE inhibitory activity were sequenced by MALDI-TOF and matched to that of previously identified ACE inhibitory peptides. The results corroborate the ACE inhibitory effect of the peptides. Hence, these highly bioactive protein hydrolysates produced from waste sesame meals can be successfully employed in various functional food formulations.     

Synthesis of 7-amino-4-methylcoumarin (AMC) derivatives and their hydrolysis by plant cysteine proteinases

A number of derivatives of citrullyl-7-amino-4-methylcoumarin (Cit-AMC) have been synthesised and characterised. Their hydrolysis, releasing the fluorescent 7-amino-4-methylcoumarin, by the plant cysteine proteinases papain, ficin and bromelain was examined. Two derivatives in particular, Z-Phe-Cit-AMC and Z-Gly-Phe-Cit-AMC, were shown to be good substrates for these enzymes. The possible use of the fluorogenic peptides in assays for these enzymes is discussed.     

Analysis of products, mechanisms of reaction, and some functional properties of soy protein hydrolysates

Soybean protein isolates were hydrolyzed with papain, bromelain, cucurbita, hieronymin, and pomiferin. The highest hydrolysis rate for cucurbita and the lowest for papain was detected at 720 min. Gel filtration, reversed-phase liquid chromatography, and electrophoresis showed that the action of each protease was different. Pomiferin acted on the native structure of β-conglycinin and glycinin, generating a large number of small hydrolysis products with hydrophilic and hydrophobic characteristics. The hydrolysates obtained with cucurbita showed residual structures that were almost intact and very similar to the substrate and contained a low number of small peptides. The hydrolyzates obtained with papain, bromelain, and hieronymin had hydrolysis products with structures similar to the partially hydrolyzed native isolate. The molecular masses of these products were similar to or lower than the controls. Polypeptides of low molecular mass were also detected. The prevalence of one-by-one and zipper mechanisms was suggested for cucurbita and pomiferin, respectively. For the other proteases both mechanisms were likely to coexist. The solubility of hydrolysates and their ability to form and stabilize foams correlated well with the structural properties and with the suggested mechanisms of hydrolysis. The best properties were presented by the hydrolysates prepared with cucurbita. Foaming ability for pomiferin hydrolysates was as high as that for unhydrolyzed soy isolate, but the foams were extremely unstable.     

Biological quality of the protein isolated from the leaves of Atriplex numularia

Plant leaf proteins have acquired great relevance during the last two decades because of their well-balanced amino acid composition. A study was therefore undertaken to evaluate the biological quality of the leaf protein of Atriplex numularia. The protein content of the fresh leaves from this plant was found to be 4.70 g/100, with a dry matter content of 18.70 g/100 g. A protein concentrate (PC) from the same material was then obtained by macerating the leaf in a 2% sodium sulfite solution at a pH of 10 and subjecting it to filtration and pressing. The product thus obtained had a dark greenish color and contained 55.42 g/100 g of protein. The amino acid analysis revealed that its protein has a balance similar to that of animal origin proteins, with a lysine and methionine content of 8.5 g/16 g N and 3.0 g/16 g N, respectively. Biological assays were then carried out to evaluate nitrogen utilization, with the following resulting values: net protein utilization (NPU) = 48.3 +/- 2.7; digestibility (D) = 58.0 +/- 1.4, and biological value (BV) = 83. As inferred from the NPU value, nitrogen utilization was low. Therefore, to improve digestibility values, the action of papain on the PC was assayed. The material thus treated was again submitted to biological trials, obtaining, this time a D of 75.4 +/- 1.05, and thus, an improved new NPU value of 54.8 +/- 1.1 (p less than 0.01).     

复合酶制备牛皮胶原多肽

以自制的牛皮胶原蛋白为原料,探讨复合酶制备牛皮胶原多肽的工艺。以牛皮胶原蛋白的水解度为指标,确定木瓜蛋白酶和胰蛋白酶的添加顺序和添加比例,同时单因素法确定酶解工艺参数的优化区间,响应面法优化复合酶酶法制备胶原多肽的工艺参数。实验结果表明,从牛皮胶原蛋白中制备胶原多肽的最优条件为:木瓜蛋白酶和胰蛋白酶按酶活力比3∶1同时添加,加酶量650 U/mL,酶解介质pH=7.0,酶解温度50℃,酶解时间7h,在该工艺条件下得到的牛皮胶原蛋白的水解度可达32.02%。     

利用水解酶提取文冠果油脂

采用3种酶对文冠果种仁进行水酶法提油,其中Alcalase 2.4 L蛋白酶的效果最好. 在Alcalase 2.4 L蛋白酶用量为0.02 mL/g的条件下,通过单因素实验和Box-Behnken中心组合实验,应用SAS软件分析得出水酶法提取文冠果油脂的最佳工艺条件为：温度55℃, pH 9, 固液比1 g:6 mL, 水解时间4 h,在此条件下油脂提取率和蛋白水解度分别达到78.67%和9.15%.     

Sesame cake protein hydrolysis by alcalase: Effects of process parameters on hydrolysis, solubilisation, and enzyme inactivation

We investigated the effects of process parameters (substrate concentration, enzyme concentration, temperature and pH) on the hydrolysis and solubilization of sesame cake protein as well as enzyme stability. The sesame cake protein was hydrolyzed by Alcalase enzyme (a bacterial protease produced by a selected strain of Bacillus Licheniformis) that was chosen among five commercial enzymes examined. The optimum process conditions for hydrolysis and solubilization were obtained as 15 g L^?1 substrate concentration, 3 ml L^?1 enzyme concentration, 50 °C and pH 8.5. Under these conditions, the values of degree of hydrolysis and solubilization were found as 26.3% and 82.1%, respectively, and enzyme lost its activity by approx. 56% at the end of 120 min processing time. Modeling studies were performed to determine the kinetics of hydrolysis, solubilization and enzyme inactivation. The relationship between hydrolysis and solubilization was found linear for all experimental conditions examined. The inactivation energy of Alcalase at the temperature range of 45–55 °C was determined to be 25544 J mol^?1.     

Chemical modification and immobilization of papain

Papain, an endolytic cysteine protease (EC: 3.4.22.2), from Carica papaya latex has been chemically modified using succinic anhydride. This reagent reacts with the amino group of the lysine residues in the enzyme, thereby changing its net charge from positive to negative. The resultant enzyme had its optimum pH shifted from pH 6 to 8 and there was no change in the temperature optima of 70 °C. The modified papain had a specific activity of about 62.8 IU mg^?1 of protein at pH 8.0 at 30 °C, whereas for the native enzyme it was 46.57 IU mg^?1 under same conditions. Stability of the modified papain was further increased by entrapping in alginate/starch beads. The immobilized papain retained its activity even after six cycles of hydrolysis. The wet beads, when dried at 50 ± 2 °C, increased the storage stability of the immobilized enzyme. The succinylated papain is active in various organic solvents and hence can be successfully used in biotransformations as well as being used as a proteolytic component in detergents. Copyright © 2004 Society of Chemical Industry     

Rapid enzymatic method for partial hydolysis of oilseed proteins for food uses

Plant enzymes were used to obtain partially hy-drolyzed oilseed proteins. Hydrolysis of oilseed pro-teins under different conditions of type of enzyme, enzyme concentration, temperature, and reaction time was investigated to develop optimum conditions. Pilot plant-produced defatted peanut flour treated with a 0.5% aqueous solution of papain at 45 C yielded a product that had 50% more soluble protein but with no increase in the microbial count of the original flour. Hydrolysis reached equilibrium in 10-15 min. Because of the mild reaction conditions of the method, enzymatic hydrolysis offers major advan-tages over nonenzymatic methods for modifying plant proteins for incorporation in specialty food products.     

Antioxidative peptides derived from enzyme hydrolysis of bone collagen after microwave assisted Acid pre-treatment and nitrogen protection

This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid). The highest degree of hydrolysis (DH) was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain), with an optimum condition of: (1) ratio of enzyme and substrate, 4760 U/g; (2) concentration of substrate, 4%; (3) reaction temperature, 55 °C and (4) pH 7.0. At 4 h, DH increased significantly (P < 0.01) under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen.     

Optimization of Enzymatic Process Condition for Protein Enrichment,Sugar Recovery and Digestibility Improvement of Soy Flour

Soy protein is a valuable nutritional supplement for food and animal feed. While protein constitutes ~50 % of defatted soy flour (SF), it coexists with complex carbohydrates (30–35 %) which may have anti‐nutritional effects. An enzymatic process can remove the carbohydrate and produce protein‐enriched soy products. The hydrolysate with monomerized carbohydrates is valuable fermentation feedstock. In this study, Aspergillus niger and Trichoderma reesei enzymes were compared for use in the process. Effects of pH (3.2–6.4), temperature (40–60 °C), enzyme‐to‐SF ratio (0–2 ml/g) and SF loading (150–350 g/l) were evaluated for the enzymatic conversion of SF carbohydrate to reducing sugar (Y_RS) and total soluble carbohydrate (Y_TC) in the hydrolysate. Effects of these single factors and interactions between factors were investigated. Optimal pH and temperature were similar for both enzymes: pH 4.8 and 50–51 °C for Y_TC, and pH 5.1–5.2 and 48–51 °C for Y_RS. The two enzymes also gave similar protein contents in resultant soy protein concentrates, i.e., 74–75 % with 2 ml/g enzyme broth and 150 g/l SF, which were higher than the 64–68 % protein in commercial concentrates. A. niger enzyme was significantly more effective in carbohydrate conversion, achieving Y_RS = 75 % and Y_TC = 78 % with 2 ml/g enzyme and 150 g/l SF, higher than the Y_RS (30 %) and Y_TC (64 %) obtained with T. reesei enzyme. Monomerization was essentially complete in hydrolysate produced with A. niger enzyme.     

Pigment removal from canola oil using chlorophyllase

Frost-damaged or prematurely harvested canola seed (rapeseed) may yield oil with a high chlorophyll content (50–60 μg/ml). Enzymatic hydrolysis of chlorophyll, added to buffer/surfactant, buffer/acetone or buffer/acetone/canola oil, to produce water-soluble chlorophyllide (green pigment) was studied using a crude chlorophyllase preparation (acetone-dried chloroplasts) from 15 to 20-day-old sugar beet seedlings. In buffer/surfactant, the optimum pH for enzyme activity was temperature dependent. At 30 C and 0.24% Triton X-100 (or 30% acetone), chlorophyllase showed maximum activity toward a crude chlorophyll preparation over the range of pH 8–10. At 60 C, the activity was more than twofold higher, with a sharp maximum at ∼pH 8. Mg²⁺ enhanced the activity with an optimal concentration of 50 mM. At pH 7.5, 50 C and in the presence of only 6% acetone, the enzyme showed high affinity for chlorophyll (Km=15μM or 13.5 μg/ml), suggesting that the natural chlorophyll concentrations found in green canola oils might facilitate high enzymatic efficiencies. The crude enzyme was stable in buffer/acetone at pH 7.5 and 50 C for at least two hr. With acetone concentrations as low as 6%, maximum enzyme activities in buffer and buffer/canola oil required intensive mixing (homogenization) of the various substrate, enzyme and liquid phases. In general, the rate and extent of chlorophyll hydrolysis were greater in buffer than in buffer/oil. In both reaction systems, chlorophyll hydrolysis slowed down with time due to accumulation of phytol, which proved to be a competitive inhibitor (K_i=11 μM or 3.3 μg/ml). The other hydrolysis product, chlorophyllide, did not affect enzymatic activity. Crude canola oil used in the reconstitution of green oil did not support enzymatic chlorophyll hydrolysis without prior degumming and desoaping. The optimum buffer/oil ratio of the reaction mixtures was above 2/1 (v/v).     

Papain Adsorption on Chitosan-Coated Nylon-Based Immobilized Metal Ion (Cu2+, Ni2+, Zn2+, Co2+) Affinity Membranes

A novel immobilized metal affinity membrane was prepared for papain adsorption in this article. Higher papain adsorption capacity between 43-67 mg/g was observed and the adsorption isotherm fitted the Freundlich equation. Experimental data were analyzed using two adsorption kinetic models. The pseudo-second-order kinetic model provided better correlation to the experimental results. A significant amount of the adsorbed papain was eluted by 1.0 M NaSCN at pH 5.0 for all affinity membranes. It was concluded that the novel chitosan-coated nylon-based immobilized metal ion affinity membrane could be applied for the large-scale isolation of papain without resulting in enzyme denaturation.     

Retinol inhibition of some proteolytic enzymes

Automated analyses were used to determine the effect of retinol on the activity of the following proteolytic enzymes: ficin (EC 3.4.4.12), bromelain (EC 3.4.4. 24), trypsin (EC 3.4.4.4.), chymotrypsin A (EC 3.4.4.5), papain (EC 3.4.4.10), clostridiopeptidase A (EC 3.4.4.19), pepsin (EC 3.4.4.1), cathepsin D (EC 3.4.4. 23) from rat-liver and rat-kidney lysosomes and the nonspecific proteolytic enzyme, pronase. Of these proteolytic enzymes only ficin, bromelain, and rat-kidney lysosomal cathepsin D were inhibited significantly by 1×10⁻⁴ M retinol. Some nonproteolytic enzymes not inhibited by retinol were acid phosphatase (EC 3.1.3.2), β-acetylglucosaminidase (EC 3.2.1.30), arylsulfatase (EC 3.1.6.1), and pyruvate kinase (EC 2.7.1.40). The inhibition of cathepsin D varied with the substrate used, being greater with hemoglobin than with ovalbumin or bovine serum albumin. Carotene and retinol inhibited ficin and cathepsin D to similar extents. Retinol inhibition of ficin was partially reversible. These studies of proteolytic enzyme inhibition by retinol serve as a simple model for studying retinol-protein interactions in vitro.     

A Kinetic Study on Corn Gluten Hydrolysis

In the present study, the hydrolysis of corn gluten was performed by using two commercial enzymes, Protamex and PTN, to investigate the reaction kinetics. The reactions were carried out for 10 min in 0.1 L of aqueous solutions containing 10, 20, 30 and 40 g protein L^–1 corn gluten at various temperature and pH values. The progress of the reactions was monitored by a pH‐stat method. It was found that the kinetics of corn gluten hydrolysis by Protamex and PTN obey the Michaelis‐Menten model and higher catalytic yields were obtained with Protamex than PTN at all of the experimental conditions applied.