Collagenous hydrolysates from untraditional sources of proteins

Synopsis Sufficiently pure collagenous hydrolysates, suitable for application in skin and hair care cosmetics, have been prepared through biotechnological methods with the use of commercially available enzymatic preparations from short cattle tendons (musculus extensor communis, musculus flexor digitorum, musculus flexor digitorum profundis). These hydrolysates contain neither lipoid nor aminosaccharide components, content of primary amino groups reaches around 1.1 mmol g⁻¹ and the average molecular weight of the resulting collagenous hydrolysates does not exceed 2000 g mol⁻¹ (2.0 kDa). Short cattle tendons represent a relatively pure and easily available source of collagens and are, despite their generally known low nutritional value, used only as a feeding mixture component.     

Optimisation of the medium composition for production of protease and soybean peptides by Bacillus subtilis SHZ using response surface methodology

Responses surface methodology was employed to enhance the production of protease and soybean peptides by Bacillus subtilis SHZ. For screening of medium composition significantly influencing protease and soybean peptides yield, the two-level Plackett–Burman design was used. Among thirteen variables tested; KH₂PO₄, glucose and defatted soybean flour (DSF) were selected based on their high significant effect on both protease activity and soybean peptides yield. Then, a three-level Box–Behnken design was employed to optimise the medium composition for the production of the protease and soybean peptides in submerged fermentation. Mathematical models were then developed to show the effect of each medium composition and their interactions on the production of protease and soybean peptides. The model estimated that, the maximal protease activity (320 ± 1 U mL⁻¹) could be obtained when the concentrations of glucose, KH₂PO₄, DSF were set at 8–9 g L⁻¹, 2–3 g L⁻¹, 55–65 g L⁻¹, respectively; while a maximal yield of soybean peptides (8.5 ± 0.1 g L⁻¹) could be achieved when the concentrations of glucose, KH₂PO₄, DSF were set at 7–9 g L⁻¹, 3–4 g L⁻¹ and 55–58 g L⁻¹, respectively. These predicted values were also verified by validation experiments.     

绿豆蛋白酶解物抗氧化活性与其结构、氨基酸组成的相关性

本试验以碱性蛋白酶、中性蛋白酶、胃蛋白酶及胰蛋白酶四种不同来源的蛋白酶酶解绿豆蛋白,测定酶解物的抗氧化能力,结合傅里叶红外光谱技术（FTIR）、聚丙烯酰胺凝胶电泳（SDS-PAGE）、圆二色谱（CD）以及氨基酸分析仪,分析酶解物的氨基酸组成以及抗氧化活性与结构变化的相关性。结果表明,绿豆蛋白4 h中性蛋白酶酶解物抗氧化能力最强,其DPPH自由基清除率、羟自由基清除率分别达到71.03%、51.94%,TBARS值达到0.4045 mg/L,Fe2+螯合率达到52.31%;结合SDS-PAGE、FTIR、CD等分析得出:绿豆蛋白酶解物的抗氧化能力与其分子量大小、二级结构构象及氨基酸组成变化紧密相关,与绿豆蛋白相比,绿豆蛋白酶解物的α-螺旋结构含量增加了4.12%、β-折叠结构含量降低了19.99%,而抗氧化活性与其α-螺旋含量的增加、β-折叠含量的降低密切相关;影响抗氧化活性的疏水氨基酸增加了0.208 g/100 g,芳香氨基酸增加了0.207 g/100 g,分子量低于10 kDa的小分子量酶解物具有更好的抗氧化性。综合以上研究结果证实绿豆蛋白酶解物活性与其α-螺旋、β-折叠的相关性较大。     

Determination of chloride, nitrate and calcium ions in sugar with ion selective-electrodes

Chloride, nitrate, and calcium ion-selective electrodes were tested and found satisfactory for the determination of the corresponding ions in highly refined white sugar, molasses and other impure sugar samples at different stages of sugar manufacturing or refining. The interferences by Br⁻ and I⁻ ions in Cl⁻ determination were eliminated by oxidation with 3 M HNO₃ and passing air to remove the free Br₂ and I₂ formed. The Cl⁻ concentration was then determined with a chloride-selective electrode against a calibration curve. Chloride should not be determined by ashing sugar, since this causes high losses of Cl⁻. It was found that NO₃⁻ and free Ca²⁺ ions could be determined directly in the sugar solution against corresponding calibration curves. Total calcium was determined after ashing of sugar samples. The ash was dissolved in 0.1 M HCl and passed through an anion exchange resin to remove PO³⁻₄ and SiO₂₋₃ ions, and Ca²⁺ was determined as before. Bound calcium was obtained by subtracting values of free from total calcium. Determination of bound calcium was useful to monitor the process of liming. For comparison, the three ions were also determined with acceptable precision without removing interfering ions using the method of standard addition and Gran's plot. The errors of determination for both direct and standard addition techniques were 3% for Cl⁻ and NO⁻₃ and 1–5% for Ca²⁺.     

EFFECT OF pH AND CATIONS ON THE THERMALLY INDUCED GELATION OF OVALBUMIN

The gelation of ovalbumin in the presence of salts containing Ca⁺² and Mg⁺² at various pH values was investigated. Characteristics of the gels produced were evaluated using penetration measurements on an Ottawa Texture Measuring System as well as dynamic rheology on a Bohlin VOR rheometer. Gel properties were related to cation binding (equilibrium dialysis) and to changes in protein structure (differential scanning calorimetry). Cation binding generally increased with increasing salt concentration, and Mg⁺² bound to a greater extent than Ca⁺² at the higher levels studied. The impact of this binding on gel structure was dependent on pH and the technique used to evaluate structure. At pH 5 proteins tended to coagulate regardless of the type or amount of salt. At pH 7, the highest rigidity values from penetration measurements were obtained at salt levels of 0.005 M. At pH 9, the salt concentration for maximum rigidity varied with the salt, while the storage moduli (G') from dynamic rheology were highest at the highest salt concentration used. Based on rigidity data, the inclusion of 0.005 M MgSo₄ would give a product with the greatest rigidity over a wide pH range.     

Preparation and characterisation of isoflavone aglycone‐rich calcium‐binding soy protein hydrolysates

Isoflavone aglycone‐rich calcium‐binding soy protein hydrolysates were prepared by subcritical water treatment and subsequent protease hydrolysis. Contaminated β‐glucosidase in the Protease M preparation could effectively convert glycosides into aglycones. Compared with Alcalase hydrolysates, Protease M hydrolysates exhibited higher molecular weight (>5000 Da) and more hydrophobic characteristics because of its weaker proteolytic activity. The antioxidant activity of Protease M hydrolysates was obviously improved. Initial increased DPPH and ABTS radical scavenging rate of Protease M hydrolysates may be ascribed to the conversion of isoflavones (<30 min) and a gradual release of antioxidant peptides. In the later hydrolysis, a gradual exposure of isoflavones involved in the interior of heat‐induced protein aggregates was mainly responsible for further improved antioxidant activities. Higher calcium‐binding capacity (up to 7.86%) with lower yield of peptide–calcium complex was observed for Protease M hydrolysates. These results could help researchers to develop a feasible protocol for producing nutrient‐enhanced soy protein hydrolysates.     

Chickpea, mungbean, cowpea and peanuts as substitutes for soybean curds

Acceptable beancurds could not be formed from chickpea, mungbean, cowpea and peanuts, when using the standard procedure used to make soybean curds. Firmness (rigidity) of the curd was proportional to the protein content; inversely proportional to the square of the starch content; and negatively correlated with the square of the fat content. Firmness also improved the taste of the curds. Satisfactory curds could be formed from legume sources other than soybean only after the partial removal of starch (in chickpea, mungbean and cowpea) or oil (in peanuts). Calcium sulphate was the best coagulant; optimum concentrations were 0.3% (0.02 M) of the milk; and optimal heating time was 15 min at 95°C. Stirring during coagulation aided formation of firmer gels. The rigidity moduli of the various curds were: soybean 51 000 N m⁻²; peanuts 2200 N m⁻²; chickpea 6000 N m⁻²; mungbean 11 000 N m⁻²; and, cowpea 29 000 N m⁻². Chickpea and mungbean curds were rated as liked (2.0 and 1.8 respectively), almost as well as soybean curds (1.5); cowpea (3.1) and peanut (4.2) curds were less preferred.     

PURIFICATION AND PROPERTIES OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE FROM CORIANDER (CORIANDRUM SATIVUM) LEAVES

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP⁺ oxidoreductase, EC 1.1.1.49; G6PD) was purified from coriander (Coriandrum sativum) leaves; the kinetic behavior and some properties of the enzyme were also investigated. The purification was done at 4C and involved two steps: ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 26.4% and had a specific activity of 1.826 U/mg protein. Optimum pH, stable pH, optimum temperature, molecular weight, K_M and V_max values for NADP⁺ and glucose 6-phosphate (G6-P) were also determined.
The overall purification was about 74-fold. SDS-PAGE of the purified enzyme showed a single band. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm.
The molecular mass was estimated to be 74.4 kDa by SDS-PAGE and 73.2 kDa by Sephadex G-200 gel filtration column chromatography. The enzyme had an optimum pH at 8.5 and was stable at pH 8.0 in 0.1 M Tris-HCl buffer. The optimum temperature was at 30C. The K_M values for NADP⁺ and G6-P were 0.026 mM and 0.116 mM, respectively. The V_max values for these substrates were 0.035 EU/mL and 0.038 EU/mL, respectively.     

A Rapid FTIR Spectroscopic Method for Estimation of Caffeine in Soft Drinks and Total Methylxanthines in Tea and Coffee

ABSTRACT: A rapid Fourier Transform infrared (FTIR) spectroscopic method was developed to estimate caffeine in a variety of soft drinks and total methylxanthine content in tea and coffee using a single calibration model. FTIR spectrum of pure caffeine was characterized, and the 2 spectral regions of 1500 to 1800 cm^-1 and 2800 to 3000 cm^-1 were used for quantitative estimation using Partial least square (PLS) and principal component regression (PCR). FTIR spectroscopy with chemometrics using the PLS-First derivative spectra could predict the caffeine content accurately up to an R² value greater than 0.99 and a standard error of prediction (SEP) of less than 3.2 with 5 factors in the model.     

Effect of soluble soybean protein hydrolysate-calcium complexes on calcium uptake by Caco-2 cells

Soybean protein hydrolysates (SPHs) bind with calcium, forming soluble SPH-calcium complexes via the carboxyl groups of glutamic and aspartic acid residues. However, their effect on calcium uptake is still unclear. In this study, Caco-2 cells were used to estimate the effect of SPH-calcium complexes with different molecular weights on calcium uptake in vitro. The changes in intracellular calcium ion concentration were measured by Fura-2 loading and expressed in fluorescence intensity. SPH-calcium complexes could promote calcium uptake. Improved fluorescence intensity was significantly different in SPH-calcium complexes (10 to 30 kDa), SPH-calcium complexes (3 to 10 kDa), and SPH-calcium complexes (1 to 3 kDa). The maximum levels of relative fluorescence intensity (18.3) occurred with SPH-calcium complexes (10 to 30 kDa). The effect of SPH-calcium complexes (10 to 30 kDa) on Ca(2+) increase was determined to be concentration dependent in the range of 0.5 to 4 mg/mL. Our results indicate that soybean protein itself might be responsible for promoting calcium absorption.     

Preparation and antioxidant activity of enzymatic hydrolysates from purple sea urchin (Strongylocentrotus nudus) gonad

Purple sea urchin (Strongylocentrotus nudus) gonad was treated separately with neutral protease, papain, pepsin and trypsin. The resultant hydrolysates were fractionated using a series of ultrafiltration membranes (molecular weight cut-offs of 10, 5, 3 and 1 kDa). Five fractions were prepared from each hydrolysate and the corresponding molecular weight ranges were below 10 kDa, 5-10 kDa, 3-5 kDa, 1-3 kDa and below 1 kDa. The peptide fractions were evaluated for antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and reducing power assay. Results indicated that all peptide fractions possessed DPPH radical scavenging capacity and reducing power in a dose-dependent manner. For all four hydrolysates, the below 1 kDa fractions exhibited the highest DPPH radical scavenging capacity. The below 1 kDa fractions prepared with neutral protease, papain and pepsin, and the 1-3 kDa fraction prepared with trypsin showed the highest reducing capacity among corresponding hydrolysates.     

花生肽钙复合物钙结合特性及氨基酸组成研究

利用花生分离蛋白通过酶解法制备花生肽,将花生肽脱酰胺处理,制备花生肽钙复合物。研究脱酰胺处理对花生肽钙结合量的影响,并分析花生肽钙复合物钙结合特性及氨基酸组成。结果表明:脱酰胺处理240 min后花生肽的钙结合量由82. 05 mg/g显著增加到135. 26 mg/g,表明脱酰胺处理可以显著提高花生肽的钙结合量;扫描电镜分析结果表明加钙反应后花生肽微观结构发生明显变化,由疏松片状结构聚合为颗粒状结构;傅里叶红外光谱分析结果表明花生肽肽链上的氨基及羧基是钙离子的主要结合位点,经脱酰胺处理羧基伸缩振动带进一步发生移动,表明脱酰胺后羧基上的氧原子与钙的配位作用得到增强,促进了花生肽钙结合量的提高;花生肽钙复合物中天冬氨酸含量为10. 76%,谷氨酸含量为23. 49%,表明酸性氨基酸含量是花生肽具有高钙结合量的重要因素。     

BULK THERMAL CONDUCTIVITY AND DIFFUSIVITY OF SOYBEAN

Bulk thermal conductivity of soybean, determined by the transient heat flow method, exhibited positive linear correlation with moisture content. The bulk thermal conductivity values increased from 0.1157 to 0.1756 W/m-K in the moisture range of 8.1 to 25% d.b. Further, thermal diffusivity of soybean, computed from the values of thermal conductivity, specific heat and bulk density showed linear increase from 2.94 × 10⁻⁴ to 3.07 × 10⁻⁴ m²/h in the specified range of 8.1 to 25% d.b. moisture content.     

Purification of a histidine-containing peptide with calcium binding activity from shrimp processing byproducts hydrolysate

The shrimp processing byproducts were hydrolyzed by various proteases, and calcium binding activity of the hydrolysates was examined. Among the digests, trypsin digest showed the most potent calcium binding activity (0.294 mmol/g-protein) and highest degree of hydrolysis (18.4%). The trypsin hydrolysate was fractionated according to the molecular weights using ultrafiltration membrane system. The lowest molecular weight fraction (<1 kDa) showed the highest calcium binding activity. Then, the lowest molecular weight fraction was isolated and purified by ion-exchange chromatography, gel filtration, and ODS reversed high-performance liquid chromatography. The purified peptide showed the highest calcium binding activity of 2.70 mmol/g-protein, and its structure was identified as Thr-Cys-His by ESI/MS/MS. Therefore, these results suggested that the peptide derived from shrimp processing byproducts protein hydrolysates is responsible for higher calcium binding properties and may be as natural functional additive in food industry.     

脱酰胺对葵花籽蛋白酶解肽的钙结合量及体外消化性的影响

通过谷氨酰胺酶对葵花籽蛋白酶解肽进行脱酰胺,得到脱酰胺的葵花籽蛋白酶解肽,以此为试样,研究脱酰胺对葵花籽蛋白酶解肽的钙结合能力的影响。结果发现,脱酰胺的葵花籽蛋白酶解肽的钙结合量由72.97mg/g显著增加到98.20 mg/g,说明脱酰胺可以显著提高葵花籽蛋白酶解肽的钙结合量;通过傅立叶红外光谱对脱酰胺前后葵花籽蛋白酶解肽的钙结合位点进行分析,发现葵花籽蛋白酶解肽与钙结合后,氨基的特征吸收峰均发生移动,N-H的伸缩振动带由3323cm-1移动至3340cm-1,酰胺Ⅱ带由1160cm-1移动至1656cm-1,酰胺Ⅲ带由1241cm-1移动至1246cm-1,同时脱酰胺使其酰胺Ⅰ带由1660cm-1移动至1656cm-1,说明葵花籽蛋白酶解物肽链上的氨基及羧基是钙的主要结合位点,且脱酰胺后C=0伸缩振动引起的酰胺Ⅰ带进一步向低频移动,表明脱酰胺后羧基上的氧原子与钙的配位作用得到增强,促进了葵花籽蛋白酶解肽钙结合量的提高;另一方面研究还发现脱酰胺后葵花籽蛋白酶解肽钙复合物的钙结合量能保持86%以上,与未脱酰胺的花籽肽钙复合物相比消化后的钙结合量提高24%以上。表明脱酰胺能够显著提高葵花籽蛋白酶解肽钙复合物的消化稳定性,即提高葵花籽蛋白酶解肽的钙结合稳定性。     

Antioxidant Activity of Soy Protein Hydrolysates in a Liposomal System

ABSTRACT: Native and heated soy protein isolate was hydrolyzed with 3 purified (pepsin, papain, and chymotrypsin) and 3 crude (Alcalase®, Protamex^TM, and Flavourzyme^TM) proteases. The hydrolysates were incubated (37 °C, 1 h) with a liposome-oxidizing system (50 μM FeCl₃/0.1 mM ascorbate, pH 7.0) to test antioxidant activities by determining the concentrations of TBARS. Degree of hydrolysis of SPI hydrolysates ranged from 1.7 to 20.6%. Both hydrolyzed and nonhydrolyzed SPI decreased TBARS (by 28 to 65%), except for papain-hydrolyzed samples. Samples of chymotrypsin- and Flavourzyme-hydrolyzed (0.5 h) preheated SPI had the greatest inhibitory effect on lipid oxidation.     

Evaluation of Antioxidant Activities of Zein Protein Fractions

Zein protein was extracted from the by‐product corn gluten meal. The obtained zein protein was 1st hydrolyzed by 4 different proteases. The antioxidant activities of the hydrolysates or peptides were evaluated by free radical scavenging activity, metal ion chelating activity, and lipid peroxidation inhibitory capacity. Among hydrolysates produced, alkaline protease hydrolysates exhibited the highest antioxidant activity. A regression model was established by uniform design to optimize the alkaline protease hydrolysis conditions. The hydrolysates with molecular weight < 3 kDa obtained from ultrafiltration showed the highest antioxidant activities in all relevant assays. The hydrolysates with molecular weight <3 kDa were subsequently purified by gel filtration chromatography, and fraction F3 exhibited the highest antioxidant activities. Two peptides were identified from fraction F3 using LC‐ESI‐Q‐TOF MS/MS as Pro‐Phe (263.13 Da) and Leu‐Pro‐Phe (375.46 Da). These peptides exhibited good free radical scavenging activity and lipid peroxidation inhibitory effect. The results clearly indicated that zein protein fractions are good sources for the development of natural antioxidants for the food industry.     

Characterization of Lactoferrin (LF) from Colostral Whey Using Anti-LF Antibody Immunoaffinity Chromatography

ABSTRACT: Lactoferrin (LF) in colostral whey was isolated by anti-LF immunoglobulin in yolk (IgY)-Sepharose 4B immunoaffinity chromatography, and parameters such as binding capacity (q_m) and dissociation constant (K_d, × 10⁻⁶ M) of this immunoaffinity gel for LF were discussed. Purification folds for colostral whey I (from colostrum collected within 6 d of postpartum) and colostral whey II (from colostrum collected within 1 d of postpartum) by anti-LF IgY-immunoaffinity chromatography were 135.80 and 103.60, respectively. The recovery for LF in the same colostral whey sample by anti-LF IgY-immunoaffinity chromatography was 82 to 99 %. q_m of anti-LF IgY-immunoaffinity gel for LF in colostral whey I and whey II were 0.372 and 0.272 mg LF/mL wet gel, respectively. K_d of anti-LF IgY-immunoaffinity gel for LF in colostral whey I was 1.594 × 10⁻⁶ M and II was 1.587 × 10⁻⁶ M.     

KINETIC AND THERMODYNAMIC CHARACTERISTICS OF A DIGESTIVE PROTEASE FROM ATLANTIC COD, GADUS MORHUA

A 23,500 dalton protease was isolated from the pyloric ceca of Atlantic cod by the successive steps of ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The protein fraction recovered after affinity chromatography migrated as one band in both Davis and Laemmli gels. The protease was classified as trypsin (EC 3.4.21.4) on the basis of its substrate specificity, molecular weight and response to known trypsin inhibitors. For trypsin hydrolysis of benzoyl-DL-arginine p-nitroanilide, the substrate turnover number was 250 BAPA units per micromole trypsin (25°C), Km¹ was 1.48 mM, the Arrhenius energy of activation (Ea) was 8.9 kcal/mol, and the free energy of activation (ΔG*) was 12.8 kcal/mol. The Vmax for the hydrolysis of tosylarginine methyl ester was 18,210 TAME units per micromole trypsin and the Km¹ for the same reaction was 0.22 mM at 25°C.     

Quenching Mechanisms and Kinetics of α-Tocopherol and β-Carotene on the Photosensitizing Effect of Synthetic Food Colorant FD&C Red No. 3

ABSTRACT: Effects of FD&C Red No. 40, Red No. 3, Yellow No. 5, Yellow No. 6, Green No. 3, Blue No. 1 and Blue No. 2 on 0.03M soybean oil oxidation in acetone at 25 °C under light were studied by measuring headspace oxygen depletion. As Red No. 3 increased from 0 to 5, 20, 100 and 200 ppm, the headspace oxygen was reduced by 2 to 70, 73, 77 and 77%, respectively, for 4 h. Only Red No. 3 acted as a photosensitizer to produce singlet oxygen in the oil. The quenching rates of α-tocopherol and β-carotene for the singlet oxygen by Red No.3 were 4.1 × 10⁷ M⁻¹s⁻¹ and 7.3 × 10⁹ M⁻¹s⁻¹, respectively. When β-carotene was below 1.86 × 10⁻⁶ M, β-carotene quenched singlet oxygen, but it quenched both singlet oxygen and Red No. 3 at or above 3.72 × 10⁻⁶ M. However, α-tocopherol quenched singlet oxygen only.