Enzymatic hydrolysis of heat-induced aggregates of whey protein isolate

The effects of heat-induced denaturation and subsequent aggregation of whey protein isolate (WPI) solutions on the rate of enzymatic hydrolysis was investigated. Both heated (60 °C, 15 min; 65 °C, 5 and 15 min; 70 °C, 5 and 15 min, 75 °C, 5 and 15 min; 80 °C, 10 min) and unheated WPI solutions (100 g L(-1) protein) were incubated with a commercial proteolytic enzyme preparation, Corolase PP, until they reached a target degree of hydrolysis (DH) of 5%. WPI solutions on heating were characterized by large aggregate formation, higher viscosity, and surface hydrophobicity and hydrolyzed more rapidly (P < 0.001) than the unheated. The whey proteins exhibited differences in their susceptibility to hydrolysis. Both viscosity and surface hydrophobicity along with insolubility declined as hydrolysis progressed. However, microstructural changes observed by light and confocal laser scanning microscopy (CLSM) provided insights to suggest that aggregate size and porosity may be complementary to denaturation in promoting faster enzymatic hydrolysis. This could be clearly observed in the course of aggregate disintegration, gel network breakdown, and improved solution clarification.     

Enzymatic hydrolysis of soil organic phosphorus by immobilized phosphatases

 In order to estimate the role of phosphatases in maintaining the potential bioavailable P pool in soils, water and 0.4 M NaOH soil extracts were incubated with immobilized acid phosphatase, alkaline phosphatase, phospholipase and nuclease, separately, and in combinations. Immobilized nuclease at an optimum pH of 7.0 hydrolyzed the most soluble unreactive P (SUP) both in water and 0.4 M NaOH extracts. The combination of immobilized alkaline phosphatase and nuclease increased the hydrolysis of SUP at pH 7.0 by up to 61% in 0.4 M NaOH extracts relative to that due to immobilized nuclease alone. The combination of immobilized acid phosphatase and nuclease, however, did not increase the hydrolysis of SUP in either extract relative to that due to immobilized nuclease alone. Immobilized alkaline phosphatase and phospholipase increased the hydrolysis of SUP at pH 7.0 by up to 62% in 0.4 M NaOH extracts relative to that due to immobilized phospholipase alone. Similarly, immobilized acid phosphatase and phospholipase increased the hydrolysis of SUP at pH 7.0 by up to 49% in 0.4 M NaOH extracts relative to that due to immobilized phospholipase alone. The similarities in the optimum pH of indigenous phosphatases in soils and the immobilized phosphatases used in this study, immobilized on positively charged supports, suggests that indigenous phosphatases could be immobilized on positively charged surfaces in soils. Received: 17 November 1998     

细微玉米粉的低温酶解

为了开发玉米粉低温酶解新工艺,采用双酶法对粒度不同的市售玉米粉(中位粒径273.6 μm)和细微玉米粉(中位粒径17.1 μm)进行液化、糖化处理,调查了30～70℃范围内的液化温度对液化速度和葡萄糖收率的影响。试验结果表明,市售玉米粉在40～70℃的温度范围内,细微玉米粉在30～70℃的温度范围内,液化速率常数与温度的关系可用Arrhenius方程式表示。细微粉碎使液化反应活化能从市售玉米粉的4.63×10⁴ J/mol降低到2.15×10⁴ J/mol。40℃时,细微玉米粉的液化速度大约是市售玉米粉的2.5倍。液化温度对细微玉米粉的葡萄糖收率没有显著影响。细微玉米粉的葡萄糖收率可达95.4%,大大高于市售玉米粉的79.2%。由此可见,通过细微粉碎可以降低玉米粉的液化温度,同时提高液化速度和葡萄糖收率。     

Enzymatic hydrolysis of ester sulphate in soil organic matter extracts

Summary A method of assessing the enzymatic hydrolysis of ester sulphate in soil organic matter was developed. Soil organic matter extracted using a mild, chelating resin extraction procedure was incubated with a sulphatase from Helix pomatia in 0.05 M sodium acetate buffer (pH 4–8) at 37°C for 2h and the sulphate released was determined by a high performance liquid chromatography-conductivity detector system. The effect of some soil factors on the enzymatic hydrolysis of ester sulphate was examined. The study showed that part of the ester sulphate in soil organic matter was biochemically reactive. In the three Podzols studied, the ester sulphate hydrolysed accounted for 2%–12% of the hydriodic acid-reducible organic sulphate extracted. The largest amount of hydrolysable ester sulphate was found in the soil with a low pH, high inorganic sulphate and high hydriodic acid-reducible organic sulphate.     

Enzymatic hydrolysis of biological and environmental samples as pretreatment for analysis

Four commercially available proteases were tested, in conjunction with a lipase, for efficacy in hydrolyzing 3 tissue substrates: cod fillet, chicken egg, and bovine liver. Enzymatic hydrolysis of tissues minimizes the formation of emulsions during liquid-liquid extraction and does not accelerate the decomposition of acid- or base-labile analytes. Recovery of hexane and benzene phases from the hydrolysates was also evaluated. Protease from Streptomyces griseus combined with lipase from Candida cylindracea (available commercially) produced the highest percent hydrolysis (relative to classical acid hydrolysis) in all 3 tested tissues (60-95%) and the greatest recovery of hexane (100%) and benzene (92-100%) solvent phases.     

Enzymatic hydrolysis of brewers' spent grain proteins and technofunctional properties of the resulting hydrolysates

Brewers' spent grain (BSG) is the insoluble residue of barley malt resulting from the manufacture of wort. Although it is the main byproduct of the brewing industry, it has received little attention as a marketable commodity and is mainly used as animal feed. Our work focuses on one of the main constituents of BSG, i.e., the proteins. The lack of solubility of BSG proteins is one of the limitations for their more extensive use in food processing. We therefore aimed to generate BSG protein hydrolysates with improved technofunctional properties. BSG protein concentrate (BPC) was prepared by alkaline extraction of BSG and subsequent acid precipitation. BPC was enzymatically hydrolyzed in a pH-stat setup by several commercially available proteases (Alcalase, Flavourzyme, and Pepsin) for different times and/or with different enzyme concentrations in order to obtain hydrolysates with different degrees of hydrolysis (DH). Physicochemical properties, such as molecular weight (MW) distribution and hydrophobicity, as well as technofunctional properties, such as solubility, color, and emulsifying and foaming properties, were determined. Enzymatic hydrolysis of BPC improved emulsion and/or foam-forming properties. However, for the hydrolysates prepared with Alcalase and Pepsin, an increasing DH generally decreased emulsifying and foam-forming capacities. Moreover, the type of enzyme impacted the resulting technofunctional properties. Hydrolysates prepared with Flavourzyme showed good technofunctional properties, independent of the DH. Physicochemical characterization of the hydrolysates indicated the importance of protein fragments with relatively high MW (exceeding 14.5 k) and high surface hydrophobicity for favorable technofunctional properties.     

莲子酶解动力学分析与酶解产物多糖的表征

为开发更温和与简便的高纯度植物多糖新型酶法水解提取工艺,该研究以胃蛋白酶（内肽酶）和曲蛋白酶（端肽酶）作为复合酶,建立酶解过程的动力学模型。对上述工艺所提取制备的多糖与45 ℃水提法、90 ℃水提法、胃蛋白酶提取法所制得的莲子多糖进行营养成分分析与结构（单糖组成、红外光谱、热特性、低场核磁和动态热机械）表征。结果显示,单酶（胃蛋白酶）/双酶分段酶解的动力学模型分别为：D_H=2.101ln[1+(0.6133(E₀/S₀)+0.1441)t]和D''_H-D_H1=2.439ln[1+(3.923(E₀/S₀)+1.1756)t];双酶法提取的莲子多糖中多酚浓度（（0.42±0.008）mg/mL）和糖醛酸含量（15.65%±0.98%）最高,而以双酶法制得的莲子多糖蛋白质含量（0.73%±0.24%）最低;4种莲子多糖均含有葡萄糖 (Glc)、阿拉伯糖（Arab）、甘露糖（Man）和鼠李糖（Rha）,其中双酶法提取的多糖中半乳糖醛酸（Gal-UA）和Man的含量较多,分别为10.700%和10.752%;4种多糖均为α-型吡喃糖;双酶提取法相比水提法可有效降低莲子多糖中的蛋白质含量和玻璃化温度,提高结合水含量和亲水能力。酶解动力学模型可为莲子多糖纯化机制提供有效参考,尤其是双酶分段酶解法的特异性强、步骤简便,有利于提取和纯化莲子多糖成分,该研究可为动植物非淀粉类多糖的高质量提取和工业化生产提供理论基础。     

糠醛渣的纤维素酶水解及其最优纤维素转化条件

该文对糠醛渣的纤维素酶水解特性进行了研究,探索利用玉米芯制糠醛联产燃料乙醇工业化生产的可行性。分析糠醛渣组分,表明其半纤维素质量分数为3.1%,纤维素为31.6%,说明糠醛生产过程对玉米芯的预处理基本满足高效酶解糖化糠醛渣并转化乙醇的要求;通过纤维素酶用量、温度、pH值、固液比、转速等因素进行条件优化,确定最佳水解条件：每克底物酶用量为6.7FPU,固液质量体积比1︰6,pH5.2,转速80 r/min;在糠醛渣水解体系中加入吐温80,结果表明在酶施用量较低情况下（6.7 FPU/g）,吐温80对提高糠醛渣水解转化率效果更为明显;通过最优化水解条件,使糠醛渣纤维素转化率达到78%,据此初步判定以糠醛渣为原料转化乙醇的工业化生产具有较大潜力。     

Enzymatic hydrolysis as a means of expanding the cold gelation conditions of soy proteins

Acid-induced cold gelation of soy protein hydrolysates was studied. Hydrolysates with degrees of hydrolysis (DH) of up to 10% were prepared by using subtilisin Carlsberg. The enzyme was inhibited to uncouple the hydrolysis from the subsequent gelation; the latter was induced by the addition of glucono-delta-lactone. Visual observations, confocal scanning laser microscopy images, and the elasticity modulus showed that hydrolysates gelled at higher pH values with increasing DH. The nonhydrolyzed soy protein isolate gelled at pH approximately 6.0, whereas a DH = 5% hydrolysate gelled at pH approximately 7.6. Gels made from hydrolysates had a softer texture when manually disrupted and showed syneresis below a pH of 5-5.5. Monitoring of gelation by measuring the development of the storage modulus could be replaced by measuring the pH onset of aggregate formation (pH(Aggr-onset)) using turbidity measurements. The rate of acidification was observed to also influence this pH(Aggr-onset). Changes in ionic strength (0.03, 0.2, and 0.5 M) had only a minor influence on the pH(Aggr-onset), indicating that the aggregation is not simply a balance between repulsive electrostatic and attractive hydrophobic interactions, but is much more complex.     

速溶龙眼粉加工的酶解提取与喷雾干燥工艺优化

为了建立速溶龙眼粉的加工技术,运用均匀设计法优化了龙眼干中可溶性固形物酶解提取工艺条件,采用响应面分析法优化了其提取液的喷雾干燥工艺条件,结果表明：以龙眼果肉干（含水率13.62 %）为原料,用果胶酶和纤维素酶同时酶解提取,pH3.1,酶解温度52℃,酶解时间160 min,果胶酶用量0.6‰,纤维素酶用量0.15‰,龙眼干中可溶性固形物得率达85.26%;当酶解提取液中可溶性固形物浓度达25%时,采用喷雾干燥法,选取麦芽糊精为助干剂,其与可溶性固形物含量比0.8：1,热风温度185℃,热风流量26.47 m3/h,入料流量0.20 L/h,在此条件下,龙眼粉得率为48.58%,含水率＜5%,水溶性良好,色泽风味佳。由此说明,以龙眼果肉干为原料采取酶解提取与喷雾干燥相结合的工艺可有效加工速溶龙眼粉。     

鸡肉蛋白酶水解工艺条件的研究

为探索鸡肉酶水解工艺条件及其动力学特性，分别采用木瓜蛋白酶、胃蛋白酶、中性蛋白酶、菠萝蛋白酶对鸡肉进行了水解试验，得出最优酶为木瓜蛋白酶；由此选用木瓜蛋白酶做正交试验，考察水解温度、时间、加酶量、pH值和固液比5个因素对酶水解的影响，并确定出最优水解条件为温度50℃，时间7 h，加酶量2.4%(以鸡肉的质量百分数计)，pH 7.0，固液比1∶4；在此条件下，水解度可达26.07%。在此基础上由试验数据推导出描述木瓜蛋白酶水解鸡肉的动力学方程，可为鸡肉酶解生化反应器的设计和鸡肉水解蛋白液或蛋白粉的开发提供一定的理论依据。     

Enzymatic hydrolysis of organic phosphorus in extracts and resuspensions of swine manure and cattle manure

Animal manure can be a valuable resource of P for plant growth. Organic phosphates (P_o) are considered bioavailable if they can be hydrolyzed to inorganic P (P_i). Therefore, investigation of the susceptibility of manure P_o to hydrolysis may increase our understanding of manure P_o bioavailability. In this study, we demonstrate that three orthophosphate-releasing enzymes, acid phosphatase from wheat germ, alkaline phosphatase from bovine intestinal mucosa, and fungal phytase from Aspergillus ficcum, were able to hydrolyze certain amounts of P_o in animal manure. A scheme of sequential enzymatic release of P_o in manure was developed and then used to investigate changes in swine and cattle manure P distribution after storage at –20°C, 4°C or 22°C for about a year. Assuming that the P distribution in manure maintained at –20°C remained unchanged (i.e., similar to fresh manure), bioavailable P (P_i and enzyme-hydrolysable P_o) in swine manure remained relatively constant [72.8–76.3% of total P (P_t)]. Soluble but enzymatically unhydrolysable P_o (P_ue) increased from 7.2% to 32.1% of P_t during storage at 4°C. In cattle manure, bioavailable P decreased from 71.6% to 62.9% of P_t, and P_ue increased from 21.7% to 37.2% of P_t during storage at 22°C. These data indicated that the major change during the storage of animal manure for a year was the increase in P_ue, so manure P solubility may increase with storage, but the increase would not produce more bioavailable P in the manure. The effects of storage on the bioavailability of manure P should be further investigated to develop an efficient manure-P management strategy.Trade or manufacturers' names mentioned in the paper are for information only and do not constitute endorsement, recommendation, or exclusion by the USDA-ARS     

Enzymatic hydrolysis of flavonoids and pectic oligosaccharides from bergamot (Citrus bergamia Risso) peel

Pectinolytic and cellulolytic enzymes (Pectinase 62L, Pectinase 690L, and Cellulase CO13P) were used to evaluate the solubilization of carbohydrates and low molecular weight flavonoids from bergamot peel, a major byproduct of the essential oil industry. The enzymes were characterized for main-chain and side-chain polysaccharide hydrolyzing activities and also against pure samples of various flavonoids previously identified in bergamot peel to determine various glycosidase activities. The addition of Pectinase 62L or 690L alone, or the combination of Pectinase 62L and Cellulase CO13P, was capable of solubilizing between 70 and 80% of the bergamot peel, and up to 90% of the flavonoid glycosides present were cleaved to their aglycones. Cellulase CO13P alone solubilized 62% of the peel but had no deglycosylating effect on the flavonoid glycosides. Over a 24-h time course, a rapid release of cell wall carbohydrates was observed after treatment with Pectinase 62L, with a concurrent gradual hydrolysis of the flavonoid glycosides. Size-exclusion chromatography of the solubilized extract showed that after 24-h incubation, the majority of the solubilized carbohydrates were present as monosaccharides with a smaller proportion of oligosaccharides.     

Enzymatic hydrolysis, grease permeation, and water barrier properties of zein isolate coated paper

An inexpensive zein-lipid mixture was isolated from yellow dent, dry-milled corn. Grease permeation through zein isolate applied to brown Kraft paper was found to be independent of loading levels at zein isolate levels above 30 mg/16 in.(2). The data shows that water vapor transmission rates depended on the amount of coating applied. Triacylglycerols were the most abundant lipid in milled corn but were absent in the zein isolate (perhaps due to hydrolysis by lipases). Zein from the paper was hydrolyzed enzymatically and the hydrolysis monitored by SDS-capillary electrophoresis. At an E:S ratio of 1:100 no further increase in the hydrolysate peak occurred after 10 and 30 min for alpha-chymotrypsin and pancreatin 8 x; however, zein and lipid were still present 1 h after hydrolysis by pancreatin 1 x.     

瓦克青霉F10-2对川楝树皮残渣纤维素酶解研究

以川楝树皮残渣为原料进行纤维素酶解研究,测定了不同培养时间培养基质中主要胞外酶活性的变化,并对发酵前后残渣结构进行扫描电镜观察和红外光谱分析。结果表明:瓦克青霉F10-2具有木质纤维素降解能力,酶解液中纤维素酶、半纤维素酶、木质素过氧化物酶和锰过氧化物酶活性在发酵的8～16d内分别达到最大值6.42U·g-1、7.62U·g-1、6.55U·g-1和3.33U·g-1。利用扫描电镜对降解后底物表面结构进行观察,可看到残渣表面变得疏松、柔软,且具有部分微孔。底物残渣傅立叶红外光谱分析表明,该菌株对残渣中各组分均有一定降解。固态发酵培养试验表明,培养24d后残渣中纤维素、半纤维素和木质素的降解率分别达到42.70%、33.96%和24.62%。     

羊奶乳清蛋白水解物的酶解制备

采用Alcalase与Flavourzyme两种酶对羊奶乳清蛋白进行水解,以水解度为指标,对两种酶单独使用及复合使用水解羊奶乳清蛋白的工艺条件进行了研究。试验结果显示:采用Alcalase与Flavourzyme复合水解羊奶乳清蛋白的效果较好,特别是采用先添加Flavourzyme后加入Alcalase进行水解,不仅能提高羊奶乳清蛋白的水解度,使其达到32.81%,而且对改善水解液的口感有较大的作用。     

Determination of total gossypol in cottonseed and cottonseed meals by derivative UV spectrophotometry

A new method for the determination of total gossypol in cottonseed and cottonseed meals has been developed. The method involves oxalic acid hydrolysis of the bound gossypol in a methyl ethyl ketone-water azeotrope, partitioning the liberated gossypol into chloroform, and quantification by 2nd derivative UV spectrophotometry. The 2nd derivative transformation and measurement of the conventional analytical band around 300 nm permits direct quantification of all compounds containing naphthalene nuclei; chromogenic reaction is not required. The method was tested at concentration levels of total gossypol normally expected for cottonseed and cottonseed meal. Precision and accuracy data suggested an overall relative standard deviation of 4.0% and an overall recovery of 89.5%. Although results for cottonseed and solvent-extracted cottonseed meal analyses were comparable to those obtained by use of American Oil Chemists' Society methods, results were lower for screw-pressed meals. The lower results were attributed to the partial conversion of gossypol, during the cooking process of these meals, to compounds that differ in composition and structure from gossypol and which react with aniline to give false readings.     

Aroma components of acid-hydrolyzed vegetable protein made by partial hydrolysis of rice bran protein

Hydrolyzed vegetable protein (HVP) was prepared from rice bran protein concentrate (RBPc) by partial hydrolysis with aqueous 0.5 N HCl at 95 degrees C for 12 or 36 h (H-RBPc-12 and H-RBPc-36, respectively). Aroma components of the RBPc and the HVPs were characterized by gas chromatography-olfactometry, gas chromatography-mass spectrometry, aroma extract dilution analysis, and calculation of odor activity values (OAVs). The predominant odorants in RBPc were 3-methylbutanal, hexanal, 2-aminoacetophenone, (E)-2-nonenal, phenylacetaldehyde, and beta-damascenone. Among these, the odor of 2-aminoacetophenone, present at 59 ng/g in RBPc, was reminiscent of the typical odor of RBPc. Most of the predominant odorants had higher log3FD factors in the H-RBPc-36 as compared to H-RBPc-12. Aroma impact compounds of H-RBPc-12 and H-RBPc-36 were 2-methoxyphenol (guaiacol), 4-hydroxy-2,5-dimethyl-3(2H)furanone, 3-hydroxy-4,5-dimethyl-2(5H)furanone (sotolon), vanillin, 3-methylbutanal, (E)-2-nonenal, 4-vinyl-2-methoxyphenol (p-vinylguaiacol), and beta-damascenone. Guaiacol had the highest OAV values of 2770 and 17650 in H-RBPc-12 and H-RBPc-36, respectively.     

白果活性蛋白的酶法水解及抗氧化活性研究

白果活性蛋白(GAP)是华中农业大学天然产物化学实验室首次从白果中分离得到的一种以清蛋白为主的且具有显著生物活性的蛋白,为扩大该蛋白在食品中的用途,提高其生物活性,研究了酶法水解制备白果肽段的方法及其抗氧化活性的变化。通过单因素分析、正交试验以及对试验结果的分析,确定了2709碱性蛋白酶水解白果活性蛋白的最佳水解条件为：底物浓度1%,pH值8.5,水解温度45℃,酶浓度7000 U/g,反应时间6 h。以Fenton体系和邻苯三酚体系检验其抗氧化活性,结果表明白果肽段较酶解前的活性蛋白,抗氧化活性有不同程度的提高,但两者均具有较强的抗氧化活性。