Enzymatic and chemical treatment limits on the controlled solubilization of brewers' spent grain

The enzymatic hydrolysis of brewers' spent grain (BSG) has been investigated through treatment with commercial carbohydrases and proteases. Resultant residues were then chemically fractionated and delignified. Enzymatic treatments released 25-30% of the BSG mass and yielded precursors suitable for subsequent conversion to potentially value-added products. Controlled chemical fractionation selectively solubilized arabinoxylan but with no differences apparent due to prior enzyme treatment. The loss of non-polysaccharide components during alkali treatment suggests the presence of a high proportion of alkali-soluble lignin. Further delignification of the alkali-insoluble residues and further chemical fractionation released the remaining hemicellulose, to yield a residue which was >90% cellulose. Further knowledge of the properties and interaction between BSG polymers will facilitate an improved enzyme-assisted total deconstruction of BSG and hence the exploitation of its biomass.     

Enzymatic solubilization of proteins in brewer's spent grain

Brewer's spent grain (BSG) is an abundant, protein-rich coproduct from the beer industry. There is a growing interest in increasing and diversifying the exploitation of BSG and related coproducts for economic and environmental reasons. In this paper, we report on a study of the solubilization of proteinaceous material from BSG using several commercial peptidase preparations. Our data show that Alcalase is the most effective peptidase for solubilization of BSG proteins, with an ability to release up to 77% of total protein. The peptides produced by Alcalase had lower average molecular weight than peptides produced by the less effective enzymes. Processes that combined peptidase treatment with carbohydrate-degrading enzyme preparations such as Depol740 increased the solubilization of dry matter (from 30 to 43% under optimal conditions). However, such additional treatment had little effect on the solubilization of protein. The choice of enzyme dosage depends on the desired hydrolysis time and was assessed through several experiments. Protein solubilization was consistently better at pH 8.0 as compared to pH 6.8. Maximum protein solubilization at pH 8.0 within 4 h required the use of 10-20 microL Alcalase per g of dry matter. However, a considerable degree of solubilization (64%) and hydrolysates with high protein content could be obtained using doses down to only 1.2 microL. Amino acid composition analyses showed that Alcalase treatment solubilizes proline and glutamine (constituents of barley hordein) slightly more efficiently than the other amino acids in BSG.     

Effect of pH on the solubilization of brewers' spent grain by microbial carbohydrases and proteases

The potential for enzymatic solubilization of brewers' spent grain by carbohydrases and proteases was examined over a broad pH range (pH 3.2-11.2). Enzymes from Trichoderma (Depol 686) were most efficient at a lower pH, while enzymes from the Humicola preparation (Depol 740) were the best performer over the whole range. Profiling of key glycoside hydrolase, esterase and protease activities across the pH range demonstrated that solubilization of spent grain by the Trichoderma enzymes corresponded to the range of maximum activities. This was not the case with the Humicola enzymes, where maximum solubilization of the substrate occurred at pH 9.1, at which pH the determined activities were low. Protease activity in Depol 740 was associated with a high solubilization, but inhibition of proteolytic activity resulted in only a 5% decrease in spent grain solubilization. These results suggest that while enzymes can be used to exploit agro-industrials byproduct, the use of high pH increases the extent of hydrolysis and an unidentified factor produced by Humicola improves the enzyme-catalyzed solubilization of lignocellulosic material.     

微射流均质预处理提高大豆分离蛋白酶解效率及酶解产物乳化性能

为了探讨微射流均质预处理对大豆分离蛋白酶解效率及酶解产物乳化性能的影响,该文研究比较了微射流均质预处理前后大豆分离蛋白酶解产物的理化性质(水解度、亚基组成、蛋白溶解性、表面疏水性和分子量分布)和乳化性能(通过测定分析样品乳状液的平均粒径和微观结构评估样品的乳化性能)的变化。研究表明:大豆分离蛋白经过微射流均质预处理后采用木瓜蛋白酶水解,其酶解产物(水解度为1.7%)与对照大豆分离蛋白和未经预处理的酶解产物相比,在较低浓度下(30 g/L)制备出粒径细小的稳定乳状液(体积平均粒径≈1.6μm)。微射流均质预处理提高了大豆分离蛋白中α-7S和A-11S亚基的酶解敏感性,使酶解产物在水解度1.3%~1.7%范围内蛋白溶解性显著增加(P0.05),同时保持较高的表面疏水性值,与未经预处理的酶解产物相比形成了更多具有界面活性的可溶性多肽(分子量主要分布在11.3 k Da左右),在乳化过程中可有效防止乳液滴间发生桥联絮凝。因此微射流均质预处理是一种辅助提高大豆蛋白酶解效率和酶解产物乳化性能行之有效的方法。研究结果可为大豆蛋白深加工蛋白乳化剂提供理论和方法参考。     

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.     

Effects of ultrasound pretreatment on the enzymatic hydrolysis of soy protein isolates and on the emulsifying properties of hydrolysates

Soy protein isolate (SPI) was modified by ultrasound pretreatment (200 W, 400 W, 600 W) and controlled papain hydrolysis, and the emulsifying properties of SPIH (SPI hydrolysates) and USPIH (ultrasound pretreated SPIH) were investigated. Analysis of mean droplet sizes and creaming indices of emulsions formed by SPIH and USPIH showed that some USPIH had markedly improved emulsifying capability and emulsion stabilization against creaming during quiescent storage. Compared with control SPI and SPIH-0.58% degree of hydrolysis (DH), USPIH-400W-1.25% (USPIH pretreated under 400W sonication and hydrolyzed to 1.25% DH) was capable of forming a stable fine emulsion (d43=1.79 μm) at a lower concentration (3.0% w/v). A variety of physicochemical and interfacial properties of USPIH-400W products have been investigated in relation to DH and emulsifying properties. SDS-PAGE showed that ultrasound pretreatment could significantly improve the accessibility of some subunits (α-7S and A-11S) in soy proteins to papain hydrolysis, resulting in changes in DH, protein solubility (PS), surface hydrophobicity (H0), and secondary structure for USPIH-400W. Compared with control SPI and SPIH-0.58%, USPIH-400W-1.25% had a higher protein adsorption fraction (Fads) and a lower saturation surface load (Γsat), which is mainly due to its higher PS and random coil content, and may explain its markedly improved emulsifying capability. This study demonstrated that combined ultrasound pretreatment and controlled enzymatic hydrolysis could be an effective method for the functionality modification of globular proteins.     

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。     

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.     

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 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 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.     

beta-lactoglobulin hydrolysis. 1. Peptide composition and functional properties of hydrolysates obtained by the action of plasmin, trypsin, and Staphylococcus aureus V8 protease.

beta-Lactoglobulin (betaLg) was subjected to limited hydrolysis by trypsin, plasmin, and endoproteinase from Staphylococcus aureus V8 (S.aur.V8) to degrees of hydrolysis (DH) of 1, 2, and 4%. The several hydrolysates had different peptide compositions (determined by reversed-phase HPLC and gel-permeation chromatography [GPC]). GPC under nondenaturing, denaturing, and denaturing plus reducing conditions showed that the peptides formed were linked by hydrophobic interactions or by disulfide bonds or were not linked at all. At very low protein concentration, some differences in emulsion-forming properties were observed: only the plasmin hydrolysates could form emulsions with a uniform particle-size distribution. The emulsions formed with S.aur.V8 hydrolysates had poor emulsion-stabilizing properties. Some hydrolysates showed increased foam-forming properties in comparison with the intact protein. All foams formed were stable. Overall, the plasmin hydrolysate (DH4) contained relatively much larger molecules and/or hydrophobic molecules. Many molecules were disulfide-linked peptides. This hydrolysate also had the best functional properties.     

Caseins and casein hydrolysates. 1. Lipoxygenase inhibitory properties

Whole casein from bovine origin, the different casein subtypes alpha, beta, and kappa, and the related dephosphorylated proteins were assayed as modulators of soybean lipoxygenase 1 activity and were found to inhibit it. To define the lipoxygenase inhibitory domain, whole casein and beta-casein were digested by proteases (trypsin, clostripain, and subtilisin). The beta-casein tryptic digest and the tryptic and subtilisin digests of whole casein retained their inhibitory properties. The tryptic beta-casein digest was the most potent inhibitor of lipoxygenase activity and was further fractionated by FPLC or HPLC. The collected peptides inhibited the lipoxygenase-catalyzed reaction to different extents. The active fractions were analyzed by ESI-MS, and the sequences of several lipoxygenase inhibitory peptides, corresponding mainly to the C-terminal moiety of beta-casein, were identified.     

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。     

Emulsion properties of casein and whey protein hydrolysates and the relation with other hydrolysate characteristics

Casein and whey protein were hydrolyzed using 11 different commercially available enzyme preparations. Emulsion-forming ability and emulsion stability of the digests were measured as well as biochemical properties with the objective to study the relations between hydrolysate characteristics and emulsion properties. All whey protein hydrolysates formed emulsions with bimodal droplet size distributions, signifying poor emulsion-forming ability. Emulsion-forming ability of some casein hydrolysates was comparable to that of intact casein. Emulsion instability was caused by creaming and coalescence. Creaming occurred mainly in whey hydrolysate emulsions and in casein hydrolysate emulsions containing large emulsion droplets. Coalescence was dominant in casein emulsions with a broad particle size distribution. Emulsion instability due to coalescence was related to apparent molecular weight distribution of hydrolysates; a relative high amount of peptides larger than 2 kDa positively influences emulsion stability.     

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 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