Optimization of a novel backward extraction of defatted wheat germ protein from reverse micelles

In this work, a novel backward extraction procedure of defatted wheat germ protein (DWGP) from reverse micelles was explored. Isooctane was recovered by vaporization firstly. Then the remained residue was dissolved in a small amount of KCl solution. The recovery of DWGP was easily performed by the ternary liquid system (acetone: deionized water: isooctane = 15:5:1) precipitation, while most of sulphosuccinic acid bis (2-ethylhexyl) ester sodium salt (AOT) remained in the ternary liquid system. In the end, the precipitation of DWGP was washed with 65% ethanol solution to further remove any residual AOT. The effects of KCl concentration, the amount of KCl solution and pH on the backward extraction efficiency of DWGP were tested. On the basis of single-factor experiments, the optimum backward extraction was achieved by response surface methodology (RSM). When the operation ran under optimized conditions, the backward extraction efficiency of DWGP achieved 80% and the end protein product was completely free of AOT.Industrial relevanceThis experimental result confirmed that this novel backward extraction method had many advantages on the extraction of protein compared to the traditional backward extraction method (changing the conditions of pH and ionic strength in a fresh aqueous phase). This method increased the backward extraction efficiency of defatted wheat germ protein (DWGP) from 57% to 80%, saved the water resource and offered the possibility of precipitating nearly pure DWGP, completely free of surfactant. On the basis of these advantages, it appears that this novel backward extraction technique may have great potential for being scaled-up to a commercially extraction process of protein.     

Egg White Lysozyme Purification by Ultrafiltration and Affinity Chromatography

Isolation and purification of lysozyme from hen egg white was studied using a two-step procedure. The egg white was diluted 5- to 9-fold with sodium phosphate buffer, and then processed by sequential dilution diafiltration using a UF membrane (molecular weight cut-off 300,000 dalton). The membrane process increased the specific activity of lysozyme 6-fold, and recovered 96% of lysozyme activity. The permeate from diafiltration was further purified by affinity chromatography using chitin as adsorbent. The second step of the process yielded a product of specific activity of 70,400 units/mg protein. The overall lysozyme recovery was 79%.     

表面活性剂沉淀鱼精蛋白及复溶回收过程研究

采用两种阴离子型表面活性剂：十二烷基硫酸钠(SDS)和2- 乙基己基琥珀酸酯磺酸钠(AOT)沉淀鱼精蛋白并用极性有机溶剂从沉淀中复溶回收鱼精蛋白。结果表明：SDS 可以完全沉淀鱼精蛋白,而表面活性剂AOT 最高只能沉淀75.02% 的鱼精蛋白;极性有机溶剂的种类及辅助无机盐的添加量,对从鱼精蛋白- 表面活性剂难溶复合物中回收鱼精蛋白有很大的影响;对于SDS- 鱼精蛋白复合物,正丙醇为最佳回收剂,鱼精蛋白最高回收率可达83.33%;对于AOT- 鱼精蛋白复合物,丙酮为最佳回收剂,鱼精蛋白最高回收率达到85.71%。回收后的鱼精蛋白抗菌活性同原始鱼精蛋白相比没有明显变化。     

Simultaneous Isolation of Avidin and Lysozyme from Egg Albumen

A single column cation exchange method was developed which allowed simultaneous recovery of lysozyme and avidin from undiluted egg white. A unique application-elution sequence was developed, involving accumulation of avidin on the column through several cycles of egg white application and lysozyme elution. Lysozyme was recovered with higher yields than reported for the isoelectric precipitation methods often used in the industry (86% vs 60–80%). Lysozyme peaks appeared homogeneous on SDS-PAGE. Avidin recovery was also as good or better than that of previously reported ion exchange methods (74%–80%). The purity of the avidin fraction (up to 40.9%) was superior to that of other reported primary avidin fractions.     

Lysozyme Separation from Egg White by Cation Exchange Column Chromatography

Cation exchange column chromatography was investigated for simple and efficient separation of lysozyme from homogenized egg white. A macroporus weak acid type resin, Duolite C-464, was selected based on high lysozyme recovery in the90–95% range, retention of whipping and gelling properties in the lysozyme-separated egg white, and ease of column operation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated high purity of the recovered lysozyme and simultaneous recovery of avidin under the eluting conditions used. The process for lysozyme separation from egg white using Duolite C-464 is suitable for operation in an automated, continuous mode.     

Immunoreactivity of hen egg allergens: Influence on in vitro gastrointestinal digestion of the presence of other egg white proteins and of egg yolk

Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicochemical characteristics and relative abundance. We aimed to identify potential undiscovered egg allergens within the egg white proteome and investigated the existence of matrix effects on the proteolytic stability and resultant IgE-binding of the allergenic proteins. In addition to the main egg allergens: ovalbumin (OVA), ovomucoid (OM) and lysozyme (LYS), two minor egg white proteins, tentatively identified as ovoinhibitor and clusterin, were found to react with serum IgE from egg-allergic patients. Egg white exhibited residual immunoreactivity after gastrointestinal digestion due to the presence of intact OVA and LYS, as well as of several IgE-binding peptides derived from OVA. The presence of egg yolk slightly increased the susceptibility to hydrolysis of egg white proteins and abrogated bile salt-induced precipitation of LYS in the duodenal medium. However, the resultant immunoreactivity against IgE of egg white proteins after in vitro digestion was not significantly modified by the presence of yolk components.     

Egg white lysozyme purification with a chitin–silica-based affinity chromatographic matrix

A composite biosorbent retaining non-covalently bound chitin in between layers of a silicon oxide matrix was assessed for lysozyme purification from undiluted egg white. The matrix can be shaped with big size and high density, thus allowing its efficient separation from the egg white after the adsorption step. The lysozyme-depleted egg white can follow its usual commercialisation route. A surface area of 142 m²/g and a total pore volume of 0.295 cm³/g were calculated from the nitrogen sorption isotherms. Its water content was 78.3%. The matrix structure is the result of the polysaccharide addition to the polymerisation mixture, which is known to influence the condensation process, leading to a material with characteristic properties. A maximum capacity of 117.1 ± 9 mg lysozyme/g and a dissociation constant of 0.73 ± 0.15 mg/mL were calculated from the Langmuir isotherm. A lysozyme purification batch process from undiluted egg white was developed, where 87% of the lysozyme was removed from the egg white and the matrix was easily recovered by a simple filtration through a strainer. The overall yield of the process was 64% with a purification factor of 20.     

鸭蛋清溶菌酶的分离纯化及致敏性评估

鸭蛋中含有丰富的蛋白质和营养成分,鸭蛋清中含有溶菌酶,溶菌酶具有致敏性,食用鸭蛋后有可能产生过敏的风险。采用超滤法与离子交换层析法结合的方法分离纯化鸭蛋清溶菌酶,利用聚丙烯酰胺凝胶电泳法比对,能得到纯度较高的溶菌酶。采用免疫印迹的方法,分析抗溶菌酶的兔多抗血清和禽蛋过敏患者混合血清池分别与鸭蛋清溶菌酶IgE的结合能力,进行鸭蛋溶菌酶的致敏性评估,实验证明IgE结合能力非常显著,溶菌酶存在交叉反应。同时证实了食用鸭蛋会引起过敏反应,也为过敏患者能否食用鸭蛋提供了有效的信息。     

Effect of pasteurisation on the chemical composition of liquid whole egg. I. Development of a scheme for the fractionation of the proteins of whole egg

The proteins of whole egg may be separated into soluble and insoluble portions by dialysis against glycine solution followed by centrifugation at a high speed. The soluble proteins can then be resolved into at least twelve fractions by ion-exchange chromatography on diethylaminoethyl-cellulose, using stepwise elution with dilute glycine-phosphate buffer solutions containing increasing concentrations of sodium chloride. Further examination has shown that most of the fractions contain more than one protein, and some tentative identifications have been made. The insoluble proteins can be dissolved in a relatively strong phosphate buffer and fractionated on diethylaminoethyl-cellulose in a similar way. Separate examinations of white and yolk indicated that the soluble proteins of whole egg are predominantly the same as those of the white, and also showed certain differences between whole egg and the separated white and yolk that could be attributed to some form of protein association in mixed whole egg. An initial experiment has suggested that the method described will reveal differences between raw and pasteurised egg.     

聚乙二醇沉淀蛋清蛋白质的规律及在卵白蛋白分离中的应用

研究3 种pH值条件下4 种聚乙二醇（polyethylene glycol,PEG）对卵白蛋白（ovalbumin,OVA）、卵转 铁蛋白（ovotransferrin,OVT）与溶菌酶（lysozyme,LYZ）的沉淀效率,根据变化规律建立无需后续脱盐的OVA 分离新工艺。结果表明：PEG 4000、6000、8000及10000均可有效沉淀OVA、OVT及LYZ,沉淀率受PEG质量分 数及pH值影响。其中pH 7.5、PEG 4000质量分数12%时,OVA、OVT及LYZ的沉淀率相差最大,当pH值从7.5调至 5.5时,3 种蛋白质沉淀率的变化幅度相差也最大。由此建立如下分离工艺：首先向pH 7.5的蛋清液中加PEG 4000至 质量分数为12%,离心收集上清液,即得纯度88.1%的OVA,提取率为95.1%;再将所得上清液pH值调至5.5,离心 收集上清液,所得OVA纯度提高至99.7%,提取率为87.3%。该工艺简便易行,有利于OVA的大规模制备及在食品 及医药领域的应用。     

新型离子液体亲和萃取鸡蛋清中的溶菌酶

建立并优化利用辛巴蓝(Cibacron Blue F-3GA,CB)修饰[C₄MIM]Cl的新型离子液体[C₄MIM]₃CB特异性萃取分离鸡蛋清中溶菌酶的方法,并采用AutoDock Vina分析[C₄MIM]₃CB与蛋白的结合能,研究溶菌酶与[C₄MIM]₃CB相结合的能力。通过亲和萃取和反萃取并进一步脱盐、浓缩、冷冻干燥,得到溶菌酶产品。结果表明,通过对影响因素的研究,优化出最佳的萃取分离条件:蛋清粉溶液pH为7,新型离子液体中[C₄MIM]₃[CB]浓度为8 mg/mL,反应震荡时间为15 min,反萃取KCl溶液pH为7.0,浓度为1.5 mol/L。最终250 mg蛋清粉可得12.3 mg的溶菌酶产品,纯度为97.56%,酶活力为35000 U/mg,纯化倍数达到37.39,酶活回收率89.74%,且与其他蛋白达到完全分离。该方法具有选择性高、操作简便、产物纯度和活性高等优点。     

Heat Denaturation of the Ovomucin-Lysozynie Electrostatic Complex-A Source of Damage to the Whipping Properties of Pasteurized Egg White

SUMMARY– A longer whip time is usually required to obtain a meringue of the same specific gravity from pasteurized egg white as from unpasteurized egg white. We have determined the rate at which this change in whipping properties occurs as a function of heating time and pH. The rate of damage is minimal at neutral pH. The activation energy for whipping property damage at pH 7.5 is 140 kcal. Experiments in which either ovomucin or lysozyme concentration of egg white was increased and decreased showed that the reaction producing damage to the whipping properties is first order with respect to both ovomucin and lysozyme concentration. Since an increase of 0.33 in the ionic strength of egg white produces a ten-fold decrease in the rate of whipping property damage, the reactants are probably present as the ovomucin-lysozyme electrostatic complex. The product appears to be an irreversibly denatured ovomucin-lysozyme aggregate or network. Removal of the product restores the whipping properties of the egg white. The whipping property damage is a decrease in the mechanical stability of the foam. For this reason a longer time is needed to whip pasteurized egg white to a satisfactory meringue. Whipping aids such as triethyl citrate or triethyl phosphate compensate for the damage to the whipping properties, but do not appear to reverse the reaction producing damage to the whipping properties of the egg white.     

一株耐受蛋清蛋白的鸡蛋腐败菌S菌株的分离鉴定及其De novo测序分析

本实验采用传统微生物培养法从鸡蛋中分离出一株革兰氏阳性腐败菌S菌株,经形态学观察、生理生化分析及16S rDNA序列比对,对其进行了鉴定,并构建系统发育树。采用抑菌圈法及存活率试验探究该菌株对蛋清环境的耐受性,并基于Illumina HiSeq测序平台对该菌株的全基因组进行了De novo测序,获得基因组框架图。结果表明,该菌株生理生化反应特征与皮生球菌属相似,且16S rDNA序列与Dermacoccus abyssi(登录号AY894323)的同源性高达99%,鉴定为深渊皮生球菌(Dermacoccus abyssi)。该菌株在含有蛋清或溶菌酶的试管中能生长,在添加了蛋清或溶菌酶的孔径中没有抑菌圈产生。对蛋白编码基因的功能注释结果显示,细菌参与膜转运、离子结合、氨基酸和碳水化合物代谢及能量转换相关的基因丰富,推测其在鸡蛋中可能通过调节相关基因来耐受蛋清抑菌环境。该菌株对蛋清及溶菌酶的耐受性,为其在鸡蛋中的存活提供了有利条件。     

Separation of Egg White Lysozyme by Anionic Polysaccharides

Anionic polysaccharides (Na-alginate, Na-carboxyl-methyl-cellulose, high methoxy pectin and κ-carrageenan) were evaluated for their suitability towards simple and more efficient separation of lysozyme from salted or fresh duck eggs and hen egg whites for use in foods. Only κ-carrageenan interacted and formed precipitates with lysozyme. Recovery of lysozyme from fivefold diluted salted duck egg whites was 60–65% and 78–81% from fresh duck and hen egg whites using 0.7%κ-carrageenan. The recovered lysozyme from the salted duck egg whites was stable during storage at 4°C for 35 days.     

Modification of Egg White Proteins with Oleic Acid

Studies on the chemical modification of egg white with oleic acid (5-50 moles/50,000g of egg white protein) revealed that the reagent partitioned equally between supernatant and precipitate. The mole ratio of oleic acid to protein in solution at the 20 mole level of treatment was 14.6:1. Oleic acid did not selectively precipitate ovalbumin, conalbumin, or lysozyme. An increase in negative charge of proteins was observed in the chromatograms of treated egg white. No difference in molecular weights of treated egg white proteins was observed. The viscosity varied with the concentration of the reagent. In cooked, frozen, and thawed egg white water retaining index was 1.5-10X greater for treated than untreated material.     

Production of corn zein microparticles with loaded lysozyme directly extracted from hen egg white using spray drying: Extraction studies

Feasibility of achieving sustained release of lysozyme by encapsulation in corn zein using spray drying was examined. To reduce the materials cost, this part of work focused on partially purifying lysozyme from hen egg white using 30–90% (v/v) aqueous ethanol, adjusted to pH 3.0–9.24. After extraction for up to 24 h and centrifugation, the purification performance was evaluated for the supernatant. Extraction was insignificantly affected by kinetics (P = 0.6186) but was inefficient at pH above 5.0 and above 60% ethanol. The optimal extraction was achieved at 50% ethanol and pH 3.5. Further, most of the lysozyme precipitated from the 50% ethanol (at pH 3.5) extract after increasing ethanol concentration to 90% but was completely recovered after diluting the precipitate back to 50% ethanol. Findings from this work may lead to low-cost encapsulation technologies using partially-purified lysozyme, such as spray drying.     

Enzymatic Hydrolysis of Recovered Protein from Frozen Small Croaker and Functional Properties of Its Hydrolysates

ABSTRACT:  Fish protein isolate were recovered from frozen small croaker using pH shift. The partial enzymatic hydrolysates were fractionated as soluble and insoluble parts. They were dried using the drum dryer and their functional properties were examined. The total nitrogen content of the enzymatic hydrolysates ranged from 12.9% to 13.7%. The degree of hydrolysis of precipitates was 18.2% and 12.2% for croaker hydrolysates treated with Protamex 1.5 MG ( Bacilllus  protease complex) and Flavourzyme 500 MG (endoproteases and exoproteases, Aspergillus oryzae ), respectively. The TCA supernatant, after centrifugation of hydrolysates, contained numerous peptides ranging from 100 to 4000 daltons. The solubility of the supernatants was higher than that of the precipitates at 0% to 3% NaCl and pH 2 to 10. The precipitate of Flavourzyme- and Protamex-treated hydrolysates showed a high emulsion activity index value compared to egg white and bovine plasma protein. In addition, the highest emulsion stability was observed for Protamex-treated precipitate hydrolysates. Emulsion stability of Protamex-treated precipitate hydrolysates was comparable to those of protein additives (egg white, bovine plasma protein, and soy protein concentrate). Water and fat binding capacity of precipitates were higher than those of supernatant. The results indicate that precipitate hydrolysate from undersized croaker can be used in processed muscle foods as a functional and nutritional ingredient.     

国产弱阳离子琼脂糖交换介质的性能及其用于蛋清溶菌酶的分离纯化

从理化性能、离子交换容量、静态和动态吸附性能等方面对国产快流速CM-琼脂糖弱酸性阳离子交换介质进行了综合考察和评价,并应用该介质进行了鸡蛋清中溶茵酶的分离.结果发现,国产介质的理化性能符合要求,离子交换容量达到了187 mmol/mL,高于国外商品介质;对模型蛋白溶菌酶的静态和动态吸附容量分别达到了107.0 mg/mL和90.3 mg/mL(高流速下),同样略高于国外商品介质,显示能够满足蛋白质的有效吸附;离子交换层析分离蛋清溶菌酶较为成功,纯度在95%以上,产量达到了4.1 mg/mL鸡蛋清.     

Lysozyme in Wine: An Overview of Current and Future Applications

Lysozyme, a muramidase enzyme from egg whites (EC 3.2.1.17), is widely used in soluble form to control lactic acid bacteria in different foods. Moreover, hen egg white lysozyme is a hydrolytic enzyme that can be used to the control malolactic fermentation (MLF) during winemaking. MLF is only desirable in red and in some white wine, this suggest that MLF is at fault and needs to be controlled in all other types of wine. Lysozyme exhibits selective antimicrobial activity based on the hydrolysis of peptidoglycan cell wall constituents in lactic acid bacteria. In the last decade, several studies identified allergic reactions due to the presence of lysozyme in food. Given this relatively high incidence of lysozyme sensitization, and in accordance with the recently changed EC food legislation (1266/2010/CE), the use of lysozyme as an additive has to be declared on the ingredient label. To overcome this problem, the immobilization of the enzyme on insoluble supports, which allows the enzyme to be removed, has been the preferred strategy. In this context, this article offers a review of reports on lysozyme enological use over the last decade. It surveys the immobilization techniques and support materials used for lysozyme food preservation. This study attempts to provide useful guidance from the wealth of available immobilization data in the literature and, more importantly, to develop an integrated perspective on how to customize lysozyme for future enological uses.     

A New Protein Band Appearing in the Electrophoretic Pattern of Egg White Heated at Below 60°C

When egg white was heated to between 50° and 60°C, a new protein band appeared in its polyacrylamide gel electrophoretic pattern. The formation of the protein band in stored egg white was almost the same as that of fresh egg white. This protein band was shown to be induced by the interaction between macroglobulin and lysozyme during the egg white heating. The optimum temperature for its formation was between 54°C and 57°C. This band formation may be used as an indicator for heat treatment of egg white.