混合菌固态发酵豆粕制备大豆活性肽

以枯草芽孢杆菌(Bacillus subtilis J3)和植物乳杆菌(Lactobacillus plantarum JNX)为发酵菌株,以发酵产物中小肽含量和挥发性盐基氮含量为检测参数,优化了混合菌固态发酵豆粕以制备大豆肽的生产工艺参数,并对发酵产生的大豆肽的性质进行了初步研究。确定的固体发酵工艺为:植物乳杆菌和枯草芽孢杆菌菌种比例为2∶1、料水比1∶0.6、接种量6%、30℃发酵24 h,该条件下发酵产物中小肽含量最高为10.64%,挥发性盐基氮含量最低为50.70 mg/100 g。SDS-PAGE电泳结果表明,发酵后豆粕提取液中的蛋白类产物的分子质量均为10k Da以下,其抗氧化活性最高可达65.76%。氨基酸组成成分分析表明,混合菌固态发酵豆粕后的提取液中甲硫氨酸、亮氨酸、缬氨酸、苯丙氨酸和异亮氨酸等必需氨基酸的含量提高了5倍以上。     

糖基化改性对大豆蛋白抗原性及结构特性的影响研究

本文以大豆分离蛋白和葡聚糖为原料,在干热条件下进行美拉德反应,制取不同时间下的糖基化复合物。以β-伴大豆球蛋白和大豆球蛋白抗原抑制率为指标,采用间接竞争ELISA方法测定糖基化产物的抗原性,在反应6 d时,糖基化产物中β-伴大豆球蛋白和大豆球蛋白的抗原性分别降低了31.94%和21.26%。糖基化产物颜色加深,且游离氨基含量降低,说明大豆蛋白与糖发生了不同程度的反应。红外光谱中糖链的引入,使蛋白质分子展开,β-转角和无规则卷曲结构含量的降低,影响了β-伴大豆球蛋白α亚基的抗原表位,从而可能使大豆蛋白的抗原性降低。糖基化反应影响抗原性的关键作用在于蛋白与糖结合部位对蛋白质结构的变化。     

枯草芽孢杆菌碱性蛋白酶基因在酿酒酵母中的表达和应用

将优化后的枯草芽孢杆菌碱性蛋白酶(serine alkaline protease,SAP)基因克隆到酿酒酵母表达载体pYES2上,在酿酒酵母Whu2d中进行表达。发酵酿酒酵母工程菌,对重组碱性蛋白酶酶学性质和降解豆粕中蛋白类抗营养因子效果进行研究。结果显示:工程菌发酵液中碱性蛋白酶酶活比出发菌株高55.2%。其最适反应温度和pH分别为45℃和8.0,在pH值为7.0~10.5时具有较好的稳定性,相对酶活可以保持在80%以上。与出发菌株相比,工程菌发酵后豆粕培养基中酸溶蛋白含量提高42.1%,粗蛋白和β-伴大豆球蛋白含量无显著差异,大豆球蛋白和胰蛋白酶抑制因子的含量分别降低41.9%和57.4%。以上结果表明:该碱性蛋白酶酿酒酵母工程菌可以用于饲料、食品等富含蛋白原料的加工。     

糖基化改性对大豆蛋白抗原性及结构特性的影响

以大豆分离蛋白和葡聚糖为原料,在干热条件下进行美拉德反应,制取不同时间下的糖基化复合物。以β-伴大豆球蛋白和大豆球蛋白抗原抑制率为指标,采用间接竞争ELISA方法测定糖基化产物的抗原性,在反应6 d时,糖基化产物中β-伴大豆球蛋白和大豆球蛋白的抗原性分别降低了36.90%和18.12%。糖基化产物颜色加深,且游离氨基含量降低,说明大豆蛋白与糖发生了不同程度的反应。红外光谱中糖链的引入,使蛋白质分子展开,β-转角和无规则卷曲结构含量降低,影响了β-伴大豆球蛋白α亚基的抗原表位,从而可能使大豆蛋白的抗原性降低。糖基化反应影响抗原性的关键作用在于蛋白与糖结合部位对蛋白质结构变化的影响。     

加热联合酶解大豆7S蛋白的分子结构及抗原性研究

选用不同加热预处理条件、不同蛋白酶处理大豆7S蛋白,SDS-PAGE考察酶解后大豆7S蛋白的降解情况,间接竞争ELISA法考察酶解物的抗原性。结果表明:加热预处理联合酶解可显著增大β-伴大豆球蛋白的降解程度,β亚基消化程度得到极大提升,α亚基、α'亚基几乎完全被降解,在相同酶解时间下,胰蛋白酶的酶解速度快于胃蛋白酶;加热预处理联合酶解大幅降低了β-伴大豆球蛋白的抗原性,胰蛋白酶的效果优于胃蛋白酶,经80℃加热10 min预处理再经胰蛋白酶处理,大豆7S蛋白的抗原性降低了79.99%,而胃蛋白酶最优条件下最多仅降低50.4%; SDSPAGE结果表明,胃蛋白酶、胰蛋白酶酶解物中出现了抗消化肽段,经过质谱鉴定,均来自于β-伴大豆球蛋白的α亚基。     

大豆过敏原在低盐固态酱油酿造过程中的降解规律

为研发低致敏酱油,探究了低盐固态酱油酿造过程中大豆蛋白致敏原的变化规律。在建立实验室的模拟低盐固态酱油酿造的基础上,采集大豆未经处理、高压灭菌(121 ℃,8 min)、制曲阶段(44 h)、发酵阶段(30 d)、灭菌前和灭菌后等样品,利用聚丙烯酰胺凝胶电泳法(SDS-PAGE)测定酿造过程中蛋白的组成变化,用兔抗大豆多克隆抗体进行酶联免疫试验和免疫印迹实验分析发酵过程中大豆致敏原抗原性变化,用过敏病人血清进行酶联免疫试验测定大豆致敏原过敏原性变化。结果表明,经高压蒸煮、制曲、发酵和加热灭菌后,原料中的大豆蛋白条带减少或消失,其中制曲变化的最大。β-伴大豆球蛋白的α、α'亚基和大豆球蛋白的酸性亚基分别在制曲阶段开始降解,β-伴大豆球蛋白的β亚基及大豆球蛋白酸性亚基在制曲之后消失,大豆球蛋白的碱性亚基在整个酿造阶段变化不大。免疫印迹结果显示相同的结果,酿造过程中大豆过敏原的过敏性和抗原性逐渐降低,在发酵30 d后的生酱油中仍能在检测到大豆球蛋白,在灭菌后也没有完全降解,但是这些残留的大豆致敏原没有检测到IgE结合能力。酶联免疫试验结果表明,和原材料大豆相比,样品中的抗原性在经过4个酿造阶段高压蒸煮、制曲、发酵和加热灭菌之后分别下降了8.13%、39.00%、69.10%和87.06%,过敏原性分别下降了8.92%、71.66%、92.26%、98.45%。在酱油酿造过程中大豆致敏原逐步降解,制曲阶段对大豆致敏原的降解最大。酱油中仍残留有大豆球蛋白,但是没有检测残留蛋白的致敏性。     

混菌株固态发酵高温豆粕工艺优化

选出最佳的菌种组合黑曲霉、酿酒酵母菌、枯草芽孢杆菌、植物乳酸杆菌后,调整混合菌种比例为2∶2∶1∶1,菌株进行固态发酵高温豆粕的最佳工艺条件:接种量10%,料水比1∶3,发酵时间96 h,发酵温度33℃,p H7.0,Mg SO4·7H2O 0.04 g,Ca Cl20.02 g,KH2PO40.04 g。粗蛋白含量为54.22%,比原料豆粕中的增加了24.36%。抗原蛋白7S球蛋白亚基和11S酸性亚基大部分被混合菌株分泌的酶系降解为分子质量更小的蛋白亚基;氨基酸含量由发酵前的41.53%提高到的48.18%;胰蛋白酶抑制剂(TI)含量由发酵前的16.91 mg/g降到0.20 mg/g,粗蛋白和氨基酸含量大幅度提高,抗原蛋白大部分被降解,TI含量显著降低,营养价值得到了很大改善,且发酵产物有豆香味、黄褐色。     

发酵工艺条件对豆粕中过敏蛋白降解的影响

为了降低豆粕的致敏性,探讨生物发酵对豆粕中主要过敏原降解的影响。通过固态法发酵豆粕,采用单因素试验优化发酵工艺条件,并利用SDS-PAGE电泳评估发酵条件对豆粕中过敏蛋白降解的影响。结果表明:枯草芽孢杆菌:干酪乳杆菌:酵母菌接种比例为2:1:1,接种量为12%,发酵温度为30℃,豆粕含水量为60%,先接种枯草芽孢杆菌发酵24 h,再接种干酪乳杆菌和酵母菌继续发酵48 h后,发酵豆粕中过敏蛋白降解显著,确定该条件为优化的发酵条件。验证试验表明,在优化发酵条件下固态发酵可较好地降解豆粕中主要过敏原。     

产蛋白酶耐酸细菌的筛选鉴定及混菌固态发酵豆粕的初步试验

为筛选出与乳酸菌混合发酵豆粕效果较佳的菌株,以透明圈法分别从霉豆腐、发酵豆粕、酱油发酵原液等样品中初筛得到13株产蛋白酶菌株,经过复筛固态发酵豆粕酶活、小肽、水解度的测定和耐酸性实验,最终得到1株耐酸性较好且蛋白酶活力较高菌株A-12,经形态观察、生理生化实验和分子生物学实验,鉴定为枯草芽孢杆菌(Bacillus subtilis)。利用此菌株与实验室保藏的植物乳杆菌NCU116混合发酵,结果表明,发酵后豆粕中胰蛋白酶抑制因子降解量为98.8%,营养物质如粗蛋白和小肽的含量分别提高了11.04%和13.71%,而且豆粕具有酸香味儿,适口性得到改善;枯草芽孢杆菌A-12可以做为与乳酸菌混和发酵豆粕的1株参考菌株。     

大豆β-伴球蛋白多肽抗氧化活性研究

以大豆β-伴球蛋白为原料,利用枯草芽孢杆菌进行发酵制备抗氧化肽,研究发酵时间和浓度对大豆β-伴球蛋白肽抗氧化活性影响。结果表明,发酵40 h时,大豆β-伴球蛋白肽抗氧化活力最高,抑制自由基能力最强;在此最佳发酵时间下,当大豆β-伴球蛋白肽浓度为1.6 mg/mL时,对羟自由基清除率最大;浓度为2 mg/mL时,对超氧阴离子自由基清除能力最强。     

微生物固态发酵菜籽粕营养特性的研究

以普通菜籽粕为原料,选用枯草芽孢杆菌、地衣芽孢杆菌、植物乳杆菌、啤酒酵母、季也蒙毕赤酵母及黑曲霉等菌种,通过单菌株与混菌株发酵试验,研究发酵对菜籽粕中粗蛋白和抗营养因子含量的影响。结果表明,芽孢杆菌的蛋白酶活性高于其他菌株;混菌株发酵效果明显优于单菌株发酵;混菌株发酵中枯草芽孢杆菌、植物乳杆菌和啤酒酵母三菌种组合发酵能较好地提高菜籽粕作为饲料蛋白的品质,此时其粗蛋白质增加率和硫代葡萄糖苷(硫苷)、唑烷硫酮(OZT)、单宁、植酸降解率分别为5.37、93.44、99.99、34.86、18.15%(干基)。     

不同预处理对低温脱脂豆粕中大豆β－伴球蛋白功能性质的影响

采用新鲜低温脱脂豆粕、干热处理脱脂豆粕和溶剂浸提脱脂豆粕为原料制备大豆β-伴球蛋白,研究预处理对低温脱脂豆粕残余脂质含量和脂肪氧合酶酶活以及制备大豆β-伴球蛋白功能性质的影响。干热处理对低温脱脂豆粕中残余脂质含量无显著影响,但可使得脂肪氧合酶酶活下降7．69％;溶剂浸提使得低温脱脂豆粕中残余脂质含量和脂肪氧合酶酶活分别下降98．08％和96．12％,表明溶剂浸提可显著抑制低温脱脂豆粕中脂质过氧化反应的发生。干热处理低温脱脂豆粕使得制备大豆β-伴球蛋白溶解性、持帕性、持油性、乳化性、起泡性以及凝胶性质变差,溶剂浸提低温脱脂豆粕可改善制备大豆β-伴球蛋白溶解性、持抽性、持油性、乳化性、起泡性和凝胶性质。这一研究结果为改善大豆蛋白功能性质提供前期研究基础。     

Improvement of soybean meal quality by one-step fermentation with mixed-culture based on protease activity

Microbial fermentation can improve the quality of soybean meal by reducing the level of anti-nutrition factors and increasing the content of protein and small peptides. In this study, Bacillus velezensis, Enterococcus faecium and Saccharomyces boulardii were used for one-step mixed fermentation of soybean meal. The fermentation of soybean meal was carried out in an anaerobic bag using fermentation liquid of strains with maximum protease activity as inoculum and water. The results of process optimization showed that the mixture ratio of B. velezensis: E. faecium: S. boulardii was 37.5%:25%:37.5%. The three microorganisms added together as a mixture. Under the conditions of 55% inoculation with microorganisms mixture, 0.8% acid protease addition, 46 ℃ and 6 days, the quality of fermented soybean meal was significantly improved. The small peptides increased from 4.48% to 47.4%, the TCA solution protein increased from 44.3 g/kg to 255.2 g/kg, the antigenic protein glycinin decreased by 94.37% from 320.12 μg/mL to 18.02 μg/mL, and β-conglycinin decreased by 85.83% from 232.11 μg/mL to 32.89 μg/mL. Other anti-nutritional factors urease, phytic acid and stachystryose all decreased. The result indicated that it was feasible to improve the feed quality of soybean meal by one-step mixed fermentation based on protease activity.Industrial relevanceFermentation is the main method to improve the feed quality of soybean meal. Our study revealed that one-step mixed-culture fermentation based on microbial protease activity showed a significant effect on increasing beneficial small peptides, reducing anti-nutrient factors and enhancing palatability of soybean meal. This process simplified the operation of mixed fermentation and could be implemented in solid fermentation system of feed and food industry to improve the beneficial properties of products.     

豆粕混菌液体发酵制种技术试验研究

利用热带假丝酵母和枯草芽孢杆菌混菌发酵进行液体制种。结果表明:受接种量、豆粕含量和培养温度的影响,两株菌种菌数的增长速度具有相对一致的变化趋势;受培养时间的影响,热带假丝酵母菌先增后降,枯草芽孢杆菌先降后增;热带假丝酵母在pH3.5时菌数最大,枯草芽孢杆菌在pH4.5～7.5范围内适宜生长。在豆粕质量分数15%、葡萄糖质量分数1%、初始pH4.5的液体培养基上,两菌分别接种10%,在发酵温度28℃、摇床转速160r/min条件下培养5d,热带假丝酵母菌和枯草芽孢杆菌菌数达到最大,分别为752.5×106和264.2×107 cfu/ml。     

天然大豆蛋白的选择性酶解

研究了几种微生物蛋白酶水解天然大豆蛋白的选择性。用SDS-聚丙烯酰胺凝胶电泳分析酶解过程中大豆蛋白各组分的变化,电泳结果显示-βconglycinin比glycinin容易被蛋白酶水解,-βconglycinin的α’亚基比α亚基更容易水解,-βconglycinin的β亚基比α’亚基和α亚基更难水解;glycinin的酸性亚基比其碱性亚基更容易被水解。     

天然大豆蛋白的选择性酶解

研究了几种微生物蛋白酶水解天然大豆蛋白的选择性。用 DDD-聚丙烯酰胺凝胶电泳分析酶解过程中大豆蛋白各组分的变化,电泳结果显示β-conglycinin 比 glycinin 容易被蛋白酶水解,βconglycinin 的α’亚基比α亚基更容易水解,β-conglycinin的β亚基比α’亚基和α亚基更难水解；glycinin 的酸性亚基比其碱性亚基更容易被水解。     

Flavor Protein Interactions: Characteristics of 2-Nonanone Binding to Isolated Soy Protein Fractions

THE ABSORPTION COEFFICIENTS at 280 nm of 1% solutions of pure soy protein, β-conglycinin, glycinin, the acidic and basic sub-units of glycinin were 6.04, 4.4, 8.04, 7.18, and 8.8, respectively. Using equilibrium dialysis the binding affinities of these proteins for the model flavor compound 2-nonanone were determined. On an equivalent weight basis soy protein, β-conglycinin and glycinin had approximately 5,2 and 3 primary binding sites per 100,000 daltons and affinity constants (K) of 570, 3050 and 540 m^-1, respectively, i.e., β-conglycinin showed a fivefold greater affinity for nonanone than the other soy protein. The acidic and basic subunits showed binding behavior similar to that of glycinin.     

Effects of Heat and Ionic Strength Upon Dissociation-Association of Soybean Protein Fractions

The effects of heat and ionic strength upon dissociation or association of soybean proteins were characterized by an immunoelectrophoresis. Among thermal products of both glycinin and β-conglycinin, the dissociated subunits of β-conglycinin retained their antigenic reactivities, while the thermal products of glycinin lost their antigenic reactivities. Appearance of immunoprecipitin arcs was dependent on the ionic strength of the solutions. This is based on the conformational changes at the ionic strengths below 0.1 where the thermal denaturation of β-conglycinin transforms from association to dissociation. The immunoelectrophoretic results can be used to determine the ionic strength of the solution in complex systems including whole soybean extracts and soybean milk. Relationship between the differences in textural properties of tofu and conformational changes of soybean protein is discussed.     

Effect of soy protein subunit composition and processing conditions on stability and particle size distribution of soymilk

The influence of soy protein subunit composition on the particle size distribution and solid content of soymilk and various supernatant fractions was investigated. A well-established seed variety (Harovinton), containing all protein subunits, and eleven null soybean genotypes lacking specific glycinin and β-conglycinin subunits were investigated, to determine the effect of protein composition on the physico-chemical characteristic of soymilk. Soymilk made from Harovinton and soybean lines null in glycinin showed significantly higher total solid yields than the other genotypes evaluated. Soymilks prepared from soybeans null for glycinin or lacking glycinin's group I (A₁, A₂) showed a smaller particle size distribution compared to soybean lines containing glycinin. In general, unheated soymilk made from lines having glycinin showed a bimodal size distribution with large particles, with a decrease in the particle size distribution after heating and heating with homogenization. The results of this work will help in the evaluation of breeding lines with a specific protein composition tailored to a specific processing functionality.