【作者】 余勃；

【导师】 陆兆新；

【作者基本信息】 南京农业大学 ， 食品科学， 2006， 博士

【摘要】 大豆多肽是大豆蛋白经酸、碱或酶的水解作用后得到的由不同氨基酸组成的多肽链的通称，目前一般是指平均肽链长度为3～10个氨基酸残基，主要分子量范围为300～3000Da的低分子量短链肽。由于它们不仅具有良好的加工特性，还具有多种对人体有益的生理功能，因此它们也常被叫做大豆活性肽或大豆功能多肽等，并被视为一种很有前途的功能性食品而备受科研人员的关注。目前我国对大豆多肽的生产研究虽然很多，但尚未形成大规模工业化生产。其原因有两个：一在于生产成本居高不下；二蛋白酶解液往往产生苦味，严重影响了多肽制品的口味。为克服目前我国在大豆活性肽研究和产业化中的不足，本课题建立一套生物技术生产大豆活性肽的工艺。主要是通过微生物发酵法产酶水解豆粕中的大豆蛋白，以获得具有生理活性的大豆多肽；并通过数理优化，确立了合适的发酵工艺；同时研究了膜技术在活性肽分离纯化上的应用；对发酵菌株的产酶特性及脱苦原理进行了探讨；研究了发酵所得大豆多肽的生物活性和加工特性，并在此基础上开发出一种具有抗疲劳效果的大豆多肽功能性饮料。主要研究结果如下：1在参照常规酶水解蛋白工艺中蛋白水解度的测定方法基础上建立了一种适于微生物发酵水解蛋白工艺的水解度测定方法。本方法通过测定肽键的断裂数来反映蛋白被水解的程度，从原理上符合肽键被水解的规律，计算公式上反映了水解度的定义。操作简单，快速，重复性好。2研究了菌株Bacillus subtilis SHZ在发酵豆粕过程中代谢产酶水解大豆蛋白的动态。结果表明该菌株能产蛋白酶和羧肽酶，在发酵的0～8h，菌体主要分泌蛋白酶，将豆粕中的大豆蛋白初步水解成长链肽，发酵液略有苦昧；随着发酵的继续进行（8～16h），蛋白酶将长链肽进一步水解成段中短链肽，同时在外肽酶的作用下，肽链内部的疏水性氨基酸大量暴露，使得发酵液苦味激增；发酵中后期（24～32h），菌体开始大量分泌羧肽酶，其能从肽链的羧基末端将疏水性的氨基酸切除从而脱苦；至发酵后期（40～48h），发酵液中主要为非苦味的短链肽和游离氨基酸。控制发酵时间为36h，可以得到36h水解度为25％，分子量分布为300～1000Da的短肽链。3通过对比用碱性蛋白酶水解大豆蛋白所得到酶解物和用B．subtili SHZ发酵豆粕制备所得大豆多肽之间的苦味值和氨基酸组成，发现具有显著苦味的蛋白酶解液的氨基酸37.69％为疏水性氨基酸；而豆粕水解液中，疏水性氨基酸仅占总量的11.75％。利用B．subtili SHZ来发酵具有显著苦味的蛋白酶解液，发酵到20h，蛋白酶解液的苦味值由4降到0，苦味完全脱除，证实了发酵菌株具有脱苦作用并推断其中存在具有脱苦作用的肽酶产生体系。对B．subtili SHZ产肽酶特性进行进一步研究，发现其能在生长代谢过程中产生胞外的羧肽酶。对此羧肽酶进行初步分离纯化，经硫酸铵沉淀、乙醇分级沉淀和凝胶层析后，羧肽酶比活力由4.5提高到24.5，但酶蛋白和总酶活损失较大，回收率只有12.7％。将纯化后得到的B．subtili SHZ羧肽酶直接作用于苦味的蛋白酶解物上进行酶解，其能从苦味肽的羧基末端将苯丙氨酸、亮氨酸、酪氨酸、异亮氨酸等疏水性氨基酸切除，以游离态释放出来，从而生成不苦的酶解物，这就是B．subtili SHZ脱苦的机理。4为获得最高大豆多肽产量，采用响应曲面法对B．subtili SHZ发酵豆粕产大豆多肽的培养基组成和发酵条件进行了优化。首先采用Box-Behnken实验设计对培养基进行了优化，最终获得大豆多肽产量最高的发酵培养基组成为，豆粕（氮源）52～58g／l，葡萄糖（碳源）7～9g／l，KH₂PO₄（无机盐）3g／l。以此发酵培养基可以获得约为8mg／mL的大豆多肽，比优化前的平均6mg／mL提高了33％。接着在优化培养基的基础上，采用CCD实验设计对发酵条件进行了优化，最终获得大豆多肽产量最高的发酵条件为，发酵时间36h，温度37℃，发酵液初始pH7，摇床转速140～160rpm。以此发酵条件可以获得约为14mg／mL的大豆多肽，比上述的8mg／mL又提高了65％。按照此最优工艺发酵生产大豆多肽，豆粕中的大豆蛋白转化为大豆多肽的转化率高达60％，是常规酶解法的3倍。5研究了超滤对发酵液的纯化效果，结果表明在发酵液中，仅有0.85％的肽分子质量大于30KDa；有2.42％的肽分子质量在30～10KDa之间；而5～3KDa之间和小于5KDa的多肽分别占了总肽量20.4％和63.4％。同时生物活性测定表明，高分子量（≥10KDa）的多肽液自由基清除能力较低，而≤5KDa的多肽具有显著的自由基清除效果，特别是3KDa超滤膜透过液的O^-2·自由基和·OH自由基清除率分别高达49％和82％，比同浓度的天然抗氧化剂生育酚的自由基清除力还强。对超滤各参数的研究表明，选用料液浓度50mg／mL，料液温度30℃，操作压力0.08MPa能获得最佳膜通量和分离效果。随着截留分子量的不断降低，超滤液中肽的分子量在不断下降，纯度在逐步提高。经RP-HPLC验证，凝胶层析后所得具有最高生物活性的片段为单一多肽组分。6对发酵法制备的大豆多肽生物活性研究表明，其具有显著的自由基清除能力和体外抗氧化活性，活性随着纯化步骤的进行而不断提高，其中O^2-·清除能力从最初的49％提高到62％，·OH清除能力从78％提高到90％，相对抗氧化值从2.0提高到2.8，具有最强体外抗氧化能力的是分子量约为1，300Da的短链肽。其抗氧化能力是相同浓度生育酚的1.5倍。体内抗氧化实验表明，小鼠经灌胃大豆肽后，其血液中GSH-PX活力和SOD活力都得到了提高，MDA含量均低于标准饲料对照组，存在显著性差异（P＜0.01），表明大豆肽的体内抗氧化机理与其能提高生物体内的抗氧化酶活性有关。而灌胃中高剂量的大豆肽，降低MDA的效果比天然抗氧化剂生育酚（V_E）的效果还明显（P＜0.01）。对大豆多肽的体内抗疲劳实验表明，三个剂量组小鼠游泳时间均比对照组延长，大豆多肽中高剂量组能显著提高小鼠的肝糖原含量（p＜0.05），三个剂量组的小鼠在剧烈游泳后其血乳酸升高比值显著低于对照组，说明小鼠运动时疲劳增加延缓；而血乳酸清除比值均显著高于对照组（p＜0.01），说明其运动后疲劳消除加快。根据卫生部公布的保健食品评价指标，可判断发酵法制备所得大豆多肽具有抗疲劳的功能。7对发酵法制备的大豆多肽加工特性研究表明，其具有良好的溶解性，能在广泛的pH范围内保持较高的氮溶指数（NSI＞80％）。与大豆分离蛋白相比，具有良好的凝胶性，同时亲水性大为增加。与大豆蛋白相比，大豆多肽具有良好的乳化性能，并在广泛的pH范围内（pH2～8）均表现出最好的乳化性和乳化稳定性。同时具有更多短链肽的发酵大豆多肽其起泡性和泡沫稳定性都要优于酶解法所得大豆多肽。8以发酵法制备的大豆多肽为原料，开发研制了大豆多肽功能性饮料。其最佳配方是以大豆多肽液（酸溶性多肽含量16mg／mL）为母液，各种添加剂添加量（W／V）分别为β—环糊精0.4％、柠檬酸0.2％、蔗糖9％、橙汁香精0.4％。最佳灭菌工艺采用微波灭菌法（80℃，10min）。最佳贮藏条件为冷藏（4℃），在不添加任何稳定剂的情况下冷藏可放置2个月。功能性试验表明，以不同剂量的多肽饮料喂饮小鼠，实验小鼠游泳时间显著延长，其中高剂量组效果最为显著，与对照组相比，小鼠游泳时间延长了近一倍。表明该大豆多肽饮料具有一定的抗疲劳作用，更多还原

【Abstract】 Soybean peptides, which derived from hydrolysis of soybean protein by acid, alkali or enzyme,were the general name of a group of peptides chain with different amino acid composition. Themolecular weight rang of them was defined from 300 to 1000Da with 3～10 amino acid now. They werealso defined as soybean bioactivity peptide or soybean functional peptide due to their good processfunction and bioactivity beneficial to human body and focused by researchers for their potential offunctional food.There are lots of studies about the production of soybean peptide in China but the industry practicein large scale still under the way due to two reasons: 1 the cost of production is high.2 the bitterness ofhydrolyzate harm to the taste of peptide produce badly.We established a production technology of soybean peptide with biotechnique in order to overcomethe shortage of the studies and industry practices on soybean peptide in our country. The soybeanpeptide with bioactivity was produced by fermentation defatted soybean flour with microorganism. Asuitable technology of fermentation was established by optimization with response surface methodology.The purification of soybean bioactivity peptide by membrane technique was studied. The enzymeproduction and the mechanism of debitterness of fermentation strain were discussed. The bioactivity andprocess character of soybean peptides produced by fermentation was studied and a soybean peptidebeverage with antifatigue function was developed. The main results were described as following:1. A new method which was suitable for fermentation technology and used for detect the degree ofhydrolysis （DH） was established at the base of traditional method. The DH was measured by calculatingthe number of peptide-bond split in new method. It accord to the rule of protein hydrolysis at the theoryand reflect the defining of DH. This method proposed in this paper was shown to be direct, sensitive,accurate and reproducible. 2. The hydrolysis dynamic of soybean protein in defatted soybean peptide under the effect of enzymeproduced by Bacillus subtiliss SHZ was studied. Results showed that B. subtiliss SHZ can yieldedproteinase and carboxypeptidase, at the beginning of fermentation （0～8h）, the strain mainly yieldedproteinase which can hydrolyzed the soybean protein into long chain peptide with slight bitterness;during 8～16h of fermentation, long chain peptide were further hydrolyzed into middle and short chainpeptide. The amino acids were exposed at the effect of carboxypeptidase which resulted to bitternessincreasing. During 24～32h, strain yielded Carboxypeptidase in larger number which can cut down theamino acids from the C-terminal of peptide and at the end of fermentation, there are mainly short chainpeptidase and free amino acid with low bitterness. When fermentation for 36h, the DH was 25% andmolecular weight range of soybean peptide was from 300 to 1000Da.3. The bitterness value and amino acid composition of enzyme hydrolyzate of soybean protein andsoybean peptides from the fermented liquid of defatted soybean flour were compared each other. It wasfound that there are 37.69% of hydrophobe amino acid occured in the form of peptide in enzymehydrolyzate which exhibit strong bitterness. While there is 11.75% of hydrophobe amino acid infermented liquid of defatted soybean flour and they are mainly in the form of free amino acid. Thebitterness values of enzyme hydrolyzate were reduced from 4 to 0 when fermented with B. subtilis SHZfor 20h. It proved the debitter ability of B. subtilis SHZ and it may be the effect of carboxypeptidaseproduced from B. subtilis SHZ. The enzyme analysis of strain showed that the strain can excretecarboxypeptidase during growth. After purified, the special activity of the carboxypeptidase from B.subtilis SHZ were increase from 4.5 to 24.5, but the loss of enzyme protein and total carboxypeptidaseactivity is huge, only 12.7% were reclaimed. The amino acid such as Phe, Ala, Tyr and Leu at theC-terminal of peptide of enzyme hydrolyzate can be cut down by the purified carboxypeptidase from B.subtiliss SHZ and result to nonbitternss. This is the main mechanism of debitterness of B. subtiliss SHZ.4. In order to obtain the highest yield of soybean peptides, the medium composition and fermentationconditions of B. subtiliss SHZ were optimization by the response surface methodology. First, themedium composition were optimized used Box-Behnken design and the optimal medium compositionwere defatted soybean flour 52～58 g/l, glucose 7～9g/l, KH₂PO₄ 3g/l. It can obtain 8mg/mL soybeanpeptide at this medium and it was increased about 33% compared with the 6mg/mL before optimization.The fermentation conditions were also optimized used central composition design （CCD） and theoptimal conditions were fermentation time 36h, temperature 37℃, initial pH 7 and rotary speed was140～160rpm. The yield of soybean peptides can attain 14mg/mL at this optimal condition and it was increased about 65% compared with the 8 mg/mL which attained before. The transferring ratio ofsoybean protein to soybean peptides was 60% at optimal medium composition and fermentationconditions which is three times compared with traditional enzyme technique.5. The purification effect of ultrafiltration of fermented liquid was studied. Results showed that, thereare 0.85% of peptide with molecular weight over 30KDa, 2.42% of peptide with molecular weight from30KDa to 10KDa, 20.4% of peptide with molecular weight from 5KDa to 3KDa and 63.42% of peptidewith molecular weight less than 3KDa. The bioactivity analysis showed that the peptide with highmolecular weight （≥10KDa） exhibit low free radical scavenging ability. The peptide with molecularweight≤5KDa exhibited strong free radical scavenging ability. The scavenging ability to O^-2·and·OHof peptide with molecular weight≤3KDa were 49%and 82%, respectively. The optimal flux andisolation effect can be attained at liquid concentration 50mg/mL, liquid temperature 30℃, operatedpressure 0.08MPa. The molecular weight distribution of ultrafiltration fraction showed that the peptidechain was shorter as the cut-off molecular weight decrease and the purity was also increased. The gelfraction of soybean peptides with the highest bioactivity was a single peptide proved by RP-HPLC.6. The study of bioactivity of soybean peptides from fermentation showed that the soybean peptides havesignificant free radical scavenging ability and antioxidant activity in vitro. As the purification developing,the scavenging ability of O2—·was increased from 49% to 62%, the scavenging ability of·OH wasincreased from 78% to 90% and the relative antioxidant value was increased from 2.0 to 2.8. Themolecular weight of the peptide with the most antioxidant activity in vitro is about 1,300Da. The resultsof antioxidant experimental in vivo showed that the activity of GSH-PX and SOD in blood of mouseincreased and the MDA content in heart of mouse decreased after being feed with different doze soybeanpeptide （P＜0.01）. The increasing of GSH-PX and SOD in blood serum indicated that the antioxidantactivity of soybean peptide were related to the increase the activity of antioxidant enzyme in vivo. Theantifatigue activity in vivo of soybean peptide produced by fermentation was examined and approved byanimal model. The swimming time of rat feed with different doze of soybean peptide were increasedmarkedly approved the anti-fatigue effect of soybean peptide. The glycogen content of rat increasedmarkedly showed that the anti-fatigue effect was related to the increasing of energy storage. Theincreasing of lactic acid in the blood of rat was deferred and the reduction of lactic acid in the blood ofrat was accelerated after being feed with different doze of soybean peptides. The conclusion can be drawthat the soybean peptides produced by fermentation have the antifatigue function according to theevaluation index of health food published by sanitation ministry. 7. The study of process character of soybean peptides from fermentation showed that the soybeanpeptides have good solubility and can keep the high NSI over 80% at abrooad pH range. Its gelation andhydrophile ability were increased compared with soybean protein. At the same time, soybean peptideshave good emulsification and can keep the good EAI and ESI at pH from 2～8. The soybean peptideswith more short chain peptides produced by fermentation have higher foaming and foaming stabilitycompared with the soybean peptides produced by enzymatic hydrolysis.8. A functional beverage based on the soybean peptides produced by fermentation was developed. Theoptimal prescription was following:β-dextrin 0.4%, citric acid 0.2%, sucrose 9%, OJ essence 0.4%.The optimal sterilization technique was microwave sterilization （80℃, 10min） and the optimal storecondition wascold storage at 4℃. The beverage can keep constant at this condition for 2 monthswithout any additive. The results of animal experiment showed that the swimming time of mice feedwith different doze of beverage increased significantly and the swimming time of high doze group wastwo times compared with check group. It can be conclude that the beverage have some antifatiguefunction.更多还原