【作者】 李萍；

【导师】 王元秀；

【作者基本信息】 济南大学 ， 生物工程， 2014， 硕士

【摘要】 大豆肽是一种生物活性肽，是大豆肽蛋白水解的产物。具有抗疲劳、抗氧化、降血压、促进脂肪代谢等生理功能，但是由于大豆蛋白水解产生的大豆肽往往带有苦味，目前只应用于养殖、化妆品、医药领域，严重限制了其在食品领域的应用。为了使其具有良好的口味，拓宽其应用领域，需对其进行脱苦处理。目前用于大豆肽脱苦的方法，虽然会大大降低大豆肽的苦味，但是会造成较大的氮损失，例如：活性炭吸附、超滤、有机溶剂提取。通过文献了解到，柑桔汁脱苦常用β-环状糊精包埋法，核桃蛋白肽脱苦常用酶解法，牛乳蛋白脱苦常用微生物法，所以本实验采用了β-环状糊精、风味蛋白酶和碱性蛋白酶复合和黑曲霉对大豆肽进行脱苦，并且通过比较脱苦前后大豆肽中多肽分子量的变化、多肽含量的变化、生理功能的变化，比较了以上三种方法的脱苦效果，得到了脱苦的同时不会造成大豆肽功能损失的脱苦工艺。实验探究了运用β-环状糊精对大豆肽进行脱苦处理。利用单因素实验和响应面设计探究了β-环状糊精脱苦大豆肽的最佳条件：pH6.7、温度40.0℃、β-环状糊精与大豆肽的质量比为1:4，反应时间40.0min。在此条件下，苦味值约为3，能够降低3~4个苦味值。与传统脱苦大豆肽的方法相比较，运用β-环状糊精对大豆肽进行脱苦不会造成功能肽的损失，即不会造成能源的浪费。但是，β-环状糊精的加入会产生一定的焦糊味，影响了其风味。实验探究了风味蛋白酶和碱性蛋白酶复合酶解对大豆肽进行脱苦处理。通过水解度实验确定了碱性蛋白酶和风味蛋白酶为实验用酶。利用单因素实验和响应面设计探究了复合酶脱苦大豆肽的最佳条件：pH7.2，温度50.0℃，加酶量6000U/g，风味蛋白酶与碱性蛋白酶酶单位配比3:2，酶解时间3h。在此条件下，大豆肽的口味得到了明显的改善。凝胶层析图谱显示，脱苦后的大豆肽分子量明显减小，同时苦味值明显下降，说明蛋白酶作用于苦味氨基酸的部位，使肽链缩短了。脱苦前后多肽含量变化很小，仅损失了2.3%。同时，抗疲劳生理功能实验显示，酶解脱苦组与未脱苦组相比较，游泳时间短5.8%（p>0.40）、肝糖原剩余量低3.8%（p>0.50）、肌糖原剩余量低13.2%（p>0.08）、血尿素氮含量高6.4%（p>0.30）、血乳酸含量高2.8%（p>0.50），说明复合酶解脱苦前后大豆肽生理功能无显著性差异。与传统酶解法脱苦大豆肽相比较，本实验运用了外切酶和内切酶联合脱苦大豆肽，实验结果表明，此种方法脱苦的效果比单独运用外切酶对大豆肽进行脱苦的效果好一些。实验探究了以黑曲霉制备酸性蛋白酶，然后运用酸性蛋白酶对大豆肽进行脱苦处理。首先用固体培养基活化黑曲霉菌株，然后用液体培养基对黑曲霉进行扩大培养，接下来用麸皮培养基对黑曲霉进行发酵使其产生酸性蛋白酶，通过得到的酸性蛋白酶滤液对大豆肽进行脱苦处理。利用单因素实验和响应面设计探究了黑曲霉产酸性蛋白酶脱苦大豆肽的最佳条件：加酶量1:10、pH6.0、时间2.1h、温度34.0℃。在此条件下，苦味值约为1，能够降低4~5个苦味值，脱苦效果非常明显。凝胶层析图谱显示，脱苦后大豆肽中产生了一些中间分子量的小肽，初步估计黑曲霉产的酸性蛋白酶作用于苦味氨基酸存在的部位，使肽链缩短了。脱苦前后几乎未造成多肽的损失，损失率为0.13%。同时，抗疲劳生理功能实验显示，黑曲霉脱苦组与未脱苦组相比较，游泳时间短3.8%（p>0.40）、肝糖原剩余量低0.42%（p>0.80）、肌糖原剩余量低7.8%（p>0.10）、血尿素氮含量高3.8%（p>0.30）、血乳酸含量高2.3%（p>0.50），说明黑曲霉脱苦前后大豆肽生理功能无显著性差异。从实验所耗资源上讲，运用黑曲霉脱苦大豆肽的方法明显优于其他的方法，而且肽损失几乎为零。以上三种方法，脱苦效果都优于传统脱苦大豆肽的方法。但是β-环状糊精脱苦会造成最终溶液中掺杂一定的焦糊味，所以说效果不是很好。黑曲霉产酸性蛋白酶脱苦效果不错，而且成本也比酶解法低很多，多肽的损失较少且小于酶解法，所以确定微生物法是脱苦大豆肽的最好方法。更多还原

【Abstract】 Soybean peptide is a kind of biological active peptide from hydrolyzing soyabean protein.But because of its heavy bitter taste, only be used in aquaculture, cosmetics, medicine fieldcurrently, limitting its application in the field of food severely. In order to making it havegood taste and improving their application, taking off its bitter taste is a very important work.Traditional methods of debitterizing soybean peptides can cause of soybean peptide functionloss. This experiment adopted three methods to take off the bitter of soybean peptide:β-Cyclodextrine method, enzymatic hydrolysis and microbial method. And by comparing thepeptide molecular weight in the soybean peptide, peptide content changes, the change ofphysiological function between before and after being debitterized, compareing thedebitterizing effect of three methods above. Getting to the debitterizing process that will takeoff the bitter at the same time will not result in a loss of the function of soybean peptide.Experiment explores the method of debitterizing uesd by β-Cyclodextrine. Using singlefactor experiment and response surface experiment explores the best condition of debitterizingsoybean peptides uesd by β-Cyclodextrine: pH6.7, hydrolysis temperature40℃, quality ratioof β-Cyclodextrine to soybean peptides1:4, and time40min. Under this condition, the tasteof the soybean peptide get some improvement. The downside is that, the addition ofβ-Cyclodextrine will produce certain anxious burnt flavour, affectting its flavor.Experiment explores the method of debitterizing uesd by composite enzyme solutionwith Protex51FP and Protex6L. First of all, determing the Protex6L and Protex51FP forexperiment through the experiments about degree of hydrolysis. Using single factorexperiment and response surface experiment explores the best condition of debitterizingsoybean peptides uesd by complex enzyme: pH7.2, hydrolysis temperature50℃, activity ratioof Protex51FP to Protex6L3:2, total enzyme dose6000U/g, and hydrolysis time3h. Underthis condition, the taste of the soybean peptide get some improvement obviously. Thespectrogram of gel chromatography show that, the molecular weight of soybean peptide afterdebitterizing has been smaller obviously, instructing that, the protease play a role to the aminoacids about of bitter taste, shortening the peptide chain. The peptide content changed little between before and after being debitterized, lost only2.3%. At the same time, the anti-fatiguephysiological experiment also showed that, compared with no debitterizing group, compositeenzyme bitter group show: swimming short time by5.8%(p>0.40), low residual amount ofhepatic glycogen of3.8%(p>0.50), low muscle glycogen amount remaining13.2%(p>0.08),blood urea nitrogen content is6.4%(p>0.30), blood lactic acid content is2.8%(p>0.50), Thissuggests that there was no significant difference of aoybean peptide physiological functionbetween before and after being debitterized used by composite enzyme. Compared with thetraditional debitterizing soybean peptide enzyme method, this study used a combination ofenzyme and enzyme debitterizing soybean peptide, the experimental results show that thedebitterizing effect of this method is better than the single use of external enzyme for soypeptideExperiment explores the method of debitterizing: preparing acid protease fromfermenting Aspergillus Niger, then using the acid protease to debitter soy peptide. First of all,activating the Aspergillus Niger strains used by the solid medium. Then, enlarging theAspergillus Niger strains used by the fluid medium. Next, fermenting Aspergillus Nigerstrains used by the bran to produce the acid protease. Finally, debitterizing the soybeanpeptides with the acid protease filtrate. Using single factor experiment and response surfaceexperiment explores the best condition of debitterizing soybean peptides used by acidprotease from fermenting Aspergillus Niger strains: pH6.0, hydrolysis temperature50℃andhydrolysis time2.1h. Under this condition, the bitter taste value is about1, reducing4~5bitter taste value, the effect of debitteration is very obvious. The spectrogram of gelchromatography show that, the molecular weight of soybean peptide after debitterizing hasbeen smaller obviously, instructing that, the acid protease from Aspergillus Niger play a roleto the amino acids about of bitter taste, shortening the peptide chain. Debitterizing almost didnot cause loss of polypeptide between before and after being debitterized, lost only0.13%. Atthe same time, the anti-fatigue physiological experiment also showed that, compared with nodebitterizing group, Aspergillus niger debitterizing group show: swimming short time by3.8%(p>0.40), low residual amount of hepatic glycogen of0.42%(p>0.80), low muscleglycogen amount remaining7.8%(p>0.10), blood urea nitrogen content is3.8%(p>0.30),blood lactic acid content is2.3%(p>0.50), This suggests that there was no significant difference of soybean peptide physiological function between before and after beingdebitterized used by Aspergillus niger. From the energy consumption into consideration,using Aspergillus niger to debittere soybean peptide has an obvious advantage over othermethods. But says from the debitterizing effect, this method is a little lower than directenzymatic hydrolysis. This is due to the fermentation of acid protease enzyme activity is low.If optimizing the conditions of enzyme production further, then we can get the Aspergillusniger strain of producing higher enzyme activity. Believe that, microbial debitterizing methodwill be one of the best debitterizing methods, not only debitterizing effect is good, but alsoconsume less energy and experiments.The three methods above, effect of debitterizing are superior to traditional methods. Butthe addition of β-Cyclodextrine can dope certain anxious burnt flavour, so the effect is notvery good. The debitterizing effect of acid protease from Aspergillus Niger is good. And thecost is much lower than that of enzymatic hydrolysis. The loss of the polypeptide is less andless than enzymatic hydrolysis. So make sure the microbial method is the best way todebitterizing soy peptide.更多还原