摘要
花生粕是花生仁经压榨提炼油料后的副产品,目前主要应用在饲料中,有时甚至作为废料扔掉。抗氧化肽作为一种天然抗氧化剂,具有较好的抗氧化性和安全性,吸引了越来越多的关注。因此,充分利用花生粕制备抗氧化肽是重要的研究课题,具有较高的应用价值。
本文以花生粕为原料,利用筛选得到的枯草芽孢杆菌20029对花生粕进行发酵处理,得到了花生粕发酵液,通过响应面分析优化得到了制备抗氧化肽的最佳条件:pH值8、温度30℃、发酵时间49.5h,在此条件下,发酵液的DPPH自由基(DPPH?)清除率为63.28%。并对不同发酵时间的DPPH?清除率与多肽含量的相关性进行了研究,发现DPPH?清除率与多肽含量的相关性较好,P值为0.0010。
利用超滤对花生粕发酵液进行了粗分离,分离效果较好,超滤后,发酵液的抗氧化性变化不大。
采用DA201-C大孔吸附树脂对多肽进行了精制处理,发现吸附过程属于快速平衡吸附,动态吸附过程中,上样浓度、流速均对吸附的穿透点产生影响,热动力学研究发现,树脂对多肽的吸附符合Langmuir方程式,拟合相关系数均在0.98以上,利用25%～100%的乙醇对吸附后的树脂进行了洗脱,发现不同洗脱组分的氨基酸组成、分子量有较大差异,对不同组分的抗氧化活性进行了测定,发现75%乙醇洗脱组分(PMFH-75)的活性最强。
利用凝胶过滤、半制备RP-HPLC对PMFH-75进行了分离纯化,PMFH-75经凝胶过滤分为4个组分,其中F3的DPPH?清除率显著高于其他三个组分,并利用LC-MS对F3中的抗氧化多肽的组成和结构进行了分析。
最后对发酵后的残渣的成分进行了分析,在氨基酸组成方面,同花生粕原料进行了比较,发现残渣中的Arg的含量降低,Met的含量增加,蛋白质的营养价值得到一定程度上的提高。
Peanut meal, which is mainly applied in feed, sometimes even discarded as waste,is a by-product when peanut oil is extracted.Simultaneously.As natural antioxidants,antioxidant peptides have attracted increasing interests because of their safety and good antioxidative properties in recent years.Therefore, making full use of Peanut meal to prepare antioxidant peptide is an important research topic.It represents a significant value.
In this paper,the optimum conditions for producing the antioxidative peptide of peanut meal,using liquid state fermention by Bacillus subtilis20029 screened,were determined by response surface methodology as follows:temperature30℃,pH value 8,the period of fermentation 49.5h.Under the optimal condition,the DPPH radical-scavenging activity of fermentative product reached 63.28%.Under the different fermentation time,the correlation of DPPH radical (DPPH?) scavenging activity and the content of peptide was studied,there were strong relativities about DPPH radical (DPPH?) scavenging activity and the content of peptide,P=0.0010.
Using ultrafiltration to Peanut meal,the result showed it was a good method of separation. After ultrafiltration,there was no considerable change of the antioxidative activity of filtrate.
Peptide was desalted by DA201-C (macroporous adsorption resin).The results indicated that the adsorption of peptide belonged to rapid equilibrium adsorption.The breakthrough point was affected by the flow rate,concentration.The result of the research of thermal dynamics showed that the adsorption of peptides was in accordance with Langmuir equation,the correlation coefficient were all more than 0.98.The primary purification was carried out by ethanol with different concentration (25 %～100 %) according to their hydrophobic property,the fraction eluted by ethanol with 75% concentration showed the best antioxidant activity.the amino acid composition and molecular weight of each fraction were different with each other.
PMFH-75 was separated by Gel filtration chromatography and RP-HPLC, PMFH-75 was separated into four components by Gel filtration chromatography,The DPPH radical (DPPH?) scavenging activity of the F3 was significantly higher than the other three components. Meanwhile,the composition and structure of F3 were analyzed.
Finally,the ingredients of residue after fermentation were analyzed.In respect of the composition of amino acids,comparing to raw materials,the results showed that the content of Met increased,Arg decreased,but the nutritive value of protein was improved.
引文
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