菌酶协同处理豆粕制备饲用肽的研究
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摘要
豆粕是大豆榨油后的副产品,粗蛋白含量在43-48%之间,必需氨基酸含量高、氨基酸组成合理,是目前使用最多、最广泛的植物性蛋白质原料。饲用肽泛指寡肽和小肽,是指由2个到50个氨基酸以肽键相连的化合物,相对分子质量在1-10 kD之间。本研究采用微生物法和酶解法处理豆粕制备饲用肽,将豆粕中的大分子蛋白降解成小分子肽,有效提高发酵豆粕产品中的肽含量,提高豆粕蛋白质消化率,从而改善豆粕营养价值,对弥补我国蛋白饲料尤其是优质蛋白资源的短缺,降低养殖业生产成本,促进畜牧业增产、增效具有重大的现实意义。
本研究从实验室保藏的8株枯草芽孢杆菌中筛选到了1株能有效降解豆粕中大分子蛋白为小分子肽的菌株BSW-2,豆粕经过该菌发酵后,肽含量从原豆粕13.35mg/g提高到发酵后108.10 mg/g。同时,本研究还筛选到了1株能有效提高发酵豆粕粗蛋白含量的酵母菌YT,经过YT发酵后,粗蛋白从原料豆粕47.97%提高到55.32%。
随后,通过正交试验建立了芽孢杆菌BSW-2、酵母菌YT混菌发酵豆粕的工艺,工艺条件为混合菌液接种量1.5%,料水比1：1.0,发酵温度30℃,发酵时间36 h。发酵后样品肽含量从发酵前的13.35 mg/g提高到184.57 mg/g,粗蛋白含量从47.62%提高到56.38%,蛋白质体外消化率从81.86%提高到91.99%,干物质体外损耗率从66.00%提高到71.40%,乳酸含量为2.65%,脲酶活性从0.45 U降低到0.15 U,抗原蛋白及大分子蛋白得到有效降解。
另一方面,试验选择了中性蛋白酶作为豆粕酶解用酶,确定其酶解工艺为加酶量400u/g、料水比1：1.6、酶解温度50℃、酶解时间48 h。酶解豆粕样品肽含量从发酵前的13.35 mg/g提高到185.72 mg/g,粗蛋白含量从47.62%提高到52.59%,抗原蛋白及大分子蛋白也得到有效降解。
最后,建立了菌酶协同处理豆粕的工艺,确定工艺条件为混合菌接种量1.5%、中性蛋白酶添加酶量450u/g、料水比1：1.4、处理温度40℃、处理时间48 h。在此条件下,豆粕经菌酶协同处理后,各项指标均优于混菌发酵和中性蛋白酶酶解,其中肽含量从13.35 mg/g提高到199.65 mg/g,粗蛋白含量从47.62%提高到56.72%,蛋白质体外消化率从81.86%提高到92.79%,干物质体外损耗率从66.00%提高到72.30%,乳酸含量为2.75%,脲酶活性从0.45 U降低到0.086 U,抗原蛋白及大分子蛋白得到有效降解。
采用PCR-DGGE方法分析菌酶协同处理豆粕过程中细菌群落的变化,结果表明1.5%的接种量可以使枯草芽孢杆菌BSW-2从发酵开始就在整个发酵体系中占优势。随着发酵时间的延长,BSW-2的生长会抑制其它细菌的生长,到发酵后期整个发酵体系中,BSW-2占有绝对的优势。
Soybean meal, a by-product after oil extraction, crude protein content of 43-48%, high content of essential amino acids, is the most widely used source of dietary protein in the poultry and swine industry. Small peptide is compounds of 2 or more amino acids linked by peptide bonds, it's molecular weight between 1-2 kD. In this study, microbiological methods and enzymatically methods was used to treated soybean meal, to increase the content of peptides. The study is of great practical significance to make up of high-quality protein feed, especially the shortage of protein resources, to reduce farming cost of production, to promote livestock output.
One strain of Bacillus subtilis BSW-2 was screened to degrade protein macromolecules of soybean meal into small peptides from 8 strains preserved by our laboratory. After fermented, the content of peptide increased from 13.35 mg/g to 108.10 mg/g. One strain of yeast YT was also screened to improve the content of crude protein. After fermented, the crude protein content increased from 47.62% to 55.32%.
The process condition of soybean meal fermentation by BSW-2 and YT was established. The results showed that the optimum fermentation parameters were mixed inoculation volume 1.5%, ratio of material to water 1:1.0, fermentation temperature 30℃, fermentation time 36 h. After fermentation, the content of peptides increased from 13.35 mg/g to 184.57 mg/g. The crude protein content increased from 47.62% to 56.38%. The vitro digestibility of protein increased from 81.86% to 91.99%. The dry matter digestibility vitro increased from 66.00% to 71.40%. The content of lactic was 2.65%. The urease activity decreased from 0.45 U to 0.15 U. The results of SDS-PAGE shows that the protein macromolecular and the antigen protein was degraded.
The neutral protease was screened and the condition of enzymatic hydrolysis was established. The results showed that the optimum condition were neutral protease 400 u/g, ratio of material to water 1:1.6, reaction temperature 50℃, hydrolysis time 48 h. Under that condition, the content of peptides increased from 13.35 mg/g to 185.72 mg/g. The crude protein content increased from 47.62% to 52.59%. The results of SDS-PAGE shows that the protein macromolecular and the antigen protein was also degraded.
An orthogonal test experiment was conducted to investigate the optimal conditions of soybean meal fermentation by Bacillus subtilis BSW-2, Saccharomyces cerevisiae YT and a neutral protease for improving the content of peptides. The results showed that the optimum conditions were mixture inoculum 1.5%, neutral protease 450 u/g, ratio of material to water 1:1.4, fermentation temperature 40℃, fermentation time 48 h. Under that condition, all the indicators were better than fermentation only and neutral protease only. The content of peptide increased from 13.35 mg/g to 199.65 mg/g. The crude protein content increased from 47.62% to 56.72%. The vitro digestibility of protein increased from 81.86% to 92.79%. The vitro dry matter digestibility increased from 66.00% to 72.30%. The content of lactic acid was 2.75%. The urease activity decreased from 0.45 U to 0.086 U. The results of SDS-PAGE shows that the protein macromolecular and the antigen protein was almost degraded.
PCR-DGGE method was used to analysis the change of bacterial communities in the collaborative process, the results show that BSW-2 was in a dominant position throughout the fermentation process. With the fermentation time, BSW-2 can inhibit the growth of other bacteria, so it can have an absolute advantage.
本研究从实验室保藏的8株枯草芽孢杆菌中筛选到了1株能有效降解豆粕中大分子蛋白为小分子肽的菌株BSW-2,豆粕经过该菌发酵后,肽含量从原豆粕13.35mg/g提高到发酵后108.10 mg/g。同时,本研究还筛选到了1株能有效提高发酵豆粕粗蛋白含量的酵母菌YT,经过YT发酵后,粗蛋白从原料豆粕47.97%提高到55.32%。
随后,通过正交试验建立了芽孢杆菌BSW-2、酵母菌YT混菌发酵豆粕的工艺,工艺条件为混合菌液接种量1.5%,料水比1：1.0,发酵温度30℃,发酵时间36 h。发酵后样品肽含量从发酵前的13.35 mg/g提高到184.57 mg/g,粗蛋白含量从47.62%提高到56.38%,蛋白质体外消化率从81.86%提高到91.99%,干物质体外损耗率从66.00%提高到71.40%,乳酸含量为2.65%,脲酶活性从0.45 U降低到0.15 U,抗原蛋白及大分子蛋白得到有效降解。
另一方面,试验选择了中性蛋白酶作为豆粕酶解用酶,确定其酶解工艺为加酶量400u/g、料水比1：1.6、酶解温度50℃、酶解时间48 h。酶解豆粕样品肽含量从发酵前的13.35 mg/g提高到185.72 mg/g,粗蛋白含量从47.62%提高到52.59%,抗原蛋白及大分子蛋白也得到有效降解。
最后,建立了菌酶协同处理豆粕的工艺,确定工艺条件为混合菌接种量1.5%、中性蛋白酶添加酶量450u/g、料水比1：1.4、处理温度40℃、处理时间48 h。在此条件下,豆粕经菌酶协同处理后,各项指标均优于混菌发酵和中性蛋白酶酶解,其中肽含量从13.35 mg/g提高到199.65 mg/g,粗蛋白含量从47.62%提高到56.72%,蛋白质体外消化率从81.86%提高到92.79%,干物质体外损耗率从66.00%提高到72.30%,乳酸含量为2.75%,脲酶活性从0.45 U降低到0.086 U,抗原蛋白及大分子蛋白得到有效降解。
采用PCR-DGGE方法分析菌酶协同处理豆粕过程中细菌群落的变化,结果表明1.5%的接种量可以使枯草芽孢杆菌BSW-2从发酵开始就在整个发酵体系中占优势。随着发酵时间的延长,BSW-2的生长会抑制其它细菌的生长,到发酵后期整个发酵体系中,BSW-2占有绝对的优势。
Soybean meal, a by-product after oil extraction, crude protein content of 43-48%, high content of essential amino acids, is the most widely used source of dietary protein in the poultry and swine industry. Small peptide is compounds of 2 or more amino acids linked by peptide bonds, it's molecular weight between 1-2 kD. In this study, microbiological methods and enzymatically methods was used to treated soybean meal, to increase the content of peptides. The study is of great practical significance to make up of high-quality protein feed, especially the shortage of protein resources, to reduce farming cost of production, to promote livestock output.
One strain of Bacillus subtilis BSW-2 was screened to degrade protein macromolecules of soybean meal into small peptides from 8 strains preserved by our laboratory. After fermented, the content of peptide increased from 13.35 mg/g to 108.10 mg/g. One strain of yeast YT was also screened to improve the content of crude protein. After fermented, the crude protein content increased from 47.62% to 55.32%.
The process condition of soybean meal fermentation by BSW-2 and YT was established. The results showed that the optimum fermentation parameters were mixed inoculation volume 1.5%, ratio of material to water 1:1.0, fermentation temperature 30℃, fermentation time 36 h. After fermentation, the content of peptides increased from 13.35 mg/g to 184.57 mg/g. The crude protein content increased from 47.62% to 56.38%. The vitro digestibility of protein increased from 81.86% to 91.99%. The dry matter digestibility vitro increased from 66.00% to 71.40%. The content of lactic was 2.65%. The urease activity decreased from 0.45 U to 0.15 U. The results of SDS-PAGE shows that the protein macromolecular and the antigen protein was degraded.
The neutral protease was screened and the condition of enzymatic hydrolysis was established. The results showed that the optimum condition were neutral protease 400 u/g, ratio of material to water 1:1.6, reaction temperature 50℃, hydrolysis time 48 h. Under that condition, the content of peptides increased from 13.35 mg/g to 185.72 mg/g. The crude protein content increased from 47.62% to 52.59%. The results of SDS-PAGE shows that the protein macromolecular and the antigen protein was also degraded.
An orthogonal test experiment was conducted to investigate the optimal conditions of soybean meal fermentation by Bacillus subtilis BSW-2, Saccharomyces cerevisiae YT and a neutral protease for improving the content of peptides. The results showed that the optimum conditions were mixture inoculum 1.5%, neutral protease 450 u/g, ratio of material to water 1:1.4, fermentation temperature 40℃, fermentation time 48 h. Under that condition, all the indicators were better than fermentation only and neutral protease only. The content of peptide increased from 13.35 mg/g to 199.65 mg/g. The crude protein content increased from 47.62% to 56.72%. The vitro digestibility of protein increased from 81.86% to 92.79%. The vitro dry matter digestibility increased from 66.00% to 72.30%. The content of lactic acid was 2.75%. The urease activity decreased from 0.45 U to 0.086 U. The results of SDS-PAGE shows that the protein macromolecular and the antigen protein was almost degraded.
PCR-DGGE method was used to analysis the change of bacterial communities in the collaborative process, the results show that BSW-2 was in a dominant position throughout the fermentation process. With the fermentation time, BSW-2 can inhibit the growth of other bacteria, so it can have an absolute advantage.
引文
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