乳酸菌发酵豆粕及其功效研究
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摘要
豆粕是畜禽生产中重要的植物蛋白源。利用微生物发酵,可降低豆粕中抗营养因子含量,同时,可提高营养价值。本研究从自然发酵的豆粕中分离出优良乳酸菌,并采用紫外诱变技术,对筛选出的菌种进一步选育,优化该乳酸菌固态发酵豆粕的条件,测定发酵过程中各种成分的变化,分离纯化发酵豆粕中抗氧化肽,对其抗氧化特性进行了系统分析,并对发酵豆粕对小白鼠的消化酶、菌群、血液指标,以及仔猪的生长性能、日粮消化率、血液指标、肠道形态的影响进行了研究。从而,为发酵豆粕的制备,及幼龄动物专用优质蛋白饲料开发,提供理论依据和技术参考。
1.豆粕发酵菌种的选育及发酵条件优化研究
为了获得一株发酵豆粕性能优良的乳酸菌,进行豆粕发酵,本文首先利用常规的微生物技术,对发酵豆粕的乳酸菌进行了分离筛选,并对筛选出的乳酸菌进行了紫外诱变处理,提高了菌株的发酵性能,选育出乳酸杆菌B作为发酵菌株,采用单因子正交优化实验,根据试验结果,得出该菌株固态制备发酵豆粕的最佳条件是：接种量3%,含水量40%,蛋白酶添加量0.15%,PH值自然,37。C密闭发酵48h。
2.豆粕发酵过程中组分变化的研究
以乳酸菌杆菌B固态发酵豆粕,并对发酵过程中各组分的变化进行了研究。豆粕发酵后,发酵豆粕中蛋白质的体外消化率显著提高,SDS-PAGE电泳结果表明,豆粕中大分子蛋白质降解为小分子的肽。同时,发酵豆粕中游离氨基酸含量显著增加。发酵豆粕中总糖含量减少,还原糖含量增加,水苏糖和棉籽糖等低聚糖含量显著降低。去除抗营养因子的结果表明,乳酸菌发酵可显著降低豆粕中胰蛋白酶抑制因子、脲酶、大豆抗原蛋白等抗营养因子含量,降低植酸含量的效果不明显。利用超滤和凝胶色谱技术,分离纯化得到了发酵豆粕中的抗氧化肽。并利用实验室分析和体外试验相结合的方法,对其中抗氧化活性最强的抗氧化肽的抗氧化活性、氨基酸组成进行了系统分析。结果显示：大豆抗氧化肽各分离组分均表现出一定的抑制自由基的能力,其中,P3显著提高了抗氧化氨基酸含量,这一结果说明发酵豆粕中的抗氧化肽,使其具有较高的抗氧化活性。
3.发酵豆粕对小鼠血液抗氧化指标、胃肠道消化酶活性、肠道菌群等的影响
选择昆明种雄性小鼠45只,体重(15.66±1.26)g,分为3组,每组3个重复,每个重复5只。对照组小鼠饲喂含正常豆粕的基础日粮,试验1组基础日粮中发酵豆粕取代50%正常豆粕,试验2组饲喂发酵豆粕,观察发酵豆粕对小白鼠血液抗氧化指标、消化酶活性、肠道菌群和免疫器官指数的影响。结果表明,试验组胃蛋白酶活性较对照组均有极显著提高；试验组大肠杆菌数均显著低于对照组(P<0.05),而乳酸菌数较对照组有显著提高(P<0.05)；试验1组、2组双歧杆菌数较对照组分别提高5.54%(P<0.01)、3.30%(P<0.05)；试验1组、2组血清中丙二醛(MDA)的含量较对照组分别降低18.15%(P<0.05)、22.12%(P<0.01)；试验1组肝脏、脾脏、胸腺指数较对照组均有显著提高。结果表明发酵豆粕具有提高小鼠抗氧化能力、胃肠道蛋白酶含量、肠道有益菌群数量和免疫器官指数的功能,适宜在幼龄动物生产中使用。
4.发酵豆粕对仔猪生长性能、日粮营养物质消化率和血液指标的影响
本文旨在研究发酵豆粕对仔猪生长性能、日粮营养物质消化率和血液指标的影响。选取36头平均体重为(9.62±0.72kg)的二元杂交仔猪,随机分为3组,每组3个重复,每个重复4头猪,对照组仔猪饲喂28.54%的普通豆粕,试验Ⅰ组仔猪饲喂14.27%的发酵豆粕,试验Ⅱ组仔猪饲喂28.54%的发酵豆粕。试验期21天,试验结束前连续收集3天粪便,测定饲料养分的表观消化率；试验结束时前腔静脉采血,测定血液指标,同时,屠宰并测定试验猪小肠组织结构。结果表明：(1)与对照组相比,试验Ⅰ组和Ⅱ组仔猪的日增重均显著提高(P<0.05),腹泻频率均极显著降低(P<0.01)；(2)试验Ⅰ组和Ⅱ组仔猪日粮中干物质、蛋白质、有机物的消化率均较对照组显著提高(P<0.05)；(3)试验Ⅱ组均显著提高了仔猪血液中碱性磷酸酶(ALP)(P<0.05)和总蛋白(TP)的含量(P<0.05),试验Ⅰ组和Ⅱ组均显著提高了血液中白蛋白(ALB)的含量(P<0.05),显著(P<0.05)和极显著降低了尿素氮(BUN)的含量(P<0.01)；试验Ⅰ组和Ⅱ组均显著降低了血液中丙二醛(MDA)的含量(P<0.05)和IgG含量(P<0.05)；(4)显著提高了仔猪十二指肠、回肠、空肠的绒毛高度(P<0.05),绒毛高度／隐窝深度也显著提高(P<0.05),空肠和回肠的隐窝深度显著降低(P<0.05)。(5)相关性分析结果表明,添加发酵豆粕后,仔猪血液中碱性磷酸酶、总蛋白、白蛋白、超氧化物岐化酶、总抗氧化能力的含量和平均日增重之间存在着正相关(R2=0.6321、0.7795、0.9842、0.9951、0.9477),血液中尿素氮、丙二醛的含量与日增重之间存在着负相关(R2=0.9825、0.9964)。日粮中添加发酵豆粕,通过提高日粮营养物质表观消化率,改善血液指标和肠道形态结构,降低仔猪断奶应激性腹泻,从而提高仔猪的生长性能、免疫功能、抗氧化功能。
Soybean meal is an important protein source for livestock and poultry. Lactic acid fermentation can eliminate the antinutritional factors and improve nutritive value of soybean meal. However, most of studies have been studied the effect of lactic acid bacteria from other source on fermentation of soybean meal. No study has separated lactic acid bacteria from fermented soybean meal. Now, firstly, we isolated lactic acid bacterias from fermented soybean meal, and continued to screened a strain of lactic acid bacterias by UV mutagenesis. Secondly, we studied the parameters of solid state fermentaion, the changes of compositions in soybean meal during the fermentation, the isolation and purification of antioxidant peptides from the biological soybean meal. Thirdly, effect of fermented soybean meal on blood antioxidant indices, gastrointestinal digestive enzyme activities and bacteria flora in mice, on growth performance, nutrient digestibility and blood indices in piglets were studied. Therefore, we provide a theoretical basis and technical basis for that soybean meal was fermented for young animals.
1. Screen of lactic acid bacteria strains for fermented soybean meal and study of the parameters for fermentation
In order to obtain a lactic acid bacteria for preparation of fermentd soybean meal, the paper used conventional microbiological techniques.Firstly, we separated lactic acid bacterias from fermented soybean meal, and continued to screen a strain of lactic acid bacterias by UV mutagenesis.Then, We obtained the best parameters of fermentation are3%inoculum volume,40%of moisture contents,0.15%protease addition, and natural PH through one factor and orthogonal design desigen experiment.
2. Changes of compositions in fermented soybean meal during lactic acid fermentation
Soybean meal was fermented by L.plantarum B and the changes of some components in fermentdd soybean meal were studied during the fermentation. Lactic acid fermeantion can increase the digestibility of protein in fermented soybean meal in vitro.The high molecule weight proteins were degraded into small peptides. L.plantarum B fermentation can decrease the content of total sugars, increase the content of reductive sugars too, meanwhile, the content of oligosaccharide also decreased.The content of trypsin inhibitior, urease and soybean antigen decreased, but little phytate was degraded. Isolation and purification of antioxidant peptides from fermented soybean meal by ultrafiltration and gel filtration chromatography was done in order to realize both the antioxidant activity of peptides and its composition. Antioxidant activity and amino acid composition were analyzed using methods containing laboratory analysis and experiments in vitro. The results showed that all different fractioned peptides had scavenging effects on radicals. In these peptides, peptide P3has the strong inhibitory activity. These results showed that peptide P3had the good antioxidant activity.
3. Effect of fermented soybean meal on blood antioxidant indices, gastrointestinal digestive enzyme activities and bacteria flora in mice
The experiment was conducted to study the effect of fermented soybean meal on blood antioxidant indices, gastrointestinal digestive enzymes levels and bacteria flora in mice. Forty-five Kunming male mice (BW=15.66±1.26g) were randomly assigned to3treatment groups with3replicates and five mice in each replicate. The control group was fed basal diet with normal soybean meal. In treatment1and treatment2, soybean meals were replaced by50%and100%fermented soybean meal respectively. The results showed that fermented soybean meal significantly increased stomach protein enzyme activities (P<0.01), decreased Escherichia Coli counts (P<0.05). Counts of bifidobacterium in treatment1and treatment2were increased by5.54%(P<0.01) and3.30%(P<0.05), MDA level were decreased by18.15%(P<0.05) and22.12%(P<0.01) compared with control group respectively. Organic indices of liver, spleen and kidney of treatment1were higer than that of control group (P<0.05),In conclusion, fermented soybean meal could increase blood antioxidant ability, gastrointestinal digestive enzyme activities, intestinal flora amounts and immune organic indices, so that fermented soybean meal is suitable to young animals.
4. Effect of fermented soybean meal on growth performance, nutrient digestibility and blood indices in piglets
The experiment was conducted to study effect of fermented soybean meal on growth performance, nutrient digestibility and blood indices in piglets. Thirty-six piglets with average body weight of (9.62±0.72) kg were randomly allotted to3groups on the basis of litter and weight, and3replicates per group and4piglets per replicate. The control group was fed basal diet with normal soybean meal, and group Ⅰ and group Ⅱ were fed basal diet in which50%,100%soybean meal were replaced with biological soybean meal.Serum was collected at21d age for blood parameter measurement and faeces of piglets were collected during a period of3consecutive days between18d to20d for digestibilitymeasurement. Piglets were slaughtered to measure morphology of small intestine. Results showed that piglets in group Ⅰ and Ⅱ both had a significantly increase in average daily gain (ADG)(P<0.05), and both had a significantly reduction in diarrhea ratio (P<0.01) compared with control group. Fermented soybean meal significantly increases the coefficients of total tract apparent digestibility (CTTAD) of dry matter, protein and organic matter (P<0.05). The level of alkaline phosphates and total protein in serum of group Ⅱ both higher than that of control group (P<0.05).The level of blood albumin in serum in group Ⅰ and group Ⅱ were both higher than that of control group (P<0.05), and level of blood urea of group Ⅰ and group Ⅱ decreased (P<0.05) and significantly decreased (P<0.01) respectively. The levels of MDA and IgG in group Ⅰ and group Ⅱ were both lower than that of control group (P<0.05) respectively. The results of the correlation analysis showed that the average daily gain of the piglet had positive correlations with level of ALP, TP, ALB, SOD and T-AOC (R2=0.6321、0.7795、0.9842、0.9951、0.9477),and had negative correlations with level of BUN and MDA in blood (R2=0.9825、0.9964). In conclusion, fermented soybean meal is beneficial to growth performance, apparent digestibility of nutrient, blood indices, and keep the integrality of small intestine morphology, improve the immunity and antioxidation activity in piglets.
1.豆粕发酵菌种的选育及发酵条件优化研究
为了获得一株发酵豆粕性能优良的乳酸菌,进行豆粕发酵,本文首先利用常规的微生物技术,对发酵豆粕的乳酸菌进行了分离筛选,并对筛选出的乳酸菌进行了紫外诱变处理,提高了菌株的发酵性能,选育出乳酸杆菌B作为发酵菌株,采用单因子正交优化实验,根据试验结果,得出该菌株固态制备发酵豆粕的最佳条件是：接种量3%,含水量40%,蛋白酶添加量0.15%,PH值自然,37。C密闭发酵48h。
2.豆粕发酵过程中组分变化的研究
以乳酸菌杆菌B固态发酵豆粕,并对发酵过程中各组分的变化进行了研究。豆粕发酵后,发酵豆粕中蛋白质的体外消化率显著提高,SDS-PAGE电泳结果表明,豆粕中大分子蛋白质降解为小分子的肽。同时,发酵豆粕中游离氨基酸含量显著增加。发酵豆粕中总糖含量减少,还原糖含量增加,水苏糖和棉籽糖等低聚糖含量显著降低。去除抗营养因子的结果表明,乳酸菌发酵可显著降低豆粕中胰蛋白酶抑制因子、脲酶、大豆抗原蛋白等抗营养因子含量,降低植酸含量的效果不明显。利用超滤和凝胶色谱技术,分离纯化得到了发酵豆粕中的抗氧化肽。并利用实验室分析和体外试验相结合的方法,对其中抗氧化活性最强的抗氧化肽的抗氧化活性、氨基酸组成进行了系统分析。结果显示：大豆抗氧化肽各分离组分均表现出一定的抑制自由基的能力,其中,P3显著提高了抗氧化氨基酸含量,这一结果说明发酵豆粕中的抗氧化肽,使其具有较高的抗氧化活性。
3.发酵豆粕对小鼠血液抗氧化指标、胃肠道消化酶活性、肠道菌群等的影响
选择昆明种雄性小鼠45只,体重(15.66±1.26)g,分为3组,每组3个重复,每个重复5只。对照组小鼠饲喂含正常豆粕的基础日粮,试验1组基础日粮中发酵豆粕取代50%正常豆粕,试验2组饲喂发酵豆粕,观察发酵豆粕对小白鼠血液抗氧化指标、消化酶活性、肠道菌群和免疫器官指数的影响。结果表明,试验组胃蛋白酶活性较对照组均有极显著提高；试验组大肠杆菌数均显著低于对照组(P<0.05),而乳酸菌数较对照组有显著提高(P<0.05)；试验1组、2组双歧杆菌数较对照组分别提高5.54%(P<0.01)、3.30%(P<0.05)；试验1组、2组血清中丙二醛(MDA)的含量较对照组分别降低18.15%(P<0.05)、22.12%(P<0.01)；试验1组肝脏、脾脏、胸腺指数较对照组均有显著提高。结果表明发酵豆粕具有提高小鼠抗氧化能力、胃肠道蛋白酶含量、肠道有益菌群数量和免疫器官指数的功能,适宜在幼龄动物生产中使用。
4.发酵豆粕对仔猪生长性能、日粮营养物质消化率和血液指标的影响
本文旨在研究发酵豆粕对仔猪生长性能、日粮营养物质消化率和血液指标的影响。选取36头平均体重为(9.62±0.72kg)的二元杂交仔猪,随机分为3组,每组3个重复,每个重复4头猪,对照组仔猪饲喂28.54%的普通豆粕,试验Ⅰ组仔猪饲喂14.27%的发酵豆粕,试验Ⅱ组仔猪饲喂28.54%的发酵豆粕。试验期21天,试验结束前连续收集3天粪便,测定饲料养分的表观消化率；试验结束时前腔静脉采血,测定血液指标,同时,屠宰并测定试验猪小肠组织结构。结果表明：(1)与对照组相比,试验Ⅰ组和Ⅱ组仔猪的日增重均显著提高(P<0.05),腹泻频率均极显著降低(P<0.01)；(2)试验Ⅰ组和Ⅱ组仔猪日粮中干物质、蛋白质、有机物的消化率均较对照组显著提高(P<0.05)；(3)试验Ⅱ组均显著提高了仔猪血液中碱性磷酸酶(ALP)(P<0.05)和总蛋白(TP)的含量(P<0.05),试验Ⅰ组和Ⅱ组均显著提高了血液中白蛋白(ALB)的含量(P<0.05),显著(P<0.05)和极显著降低了尿素氮(BUN)的含量(P<0.01)；试验Ⅰ组和Ⅱ组均显著降低了血液中丙二醛(MDA)的含量(P<0.05)和IgG含量(P<0.05)；(4)显著提高了仔猪十二指肠、回肠、空肠的绒毛高度(P<0.05),绒毛高度／隐窝深度也显著提高(P<0.05),空肠和回肠的隐窝深度显著降低(P<0.05)。(5)相关性分析结果表明,添加发酵豆粕后,仔猪血液中碱性磷酸酶、总蛋白、白蛋白、超氧化物岐化酶、总抗氧化能力的含量和平均日增重之间存在着正相关(R2=0.6321、0.7795、0.9842、0.9951、0.9477),血液中尿素氮、丙二醛的含量与日增重之间存在着负相关(R2=0.9825、0.9964)。日粮中添加发酵豆粕,通过提高日粮营养物质表观消化率,改善血液指标和肠道形态结构,降低仔猪断奶应激性腹泻,从而提高仔猪的生长性能、免疫功能、抗氧化功能。
Soybean meal is an important protein source for livestock and poultry. Lactic acid fermentation can eliminate the antinutritional factors and improve nutritive value of soybean meal. However, most of studies have been studied the effect of lactic acid bacteria from other source on fermentation of soybean meal. No study has separated lactic acid bacteria from fermented soybean meal. Now, firstly, we isolated lactic acid bacterias from fermented soybean meal, and continued to screened a strain of lactic acid bacterias by UV mutagenesis. Secondly, we studied the parameters of solid state fermentaion, the changes of compositions in soybean meal during the fermentation, the isolation and purification of antioxidant peptides from the biological soybean meal. Thirdly, effect of fermented soybean meal on blood antioxidant indices, gastrointestinal digestive enzyme activities and bacteria flora in mice, on growth performance, nutrient digestibility and blood indices in piglets were studied. Therefore, we provide a theoretical basis and technical basis for that soybean meal was fermented for young animals.
1. Screen of lactic acid bacteria strains for fermented soybean meal and study of the parameters for fermentation
In order to obtain a lactic acid bacteria for preparation of fermentd soybean meal, the paper used conventional microbiological techniques.Firstly, we separated lactic acid bacterias from fermented soybean meal, and continued to screen a strain of lactic acid bacterias by UV mutagenesis.Then, We obtained the best parameters of fermentation are3%inoculum volume,40%of moisture contents,0.15%protease addition, and natural PH through one factor and orthogonal design desigen experiment.
2. Changes of compositions in fermented soybean meal during lactic acid fermentation
Soybean meal was fermented by L.plantarum B and the changes of some components in fermentdd soybean meal were studied during the fermentation. Lactic acid fermeantion can increase the digestibility of protein in fermented soybean meal in vitro.The high molecule weight proteins were degraded into small peptides. L.plantarum B fermentation can decrease the content of total sugars, increase the content of reductive sugars too, meanwhile, the content of oligosaccharide also decreased.The content of trypsin inhibitior, urease and soybean antigen decreased, but little phytate was degraded. Isolation and purification of antioxidant peptides from fermented soybean meal by ultrafiltration and gel filtration chromatography was done in order to realize both the antioxidant activity of peptides and its composition. Antioxidant activity and amino acid composition were analyzed using methods containing laboratory analysis and experiments in vitro. The results showed that all different fractioned peptides had scavenging effects on radicals. In these peptides, peptide P3has the strong inhibitory activity. These results showed that peptide P3had the good antioxidant activity.
3. Effect of fermented soybean meal on blood antioxidant indices, gastrointestinal digestive enzyme activities and bacteria flora in mice
The experiment was conducted to study the effect of fermented soybean meal on blood antioxidant indices, gastrointestinal digestive enzymes levels and bacteria flora in mice. Forty-five Kunming male mice (BW=15.66±1.26g) were randomly assigned to3treatment groups with3replicates and five mice in each replicate. The control group was fed basal diet with normal soybean meal. In treatment1and treatment2, soybean meals were replaced by50%and100%fermented soybean meal respectively. The results showed that fermented soybean meal significantly increased stomach protein enzyme activities (P<0.01), decreased Escherichia Coli counts (P<0.05). Counts of bifidobacterium in treatment1and treatment2were increased by5.54%(P<0.01) and3.30%(P<0.05), MDA level were decreased by18.15%(P<0.05) and22.12%(P<0.01) compared with control group respectively. Organic indices of liver, spleen and kidney of treatment1were higer than that of control group (P<0.05),In conclusion, fermented soybean meal could increase blood antioxidant ability, gastrointestinal digestive enzyme activities, intestinal flora amounts and immune organic indices, so that fermented soybean meal is suitable to young animals.
4. Effect of fermented soybean meal on growth performance, nutrient digestibility and blood indices in piglets
The experiment was conducted to study effect of fermented soybean meal on growth performance, nutrient digestibility and blood indices in piglets. Thirty-six piglets with average body weight of (9.62±0.72) kg were randomly allotted to3groups on the basis of litter and weight, and3replicates per group and4piglets per replicate. The control group was fed basal diet with normal soybean meal, and group Ⅰ and group Ⅱ were fed basal diet in which50%,100%soybean meal were replaced with biological soybean meal.Serum was collected at21d age for blood parameter measurement and faeces of piglets were collected during a period of3consecutive days between18d to20d for digestibilitymeasurement. Piglets were slaughtered to measure morphology of small intestine. Results showed that piglets in group Ⅰ and Ⅱ both had a significantly increase in average daily gain (ADG)(P<0.05), and both had a significantly reduction in diarrhea ratio (P<0.01) compared with control group. Fermented soybean meal significantly increases the coefficients of total tract apparent digestibility (CTTAD) of dry matter, protein and organic matter (P<0.05). The level of alkaline phosphates and total protein in serum of group Ⅱ both higher than that of control group (P<0.05).The level of blood albumin in serum in group Ⅰ and group Ⅱ were both higher than that of control group (P<0.05), and level of blood urea of group Ⅰ and group Ⅱ decreased (P<0.05) and significantly decreased (P<0.01) respectively. The levels of MDA and IgG in group Ⅰ and group Ⅱ were both lower than that of control group (P<0.05) respectively. The results of the correlation analysis showed that the average daily gain of the piglet had positive correlations with level of ALP, TP, ALB, SOD and T-AOC (R2=0.6321、0.7795、0.9842、0.9951、0.9477),and had negative correlations with level of BUN and MDA in blood (R2=0.9825、0.9964). In conclusion, fermented soybean meal is beneficial to growth performance, apparent digestibility of nutrient, blood indices, and keep the integrality of small intestine morphology, improve the immunity and antioxidation activity in piglets.
引文
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