CN114027435A - 一种益生菌混合发酵消除葵花粕中抗营养因子的方法 - Google Patents
一种益生菌混合发酵消除葵花粕中抗营养因子的方法 Download PDFInfo
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Abstract
本发明公开一种益生菌混合发酵消除葵花粕中抗营养因子的方法,该方法以葵花粕为原料,添加经单因素和响应面试验优化后的复合营养源,其碳源、氮源、硫酸镁及磷酸二氢钾溶于水后添加到葵花粕中。选用经定性和定量选择后获得的纤维素降解优势菌株枯草芽孢杆菌,并结合酿酒酵母和长枝木霉菌,单独进行液态发酵,取各自达到生长稳定期的菌液成混合菌液,将其添加到葵花粕中。将葵花粕混合均匀,导入发酵槽内,制成堆垛,采用覆膜式好氧发酵工艺发酵。发酵完成,经晾干、去水后,制得发酵葵花粕。本发明可降解葵花粕中粗纤维、绿原酸和植酸等抗营养因子达30%~40%,其中粗纤维的降解率可达25%~30%。同时,粗蛋白含量可提高20%~25%,小分子蛋白含量可提高约17%~20%。
Description
技术领域
本发明涉及生物发酵技术领域,尤其是一种益生菌混合发酵消除葵花粕中抗营养因子的方法。
背景技术
近年来,随着我国畜牧行业的快速发展,对蛋白饲料原料的需求较大,且对其质量要求越来越高。研发抗营养因子含量低、营养价值高、生产成本低的饲料是饲料工业发展的一大趋势。
葵花粕是以葵花籽为原料,用预压浸提法或浸提法取油后的副产物,其营养物质丰富,来源较广,是一种重要的植物蛋白资源,且蛋白中必需氨基酸的含量(除赖氨酸外)均高于或接近FAO推荐值,尤其是蛋氨酸和胱氨酸的含量较高,但相对豆粕而言,其蛋白质含量偏低。葵花粕由于没有脱壳,粗纤维含量较高,若直接制成饲料,会降低动物对饲料的消化利用率。并且,葵花粕中含有少量的酚类化合物,主要是绿原酸,含量约为0.7%~0.82%,经氧化后会变黑,是饼粕色泽灰暗的主要原因。且绿原酸对胰蛋白酶、淀粉酶和脂肪酶活性有抑制作用,与蛋白质的极性基团、氨基酸(半胱氨酸、色氨酸、组氨酸等)残基和亚甲基等基团共价结合,会使其生成非反刍动物和家禽无法消化的非营养性物质,从而影响蛋白质功能性质、降低蛋白质营养价值,限制其在动物饲料中的大量应用。若用物理、化学等方法进行消除,又存在或成本高、或破坏某些营养成分、或残留化学试剂等缺点。
因此,解决葵花粕中粗纤维和绿原酸等抗营养因子含量偏高、粗蛋白品质难以提升的问题极为重要。
发明内容
本发明的目的在于提供一种益生菌混合发酵消除葵花粕中抗营养因子的方法。本发明所述方法采用益生菌混合发酵技术,通过发酵产生纤维素酶、淀粉酶、蛋白酶、脂肪酶等,有效降解葵花粕中的粗纤维和绿原酸等抗营养因子,提高粗蛋白和可溶性蛋白含量,提高养殖动物对其的消化利用率,最终获得能大量应用于动物饲料的、富含营养物质的、低成本的发酵葵花粕饲料。
本发明的技术方案为:一种益生菌混合发酵消除葵花粕中抗营养因子的方法,该方法以葵花粕为原料,添加经单因素和响应面试验优化后的复合营养源,其碳源、氮源、硫酸镁及磷酸二氢钾分别为葵花粕重量的5~8份、0.5~2份、0.02~0.04份和0.2~0.4份,溶于水后添加到葵花粕中;选用经定性和定量选择后获得的纤维素降解优势菌株枯草芽孢杆菌,并结合酿酒酵母和长枝木霉菌,分别进行液态发酵,取各自达到生长稳定期的菌液,以2~3:4~6:2~3的比例制成混合菌液,将其添加到葵花粕中,两者比例为1~10:10~100;将葵花粕混合均匀,导入发酵槽内,制成堆垛,采用覆膜式好氧发酵工艺发酵72~96 h,每隔36~48小时翻堆一次,并定时测定垛心温度,查看温度变化情况;发酵完成,经晾干、去水后,制得发酵葵花粕。
所述葵花粕进行粉碎,过18~24目筛预处理,以便增加原料与水和菌的接触面积,促进物质降解与生成。
所述复合营养源成分包括碳源、氮源以及其他无机盐。所述碳源为葡萄糖或糖蜜,所述氮源为硫酸铵或尿素,所述无机盐包括硫酸镁和磷酸二氢钾等。所述碳源、氮源与无机盐的成分及比例均经过单因素试验和响应面优化试验而得,所述碳源、氮源、硫酸镁与磷酸二氢钾分别为葵花粕重量的5~8份、0.5~2份、0.02~0.04份和0.2~0.4份。所述复合营养源中碳氮比为10~15:1,添加所述成分和比例能使葵花粕发酵得到最优效果。
所述枯草芽孢杆菌来源于耕作土壤,是采用纤维素降解圈法和3,5-二硝基水杨酸比色法经定性和定量选择后获得的纤维素降解优势菌株,可有效降解葵花粕中的粗纤维含量。
所述枯草芽孢杆菌、酿酒酵母和长枝木霉菌分别进行固体试管斜面活化培养、液体三角瓶培养和种子罐扩大培养,分别在三种菌生长曲线接近最高值时取菌液,并按比例混合,其步骤如下:
(1)固体试管斜面活化培养:将枯草芽孢杆菌于LB试管斜面培养基中在35~37 ℃下培养20~24 h;酿酒酵母于马铃薯葡萄糖试管斜面培养基中在25~30 ℃下培养36~48 h;长枝木霉菌于马铃薯葡萄糖试管斜面培养基中在25~35 ℃下培养72~96 h;
(2)液体三角瓶培养:用接种环刮取一环步骤(1)的枯草芽孢杆菌于LB液体培养基中在35~37 ℃下培养18~27 h,此阶段菌种活性强、生长代谢旺盛,且在27 h左右菌落数达到最大;用接种环刮取一环步骤(1)的酿酒酵母于马铃薯葡萄糖液体培养基中在25~30 ℃下培养15~21 h,此阶段菌种活性强、生长代谢旺盛,且在21 h左右菌落数达到最大;用接种环刮取一环步骤(1)的长枝木霉菌于马铃薯葡萄糖液体培养基中在25~35 ℃下培养72~96小时,此阶段菌种活性强、生长代谢旺盛,且在96 h左右孢子数达到最大;
(3)种子罐扩大培养:按1%~2%比例接种步骤(2)的枯草芽孢杆菌菌液于种子罐中在35~37 ℃下扩大培养18~27 h;按1%~2%比例接种步骤(2)的酿酒酵母菌液于种子罐中在25~30 ℃下扩大培养15~21小时;按1%~2%比例接种步骤(2)的长枝木霉菌菌液于种子罐中在25~35 ℃下培养72~96小时;
(4)分别在三种菌接近生长曲线最高值时取菌液,并按比例添加到一起,制成混合菌液;
(5)所述混合菌液的比例为枯草芽孢杆菌:酿酒酵母:长枝木霉菌=2~3:4~6:2~3;
(6)所述枯草芽孢杆菌和酿酒酵母菌液的有效活菌数均为2×108~3×108 CFU/mL,所述长枝木霉菌菌液的孢子数为2×108~3×108 CFU/mL;
(7)所述混合菌液接种量与葵花粕的比例为1~10:10~100。
所述固体发酵培养基包括葵花粕、混合菌液、复合营养源和水,含水量为葵花粕重量的60%~120%。其制作步骤在于:将所述复合营养源溶于水中按比例混合到葵花粕中,再将制备好的混合菌液按比例混合接种到葵花粕中,进行搅拌混匀,最后将固体物料导入发酵槽内,制成堆垛,并覆膜正压。
所述发酵槽长为25~30 m,宽为7~10 m,高为1.5~1.6 m。发酵槽底部设置有两条送风通道,每天通风8~12小时,提供充足氧气,以便发酵料进行好氧发酵。所述堆垛的堆积高度为1.8~2.0 m,堆垛高度高于发酵槽高度。
所述覆膜中间为膨胀聚四氟乙烯膜、两侧为聚酯膜的复合膜,膨胀聚四氟乙烯膜上均布有0 .2 μm孔径的微孔。覆膜上安装温度传感器,可以实时监控堆垛的发酵温度。
所述好氧发酵时间为72~96 h,每隔36~48 h翻堆一次,并定期测定垛心温度,查看温度变化情况。
发酵后的葵花粕经晾干、去水制成干燥的发酵葵花粕,保证含水量至少低于20%。
本发明的优点在于:
1.本发明所述的枯草芽孢杆菌,来源于耕作土壤,是采用纤维素降解圈法和3,5-二硝基水杨酸比色法经定性和定量选择后获得的纤维素降解优势菌株,能有效降解葵花粕中粗纤维、绿原酸和植酸等抗营养因子。
2.本发明所述的枯草芽孢杆菌、酿酒酵母和长枝木霉菌分别进行液态发酵,且在各自生长曲线接近最高值时取菌液,再按比例制成混合菌液,此时期的菌种活性强,可保证葵花粕发酵效果最优。
3.本发明所选用的枯草芽孢杆菌、酿酒酵母和长枝木霉菌之间具有良好的协同作用,且配比为1:2:1时效果最佳。三种菌互相配合,协同发酵,共同降解葵花粕中粗纤维、绿原酸和植酸等抗营养因子可达30%~40%,其中粗纤维的降解率可达25%~30%。同时,粗蛋白含量可提高20%~25%,小分子蛋白含量可提高约17%~20%。
4.本发明所述的复合营养源及其配比,是根据所选菌株的适宜生长环境条件经单因素试验和响应面优化试验而得,可使葵花粕发酵效果达到最佳。
5.本发明采用覆膜式好氧发酵,堆垛高度高于发酵槽高度,以便发酵料与空气更好接触。同时,覆膜的孔径只有0.2 μm,可使发酵槽内具有良好的保温效果,促进葵花粕发酵。
6.本发明产品在仔鸡的养殖试验中表明,应用该产品替代豆粕、菜籽粕等植物性饲料源,部分替代鱼粉、血粉等动物性饲料源,可提高仔鸡生长性能,并降低了生产成本,取得了良好的生产效益。
具体实施方式
一种益生菌混合发酵消除葵花粕中抗营养因子的方法,该方法以葵花粕为原料,添加经单因素和响应面试验优化后的复合营养源,其碳源、氮源、硫酸镁及磷酸二氢钾溶于水后添加到葵花粕中。
所述葵花粕经粉碎后过18目筛预处理,以便增加原料与水和菌的接触面积,促进物质降解与生成。
所述复合营养源成分包括碳源、氮源以及其他无机盐。所碳源为葡萄糖,所述氮源为硫酸铵,所述无机盐为硫酸镁和磷酸二氢钾。
所述碳源、氮源与无机盐的成分及比例均经过单因素试验和响应面优化试验而得,添加所述成分和比例能使葵花粕发酵得到最优效果。
所述碳源、氮源、硫酸镁与磷酸二氢钾分别为葵花粕重量的5份、1份、0.03份和0.3份。所述复合营养源中碳氮比约为23:1,适宜微生物生长。
所述枯草芽孢杆菌来源于耕作土壤,是采用纤维素降解圈法和3,5-二硝基水杨酸比色法经定性和定量选择后获得的纤维素降解优势菌株,可有效降解葵花粕中的粗纤维含量。
本发明所述混合菌液的制备方法,所述枯草芽孢杆菌、酿酒酵母、长枝木霉菌分别进行固体试管斜面活化培养、液体三角瓶培养和种子罐扩大培养,分别在三种菌生长曲线接近最高值时取菌液,并按比例混合,具体步骤如下:
(1)固体试管斜面活化培养:将枯草芽孢杆菌于LB试管斜面培养基中在36 ℃下培养24 h;酿酒酵母于马铃薯葡萄糖试管斜面培养基中在28 ℃下培养48 h;长枝木霉菌于马铃薯葡萄糖试管斜面培养基中在32 ℃下培养96 h。
(2)液体三角瓶培养:用接种环刮取一环步骤(1)的枯草芽孢杆菌于LB液体培养基中在36 ℃下培养27 h,此阶段菌种活性强、生长代谢旺盛,且在27 h左右菌落数达到最大;用接种环刮取一环步骤(1)的酿酒酵母于马铃薯葡萄糖液体培养基中在28 ℃下培养21 h,此阶段菌种活性强、生长代谢旺盛,且在21 h左右菌落数达到最大;用接种环刮取一环步骤(1)的长枝木霉菌于马铃薯葡萄糖液体培养基中在32 ℃下培养96 h,此阶段菌种活性强、生长代谢旺盛,且在96 h左右孢子数达到最大。
(3)种子罐扩大培养:按1%比例接种步骤(2)的枯草芽孢杆菌菌液于种子罐中在36℃下扩大培养27 h;按1%比例接种步骤(2)的酿酒酵母菌液于种子罐中在28 ℃下扩大培养21 h;按1%比例接种步骤(2)的长枝木霉菌菌液于种子罐中在32 ℃下培养96 h。
(4)分别在三种菌接近生长曲线最高值时取菌液,并按比例添加到一起,制成混合菌液。
(5)所述混合菌液的比例为枯草芽孢杆菌:酿酒酵母:长枝木霉菌=2:4:2。
(6)所述枯草芽孢杆菌和酿酒酵母菌液的有效活菌数均为3×108 CFU/mL,所述长枝木霉菌菌液的孢子数为3×108 CFU/mL
(7)所述混合菌液接种量与葵花粕的比例为10:100。
所述固体发酵培养基,其特征在于:所述固体发酵培养基包括葵花粕、混合菌液、复合营养源和水。
所述的固体发酵培养基,其特征在于:所述固体发酵培养基含水量为葵花粕重量的100%。
所述的堆垛,其制作步骤在于:将所述复合营养源溶于水中按比例混合到葵花粕中,再将制备好的混合菌液按比例混合到葵花粕中,进行搅拌混匀,最后将固体物料导入发酵槽内,制成堆垛,并覆膜正压。
所述的发酵槽,其特征在于:所述发酵槽的长为25 m,宽为8 m,高为1.5 m。
所述的堆垛,其特征在于:所述堆垛的堆积高度为1.8 m,堆垛高度高于发酵槽高度,以便发酵料与空气更好接触,促进葵花粕发酵。
所述的覆膜式好氧发酵工艺,其特征在于:所述覆膜中间为膨胀聚四氟乙烯膜、两侧为聚酯膜的复合膜,膨胀聚四氟乙烯膜上均布有0 .2 μm孔径的微孔。
所述的覆膜式好氧发酵工艺,其特征在于:所述好氧发酵时间为96 h,每隔48 h翻堆一次,并定期测定垛心温度,查看温度变化情况。
所述的覆膜式好氧发酵工艺,其特征在于:发酵槽底部设置有两条送风通道,每天通风12小时,提供充足氧气,以便发酵料进行好氧发酵。
所述的覆膜式好氧发酵工艺,其特征在于:所述覆膜上安装温度传感器,可以实时监控堆垛的发酵温度。
所述的发酵葵花粕的制备方法,其特征在于:发酵后的葵花粕经晾干、去水制成干燥的发酵葵花粕,保证含水量至少低于20%。。
Claims (10)
1.一种益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:该方法以葵花粕为原料,添加经单因素和响应面试验优化后的复合营养源,其碳源、氮源、硫酸镁及磷酸二氢钾分别为葵花粕重量的5~8份、0.5~2份、0.02~0.04份和0.2~0.4份,溶于水后添加到葵花粕中;选用经定性和定量选择后获得的纤维素降解优势菌株枯草芽孢杆菌,并结合酿酒酵母和长枝木霉菌,分别进行液态发酵,取各自达到生长稳定期的菌液,以2~3:4~6:2~3的比例制成混合菌液,将其添加到葵花粕中,两者比例为1~10:10~100;将葵花粕混合均匀,导入发酵槽内,制成堆垛,采用覆膜式好氧发酵工艺发酵72~96 h,每隔36~48小时翻堆一次,并定时测定垛心温度,查看温度变化情况;发酵完成,经晾干、去水后,制得发酵葵花粕。
2.根据权利要求1所述益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:所述葵花粕进行粉碎,过18~24目筛预处理,以便增加原料与水和菌的接触面积,促进物质降解与生成。
3.根据权利要求1所述益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:所述复合营养源成分包括碳源、氮源以及其他无机盐;所述碳源为葡萄糖或糖蜜,所述氮源为硫酸铵或尿素,所述无机盐包括硫酸镁和磷酸二氢钾等;所述碳源、氮源与无机盐的成分及比例均经过单因素试验和响应面优化试验而得,所述碳源、氮源、硫酸镁与磷酸二氢钾分别为葵花粕重量的5~8份、0.5~2份、0.02~0.04份和0.2~0.4份;所述复合营养源中碳氮比为10~15:1,添加所述成分和比例能使葵花粕发酵得到最优效果。
4.根据权利要求1所述益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:所述枯草芽孢杆菌来源于耕作土壤,是采用纤维素降解圈法和3,5-二硝基水杨酸比色法经定性和定量选择后获得的纤维素降解优势菌株,可有效降解葵花粕中的粗纤维含量。
5.根据权利要求1所述益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:所述枯草芽孢杆菌、酿酒酵母和长枝木霉菌分别进行固体试管斜面活化培养、液体三角瓶培养和种子罐扩大培养,分别在三种菌生长曲线接近最高值时取菌液,并按比例混合,具体步骤如下:
(1)固体试管斜面活化培养:将枯草芽孢杆菌于LB试管斜面培养基中在35~37 ℃下培养20~24 h;酿酒酵母于马铃薯葡萄糖试管斜面培养基中在25~30 ℃下培养36~48 h;长枝木霉菌于马铃薯葡萄糖试管斜面培养基中在25~35 ℃下培养72~96 h;
(2)液体三角瓶培养:用接种环刮取一环步骤(1)的枯草芽孢杆菌于LB液体培养基中在35~37 ℃下培养18~27 h,此阶段菌种活性强、生长代谢旺盛,且在27 h左右菌落数达到最大;用接种环刮取一环步骤(1)的酿酒酵母于马铃薯葡萄糖液体培养基中在25~30 ℃下培养15~21 h,此阶段菌种活性强、生长代谢旺盛,且在21 h左右菌落数达到最大;用接种环刮取一环步骤(1)的长枝木霉菌于马铃薯葡萄糖液体培养基中在25~35 ℃下培养72~96小时,此阶段菌种活性强、生长代谢旺盛,且在96 h左右孢子数达到最大;
(3)种子罐扩大培养:按1%~2%比例接种步骤(2)的枯草芽孢杆菌菌液于种子罐中在35~37 ℃下扩大培养18~27 h;按1%~2%比例接种步骤(2)的酿酒酵母菌液于种子罐中在25~30℃下扩大培养15~21小时;按1%~2%比例接种步骤(2)的长枝木霉菌菌液于种子罐中在25~35℃下培养72~96小时;
(4)分别在三种菌接近生长曲线最高值时取菌液,并按比例添加到一起,制成混合菌液;
(5)所述混合菌液的比例为枯草芽孢杆菌:酿酒酵母:长枝木霉菌=2~3:4~6:2~3;
(6)所述枯草芽孢杆菌和酿酒酵母菌液的有效活菌数均为2×108~3×108 CFU/mL,所述长枝木霉菌菌液的孢子数为2×108~3×108 CFU/mL;
(7)所述混合菌液接种量与葵花粕的比例为1~10:10~100。
6.根据权利要求1所述益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:所述固体发酵培养基包括葵花粕、混合菌液、复合营养源和水,含水量为葵花粕重量的60%~120%;其制作步骤在于:将所述复合营养源溶于水中按比例混合到葵花粕中,再将制备好的混合菌液按比例混合接种到葵花粕中,进行搅拌混匀,最后将固体物料导入发酵槽内,制成堆垛,并覆膜正压。
7.根据权利要求1所述益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:所述发酵槽长为25~30 m,宽为7~10 m,高为1.5~1.6 m;发酵槽底部设置有两条送风通道,每天通风8~12小时,提供充足氧气,以便发酵料进行好氧发酵;所述堆垛的堆积高度为1.8~2.0 m,堆垛高度高于发酵槽高度。
8.根据权利要求1所述益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:所述覆膜中间为膨胀聚四氟乙烯膜、两侧为聚酯膜的复合膜,膨胀聚四氟乙烯膜上均布有0.2 μm孔径的微孔;覆膜上安装温度传感器,可以实时监控堆垛的发酵温度。
9.根据权利要求1所述益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:所述好氧发酵时间为72~96 h,每隔36~48 h翻堆一次,并定期测定垛心温度,查看温度变化情况。
10.根据权利要求1所述益生菌混合发酵消除葵花粕中抗营养因子的方法,其特征在于:发酵后的葵花粕经晾干、去水制成干燥的发酵葵花粕,保证含水量至少低于20%。
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