CN111748493A - 一种复合微生物制剂的制备方法及其用途 - Google Patents
一种复合微生物制剂的制备方法及其用途 Download PDFInfo
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Abstract
本发明提供一种复合微生物发酵液制备及其应用于生物发酵饲料，其由保加利亚乳杆菌、干酪乳杆菌、产朊假丝酵母和枯草芽孢杆菌单菌培养液进行混合后扩大培养，可用于食品、种植肥料和养殖饲料发酵生产等领域。本发明所述复合发酵液是由保加利亚乳杆菌、干酪乳杆菌、产朊假丝酵母和枯草芽孢杆菌按体积5：5：2：1进行混合发酵。所述混合培养温度为30‑35℃，所述混合培养pH为6.0‑7.0,所述混合培养转速为50‑100 rpm，所述混合培养时间为20‑48小时。所述方法比单个菌单独发酵获得发酵液然后混合使用要操作简单，节约物料和设备成本。
Description
技术领域
本发明属于微生物培养技术领域，尤其涉及一种混合发酵培养乳酸菌的方法。
背景技术
在现有食品、种植肥料和养殖饲料发酵生产等领域通常采用单一菌种的菌粉或多种纯种菌粉在添加有葡萄糖，氯化钠等物质的活化液活化一段时间后用于发酵生产产品。这种生产方法简单，成本较低，但发酵时有效的活菌量通常较少，很容易感染杂菌，导致腐败变质，发酵的效果很不理想。
近些年来，也有人利用大型发酵罐分别发酵生产多种活性菌，然后与原料按一定比例混合后发酵，如此发酵生产的产品，发酵时活菌含量较高，菌体能够产生较多的代谢物，可以明显提高发酵产品的营养物质（氨基酸，维生素、有机酸）的含量与多样性，具有良好的甜、酸、酒香味，提高了产品的适口性和营养物质的转化率。但不同菌种要求纯种无菌培养，在工厂规模化生产时，在需要很多的发酵罐和场地，电力、蒸气、物料消耗巨大，再则国内发酵专业人才匮乏，人员成本较大，导致生产成本高，发酵的成品价格高，市场推广难度大。
本共生混合发酵液生产方法，可以实现多菌种共生混合发酵，本方法生产的共生混合发酵液与纯菌种生产发酵液在应用于饲料发酵后，发酵的成品在活菌数含量、营养物质含量，气味等方面没有显著的差导。本共生混合发酵液生产方法，节约了发酵罐设备、人员和物料，可以实现更具规模的工业化生产。
发明内容
本发明的目的在于提供一种共生混合乳酸菌发酵液。相较于单一菌纯种发酵获得发酵液然后按一定比例混合使用，本发明所述方法要操作简单，节约物料、人员、场地和设备成本。
为了实现上述发明目的，本发明提供了以下技术方案。
1.一种共生混合发酵液制备方法如下：种子活化：用MRS培养基分别活化保加利亚乳杆菌，37℃，静置培养24小时；用MRS培养基活化干酪乳杆菌，37℃，静置培养24小时；用YPD培养基活化产朊假丝酵母，28℃，160 rpm，恒温振荡器培养20h，用牛肉膏培养基活化枯草芽孢杆菌，37℃，160 rpm，恒温振荡器培养20h，各菌接种量为1-5%。
所述保加利亚乳杆菌的单菌培养液中保加利亚乳杆菌的活菌浓度为(2-5)×108CFU/mL，所述干酪乳杆菌的单菌培养液中干酪乳杆菌的活菌浓度为(1-3)×1010CFU/mL，所述产朊假丝酵母的单菌培养液中产朊假丝酵母的活菌浓度为(1-5)×109CFU/mL，所述枯草芽孢杆菌的单菌培养液中枯草芽孢杆菌的活菌浓度为(1-5)×109CFU/mL。
混合发酵：由保加利亚乳杆菌、干酪乳杆菌、产朊假丝酵母和枯草芽孢杆菌按体积5：5：2：1进行混合，以总接种量5-10%进行发酵。所述混合培养的温度为30℃，所述混合培养的pH值为6.5-7.0，所述混合培养的时间为20-48h,所述混合培养的转速为50-100rpm ；所述混合培养基为：葡萄糖50-100g，酵母浸粉8-10g，KH2PO4 1g，MgSO4·7H2O 0.6g，三水乙酸钠1g，轻质CaCO3 25-50g，蒸馏水1000ml，调节pH至6.5-7.0，于发酵罐中115℃灭菌30min。所述混合发酵液培养20-48h时乳酸钙含量不低于25 mg/ml，蛋白酶含量不低于200U/ml。
本发明还保护所述共生混合发酵液在食品、种植肥料和养殖饲料发酵生产等领域的应用。
本发明的有益效果如下。
1.多菌种共生混合培养发酵产生较多的乳酸钙，有利于应用于食品和饲料发酵生产中，给人体或动物提供钙源。
2.多菌种共生混合培养发酵产生一定量的蛋白酶，有利于降解饲料原料如豆粕中抗营养物质含量，并提高饲料中游离氨基酸、小肽活性成分含量，有效提高动物体内蛋白质转化率。
3.在养殖饲料发酵生产时，多菌种共生混合发酵方法相对于单一菌种菌粉发酵或多个单一菌种菌粉混合后发酵，提高了发酵饲料中乳酸等有机酸含量，降解饲料中抗原物质的能力更强。
4.在食品、种植肥料和养殖饲料发酵生产时，相对于单一菌纯种培养然后按一定比例制成混合菌液，多菌种共生混合发酵生产发酵液的方法生产效率更高，设备利用率更高，所需培养基等物料成本更低，并能得到较高的转化率。
具体实施方式
以下通过具体实施例来说明本发明技术方案，但本发明的保护范围并不局限于此。
下述实施例中，所用MRS培养基、YPD培养基、牛肉膏培养基组成采用本领域常规配方即可，故不赘述。
实施例1
一种添加剂，该1000g饲料添加剂由如下组分组成：高温水解羽毛粉250g、鱼溶浆200g、胡萝巴250g、辣椒粕200g、米糠50g、角蛋白酶2g、乳酸菌2g、酿酒酵母菌1g、大豆异黄酮2g、耐高温脂肪酶4g、溶血磷脂油25g、耐高温植酸酶4g、姜黄素4g、茶多酚2g、青蒿素4g。
所述饲料添加剂由下述步骤制成：
（1）将高温水解羽毛粉250g、胡萝巴250g、辣椒粕200g、米糠50g过2.0mm筛片进行粉碎投入混合机；
（2）鱼溶浆200g溶于50℃温水，将角蛋白酶2g、乳酸菌2g和酿酒酵母菌1g投入鱼溶浆水溶液中，搅拌均匀,用1.0mm筛网进行过滤，去除筛上物；
（3）将步骤（2）制得的混合液和步骤（1）的混合料一起投入混合机中，混合均匀；
（4）将步骤（3）制得的混合物进行发酵24-48小时后低温烘干；
（5）将步骤（4）的烘干物过2.0mm筛片进行粉碎，与大豆异黄酮2g、耐高温脂肪酶4g、溶血磷脂油25g、耐高温植酸酶4g、姜黄素4g、茶多酚2g、青蒿素4g一起投入混合机，混合均匀制得添加剂。
实施例2
以发酵豆粕生产为例:豆粕80kg，实施例1制备的共生混合发酵菌液39kg，中性蛋白酶2.5 kg混合搅拌均匀，然后分装打包密封厌氧发酵7天。
对比例的制备。
对照1 采用润盈生物工程（上海）有限公司购买的保加利亚乳杆菌菌粉和干酪乳杆菌菌粉，产朊假丝酵母菌菌粉，枯草芽孢杆菌菌粉。各菌种按厂家说明书活化，活化好的保加利亚乳杆菌、干酪乳杆菌、产朊假丝酵母和枯草芽孢杆菌按体积5：5：2：1制备成混合菌液。
豆粕80kg，对照1制备的混合菌液39kg，中性蛋白酶2.5 kg混合搅拌均匀，然后分装打包密封厌氧发酵7天。
对照2 4株菌种种子液制备同实施例1，4株菌种种子液进行发酵罐纯种扩培，发酵罐扩培所采用培养基、培养条件与实施例1种子液制备条件相同。发酵罐扩培好的保加利亚乳杆菌、干酪乳杆菌、产朊假丝酵母和枯草芽孢杆菌按体积5：5：2：1制备成混合菌液。
豆粕80kg，对照2制备的混合菌液39kg，中性蛋白酶2.5 kg混合搅拌均匀，然后分装打包密封厌氧发酵7天。
由此可见，采用本发明提供的共生混合发酵培养乳酸菌、酵母和枯草芽孢杆菌的方法能够很好的保持各菌的活菌浓度以及活性，与对照1相比，发酵豆粕的酸溶蛋白、小肽、有机酸和赖氨酸等各项指标显著性更优，与对照2相比，发酵豆粕各项指标相差不大。相对于单独发酵培养各个菌液，本共生混合发酵液生产方法，节约了发酵罐设备、人员和物料，可以实现更具规模的工业化生产。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种共生混合发酵液制备方法如下：
种子活化：用MRS培养基分别活化保加利亚乳杆菌，37℃，静置培养24小时；用MRS培养基活化干酪乳杆菌，静置培养24小时；用YPD培养基活化产朊假丝酵母，28℃，160 rpm，恒温振荡器培养20h，用牛肉膏培养基活化枯草芽孢杆菌，37℃，160 rpm，恒温振荡器培养20h，各菌接种量为1-5%；
所述保加利亚乳杆菌的单菌培养液中保加利亚乳杆菌的活菌浓度为(2-5)×108CFU/mL，所述干酪乳杆菌的单菌培养液中干酪乳杆菌的活菌浓度为(5-8)×109CFU/mL，所述产朊假丝酵母的单菌培养液中产朊假丝酵母的活菌浓度为(1-5)×109CFU/mL，所述枯草芽孢杆菌的单菌培养液中枯草芽孢杆菌的活菌浓度为(1-5)×109CFU/mL；
混合发酵：由保加利亚乳杆菌、干酪乳杆菌、产朊假丝酵母和枯草芽孢杆菌按体积5：5：2：1进行混合，以总接种量5-10%进行发酵；所述混合培养的温度为30℃，所述混合培养的pH值为6.5-7.0，所述混合培养的时间为20-48h,所述混合培养的转速为50-100rpm；所述混合培养基为：葡萄糖50-100g，酵母浸粉8-10g，KH2PO4 1g，MgSO4·7H2O 0.6g，三水乙酸钠1g，轻质CaCO3 25-50g，蒸馏水1000ml，调节pH至6.5-7.0，于发酵罐中115 ℃灭菌30min；所述混合发酵液培养20-48h时乳酸钙含量不低于25 mg/ml，蛋白酶含量不低于200U/ml。
2.权利要求1所述种子活化中，其特征在于，所述各菌种种子液中，保加利亚乳杆菌活菌浓度为(2-5)× 108 CFU/mL，干酪乳杆菌的活菌浓度为(1-3)× 1010CFU/，产朊假丝酵母的活菌浓度为(1-5)×109CFU/mL，枯草芽孢杆菌的活菌浓度为(1-5)×109 CFU/mL。
3.权利要求1所述混合发酵液，其特征在于，所述步骤中，按照由保加利亚乳杆菌、干酪乳杆菌、产朊假丝酵母和枯草芽孢杆菌按体积5：5：2：1进行混合培养。
4.权利要求1所述混合发酵中，其特征在于，所述混合发酵液灭菌方法中要求115℃灭菌30min。
5.权利要求1所述共生混合发酵液在可用于食品、种植肥料和养殖饲料发酵生产等领域。
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