CN115161248A - 一种以海带酶解粉为主要基质的多菌种联合发酵生产植物乳杆菌的方法 - Google Patents
一种以海带酶解粉为主要基质的多菌种联合发酵生产植物乳杆菌的方法 Download PDFInfo
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Abstract
本发明公开了一种以海带酶解粉为主要基质的多菌种联合发酵生产植物乳杆菌的方法，属于微生物发酵技术领域。本发明利用酿酒酵母、地衣芽孢杆菌和植物乳杆菌进行联合发酵，可生产高活性饲料用微生态制剂产品。在液态培养基中，先通过酵母菌的生长消耗掉其中的氧气，创造厌氧环境，然后通过地衣芽孢杆菌生长分泌蛋白酶、淀粉酶等，水解培养基中养分可供植物乳杆菌快速生长。三种菌的联合培养充分利用了海带酶解物等培养基中的各种营养成分，将其转化成高益生活性的植物乳杆菌菌体。
Description
技术领域
本发明属于微生物发酵技术领域，尤其涉及一种以海带酶解粉为主要基质的多菌种联合发酵生产植物乳杆菌的方法。
背景技术
海带(Laminariajaponica)是多年生大型食用藻类，含有丰富的褐藻胶、岩藻糖胶、褐藻淀粉等海带多糖，并含有酸性聚糖类物质、岩藻半乳多糖硫酸酯、大叶藻素、半乳糖醛酸、昆布氨酸、牛磺酸、双歧因子等多种活性成分。据测定，每100g海带中含8g蛋白质、0.1g脂肪、0.57mg胡萝卜素、0.09mgVB、0.36mgVB1、1.6mg烟酸、1.177mg钙、1.50mg铁，2.16mg磷，还含有丰富的褐藻酸、纤维素、甘露醇以及多种微量元素。海带酶解干粉是通过多种酶制剂将海带原浆进行降解，充分释放里面的活性成分，然后喷雾干燥处理成干粉。海带酶解干粉里面含有多种氨基酸、维生素、多糖和微量元素，非常适合作为微生物发酵培养基使用。利用微生物发酵，将低值的海带酶解粉转化为高活性微生态制剂产品，可以大幅度提高其附加值。
酿酒酵母(Saccharomyces cerevisiae)，又称面包酵母或者出芽酵母。酿酒酵母是与人类关系最广泛的一种酵母，用于制作面包和馒头等食品及酿酒。酿酒酵母的细胞为球形或者卵形，直径5-10μm。其繁殖的方法为出芽生殖。酿酒酵母与同为真核生物的动物和植物细胞具有很多相同的结构，又容易培养，酵母被用作研究真核生物的模式生物。酿酒酵母被认为是最具潜力的大规模生产菌种。
地衣芽孢杆菌(Bacillus licheniformis)是一种革兰氏染色阳性菌，能够分泌蛋白酶、淀粉酶等多种酶，酶分泌的最适温度为37℃该细菌可调整菌群失调达到治疗目的，可促使机体产生抗菌活性物质、杀灭致病菌。能产生抗活性物质抑制致病菌的生长繁殖。
植物乳杆菌(Lactobacillusplantarum)是一种在自然界广泛存在的革兰氏阳性菌，普遍分布在各类发酵食品中，同时也是肠道内重要的益生微生物菌群之一。由于植物乳杆菌具有优良的耐受强酸、胆盐、高渗透压、极端温度、饥饿和氧化等胁迫的能力，使得其在工业生产、医疗保健及食品加工等领域被广泛应用。
发明内容
本发明的目的之一在于提供一种以海带酶解粉为主要基质的多菌种联合发酵生产植物乳杆菌的方法，所述方法是以酿酒酵母、地衣芽孢杆菌和植物乳杆菌作为发酵菌种，在含有海带酶解物干粉的培养基中进行发酵。
优选地，所述酿酒酵母、地衣芽孢杆菌和植物乳杆菌分别为酿酒酵母BNCC192858、地衣芽孢杆菌SDHY-2101和植物乳杆菌BNCC 138404。
更优选地，所述酿酒酵母BNCC192858、地衣芽孢杆菌SDHY-2101和植物乳杆菌BNCC138404的种子液的体积比为1:2:2或2:2:2。
更优选地，所述含有海带酶解物干粉的培养基为：海带酶解物干粉10％-19wt％、菜粕8wt％、葡萄糖3wt％、小麦麸皮8wt％、玉米粉5wt％、余量为水。
优选地，所述发酵过程为恒温发酵，温度为30℃，发酵时间为72h。
优选地，所述发酵过程分为两个阶段，第一个阶段为30℃培养24h，第二个阶段为37℃继续培养48h。
本发明的目的之二在于提供上述以海带酶解粉为主要基质的多菌种联合发酵生产植物乳杆菌的方法在生产植物乳杆菌中的应用。
与现有技术相比，本发明具有如下有益效果：
本发明利用酿酒酵母、地衣芽孢杆菌和植物乳杆菌进行联合发酵，可生产高活性饲料用微生态制剂产品。在液态培养基中，先通过酵母菌的生长消耗掉其中的氧气，创造厌氧环境，然后通过地衣芽孢杆菌生长分泌蛋白酶、淀粉酶等，水解培养基中养分可供植物乳杆菌快速生长。三种菌的联合培养充分利用了海带酶解物等培养基中的各种营养成分，将其转化成高益生活性的植物乳杆菌菌体。
具体实施方式
实施例1酿酒酵母BNCC192858种子液的制备
YPD培养基配制：酵母提取物10.0g、蛋白胨20.0g、葡萄糖20.0g，蒸馏水溶解，定容至1000mL，121℃高压灭菌20min。
取酿酒酵母BNCC192858单菌落接种于10mLYPD液体培养基，30℃、220r/min振荡培养12h。吸取100μL，转接100mLYPD培养基，继续28℃、220r/min振荡培养18-24h。
实施例2地衣芽孢杆菌SDHY-2101种子液的制备
培养基配制：葡萄糖10.0g、玉米粉12.0g、酵母粉3.0g、海带酶解干粉5.0g，定容至1000mL，121℃高压灭菌20min。
地衣芽孢杆菌SDHY-2101由本公司实验室保存。挑取地衣芽孢杆菌单菌落接种10mL培养基，37℃、220r/min振荡培养12h。吸取100μl菌液转接100mL培养基中，继续37℃、220r/min振荡培养24h。
实施例3植物乳杆菌BNCC 138404种子液制备。
MRS培养基配制：蛋白胨10.0g、牛肉膏10.0g、酵母膏5.0g、柠檬酸氢二铵2.0g、葡萄糖20.0g、吐温-801.0mL、乙酸钠5.0g、磷酸氢二钾2.0g、MgSO4·7H2O 0.50g、MnSO4·H2O0.25g，蒸馏水溶解，定容至1000mL，pH在6.2-6.6之间。121℃高压灭菌20min。
植物乳杆菌菌种(BNCC138404)购北京北纳创联生物技术研究院。挑取植物乳杆菌单菌落，接种10mLMRS液体培养基，37℃静止培养24h。吸取1.0mL，转接至100mLMRS培养基，继续37℃静止培养24h。
实施例4海带酶解物干粉的制备
鲜海带以1:1-1:1.5的比例加水粉碎后加入0.1％-0.5％的复合酶(复合酶中纤维素酶、果胶酶、蛋白酶和淀粉酶的质量百分数分别为60％、30％、5％和5％)，40-60℃酶解20-24h，获得海带酶解液，海带酶解液经喷雾干燥机(设定送风频率15-22HZ、雾化频率225-235HZ、引风频率35-45HZ、进风温度170-180℃、出风温度75-85℃)进行喷粉。
实施例5发酵培养基的制备
培养基主要成分为：海带酶解物干粉10％-19wt％、菜粕8wt％、葡萄糖3wt％、小麦麸皮8wt％、玉米粉5wt％，具体配方见表1，pH值自然。500mL三角瓶中装50g，封口膜密闭封口，121℃高压灭菌20min。
表1发酵培养基配方
| 组分 | 培养基1 | 培养基2 | 培养基3 | 培养基4 |
| 海带酶解干粉 | 10％ | 13％ | 16％ | 19％ |
| 菜粕 | 8％ | 8％ | 8％ | 8％ |
| 玉米粉 | 5％ | 5％ | 5％ | 5％ |
| 葡萄糖 | 3％ | 3％ | 3％ | 3％ |
| 小麦麸皮 | 8％ | 8％ | 8％ | 8％ |
| KH<sub>2</sub>PO<sub>4</sub> | 2％ | 2％ | 2％ | 2％ |
| NaCl | 1％ | 1％ | 1％ | 1％ |
| 蒸馏水 | 63％ | 60％ | 57％ | 54％ |
实施例6恒温发酵工艺
接种剂量组合：将培养好的酿酒酵母、地衣芽孢杆菌和植物乳杆菌的种子液按照体积比1:2:2(接种组合1)和2:2:2(接种组合2)的比例(见表2)，在超净工作台中接种(接种量为千分之一)，接种后摇匀，密封瓶口。每种培养基做3个平行处理，置于30℃培养箱中培养72h。
表2各菌株在发酵培养基中的接种比例
实施例7变温发酵工艺
接种剂量组合：将培养好的酿酒酵母、地衣芽孢杆菌和植物乳杆菌的种子液按照体积比1:2:2(接种组合1)和2:2:2(接种组合2)的比例(见表2)，在超净工作台中接种，接种总量同实施例6，接种后摇匀，密封瓶口。每种培养基做3个平行处理，先置于30℃培养箱中培养24h，调整培养温度为37℃继续培养48h。
实施例8培养物pH值及活菌计数
发酵完成后，测定培养物pH值和植物乳杆菌的活菌数(见表3、4)。植物乳杆菌的活菌计数采用GB/T 4789.35的方法进行。从结果可知，第2种接种组合中，第3种培养基的发酵效果最好，pH值最低，活菌数量最多。变温培养的活菌数高于恒温培养的活菌数。
表3恒温培养物的pH值和植物乳杆菌活菌数
表4变温培养物的pH值和植物乳杆菌活菌数
对比例1
配制第3种培养基(参见实施例5中的表1)，灭菌后，等体积比例(2:2)接种酿酒酵母和植物乳杆菌或者地衣芽孢杆菌和植物乳杆菌，接种总量同实施例6，每个接种剂量设置3个重复，然后按照变温培养方案进行发酵培养，测定pH值和植物乳杆菌的活菌数(表5)。
表5
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (9)
1.一种以海带酶解粉为主要基质的多菌种联合发酵生产植物乳杆菌的方法，其特征在于，所述方法是以酿酒酵母、地衣芽孢杆菌和植物乳杆菌作为发酵菌种，在含有海带酶解物干粉的培养基中进行发酵。
2.根据权利要求1所述的方法，其特征在于，所述酿酒酵母、地衣芽孢杆菌和植物乳杆菌分别为酿酒酵母BNCC192858、地衣芽孢杆菌SDHY-2101和植物乳杆菌BNCC 138404。
3.根据权利要求2所述的方法，其特征在于，所述酿酒酵母BNCC192858、地衣芽孢杆菌SDHY-2101和植物乳杆菌BNCC 138404的种子液的体积比为1:2:2或2:2:2。
4.根据权利要求1-3中任一项所述的方法，其特征在于，所述含有海带酶解物干粉的培养基为：海带酶解物干粉10％-19wt％、菜粕8wt％、葡萄糖3wt％、小麦麸皮8wt％、玉米粉5wt％、余量为水。
5.根据权利要求4所述的方法，其特征在于，所述发酵过程为恒温发酵，温度为30℃，发酵时间为72h。
6.根据权利要求4所述的方法，其特征在于，所述发酵过程分为两个阶段，第一个阶段为30℃培养24h，第二个阶段为37℃继续培养48h。
7.权利要求1-3中任一项所述的方法在生产植物乳杆菌中的应用。
8.权利要求4所述的方法在生产植物乳杆菌中的应用。
9.权利要求5或6所述的方法在生产植物乳杆菌中的应用。
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