CN115074290A - 一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌及其应用 - Google Patents
一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌及其应用 Download PDFInfo
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- CN115074290A CN115074290A CN202210790286.2A CN202210790286A CN115074290A CN 115074290 A CN115074290 A CN 115074290A CN 202210790286 A CN202210790286 A CN 202210790286A CN 115074290 A CN115074290 A CN 115074290A
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Abstract
本发明公开一种联产共轭亚油酸和γ‑氨基丁酸的干酪乳杆菌及其应用,该菌株于2022年5月13日保藏于中国典型培养物保藏中心,保藏编号为CCTCCM 2022616;本发明通过采用常压室温等离子体诱变技术,经过筛选得到能够高效联产苯乳酸和γ‑氨基丁酸的干酪乳杆菌MSG308,将此干酪乳杆菌MSG308接种至含有谷氨酸的培养基中发酵72h,发酵液中苯乳酸含量高达15.75g/L,γ‑氨基丁酸的含量高达180.45g/L,大大降低生产成本;本发明的干酪乳杆菌MSG308属于食品安全乳杆菌属,可广泛应用于生物发酵饲料和饲料添加剂领域,具有巨大的应用价值和市场开发潜力。
Description
技术领域
本发明具体涉及一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌及其应用,属于微生物技术领域。
背景技术
近年来,饲用抗生素滥用导致病原菌耐药性、药物残留和环境污染等生物安全问题得到了越来越多的关注,开发无毒无公害的新型微生物制剂来代替抗生素已经成为饲料行业的研究热点。微生态制剂具有能提高饲料的消化吸收率,提高动物免疫力,延长货架期等功能,利用益生菌微生物发酵生产的无毒无害、无抗无残留的绿色饲料添加剂在国内外饲料工业中应用不断增多。
乳酸菌是我国农业部公布的可直接饲喂动物且允许使用的饲料级益生菌菌种之一,其具备一系列优良的益生特性而广泛地应用于动物发酵饲料领域中,诸如:耐酸性、耐受胆汁酸、防治疾病、增强机体免疫力、提高生长性能、无毒副作用、无耐药性、无残留、促进生长、增加体重等优点。
益生菌发酵饲料的研究和开发的关键问题在于优良菌株的分离、筛选和发酵饲料的制备工艺。目前,生物发酵饲料的应用情况并不理想,应用效果欠佳,主要是由以下原因造成的:1、大多数的益生菌为不产芽孢的乳酸菌,且多属于专性厌氧菌,极易受到温度、氧气、水分及酸碱等外界环境因素的影响,菌活性保持比较困难;2、生物发酵饲料由于未进行消毒灭菌(若对发酵饲料进行消毒,会破坏饲料中的营养成分,造成其营养流失),存在易变质、易霉变、安全性差等问题,因此,需要通过在生物发酵饲料中额外添加防腐剂来解决上述问题,生产成本高;3、菌种本身的特性不足,抑菌能力差、转化效率不高及功能成分不足。
苯乳酸是一种广泛存在自然界中的小分子天然有机酸,由部分乳酸菌分泌的一种具有广谱抗菌性的新型抑菌剂,能抑制食源性致病菌、腐败菌,特别能抑制真菌的污染。研究表明,在饲料中添加苯乳酸可以促进动物生长、调节畜禽免疫功能、抑制致病菌生长,起到维持畜禽健康和提高生产性能的作用。目前苯乳酸生产方法主要有化学合成和生物合成。化学合成存在技术条件复杂、反应条件严苛、环境污染比较大等。生物合成苯乳酸是一种新方法,微生物在合成苯乳酸起到防腐效果的同时还能产生其它有机酸成分,是目前普遍认可的一种成本低、效果好、使用安全的方法。
γ-氨基丁酸是一种非蛋白质天然氨基酸,是哺乳动物中枢神经系统内最主要的抑制性神经递质,不仅对人类具有镇痛、抗焦虑、抗惊厥、调节激素分泌和营养神经细胞等作用,对畜禽也有促进动物采食增重、提高动物免疫力、抗应激能力及促进生殖等作用。近年来,γ-氨基丁酸被作为一种饲料添加剂在畜禽养殖业和饲料工业中广泛应用。
目前,现有技术是在饲料中通过外添加的方式使其富含苯乳酸或者γ-氨基丁酸,而天然或微生物合成的高纯苯乳酸或者γ-氨基丁酸成本高,限制了其在饲料添加剂中的规模化应用,因此,开发高效联产苯乳酸和γ-氨基丁酸的乳酸菌应用于饲料发酵领域具有重要意义
发明内容
为了解决上述技术问题,本发明提供一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌及其应用,本发明的干酪乳杆菌在高产苯乳酸的同时γ-氨基丁酸的产量也能达到较高的水平,达到功能活性成分多效叠加的目的,在生物发酵饲料领域具有重要的应用价值。
本发明的目的一在于一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌,所述干酪乳杆菌MSG308于2022年5月13日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCCM 2022616,保藏地址为中国武汉,武汉大学,邮编:430072。
本发明提供的干酪乳杆菌的形态学及生理化学特征如下:
菌落颜色:乳白色;
需氧方式:兼性厌氧;
适宜生长温度:35℃-37℃;
适宜生长pH:6-6.5;
菌落形态:杆状;
革兰氏染色:阳性。
本发明的目的二在于提供一种干酪乳杆菌在发酵联产苯乳酸和γ-氨基丁酸中的应用。
本发明的目的三在于提供一种联产苯乳酸和γ-氨基丁酸的方法,采用本发明提供的干酪乳杆菌MSG308,依次经过活化培养、种子培养、发酵罐补料反馈培养高效联产苯乳酸和γ-氨基丁酸。
进一步的,所述发酵罐内液体培养基pH为6.0~6.5,包括如下按质量百分比计的组份:葡萄糖0.5%,牛大骨蛋白胨2%,酵母粉2%,七水硫酸镁0.03%,一水硫酸锰0.01%,吐温-80 0.2%,谷氨酸5%,苯丙氨酸1%,其余为水。
进一步的,所述发酵罐培养周期划分为两个阶段:
(1)在发酵培养起始阶段,初始葡萄糖的浓度控制在20g/L,通过流加60%的葡萄糖控制发酵体系中的残糖(以葡萄糖计)为0.2-0.5g/L;
(2)在发酵罐内OD600达到20后,通过流加苯丙氨酸和谷氨酸控制发酵体系中的苯丙氨酸浓度保持在0.01-0.1g/L和谷氨酸的浓度保持在1-5g/L;
其中,所述发酵罐培养全过程控制溶解氧在5-10%,pH为6.0-6.5。
本发明的目的四在于提供一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌在饲料中的应用。
本发明的目的五在于提供一种饲料添加剂的制备方法,以本发明提供的干酪乳杆菌作为发酵菌株,将发酵菌株种子液接种至固态发酵培养基中,并于固态发酵池中通过固态发酵制备富含苯乳酸和γ-氨基丁酸的饲料添加剂。
进一步的,所述固态发酵培养基包括如下按质量百分比计的组份:黄豆饼粉20%,麸皮10%,玉米淀粉2.5%,菜籽粕1%,谷氨酸5%,苯丙氨酸1%,酵母提取物0.3%,硫酸铵0.2%,氯化钠1%,其余为水;所述固态发酵培养基的pH为6.0~6.5。
相较于现有技术,本发明的有益效果在于:
1、本发明采用常温常压等离子体诱变技术,首次发现能够高效联产苯乳酸和γ-氨基丁酸的干酪乳杆菌MSG308,该菌株经过分批补料发酵,发酵液中苯乳酸的浓度最高可达15.75g/L,γ-氨基丁酸的浓度最高可达180.45g/L,证实了本发明提供的干酪乳杆菌MSG308极具工业化运用价值。
本发明提供的干酪乳杆菌MSG308是乳酸益生菌,是饲料添加剂上广泛应用的一种益生菌种,同时兼具联产苯乳酸和γ-氨基丁酸能力,可广泛应用于制备富含苯乳酸和γ-氨基丁酸的复合功能饲料添加剂,大幅降低制备成本,通过饲喂试验证明,将所述饲料添加剂添加到猪饲料中,可提高猪只的采食量和日增重,降低料重比,具有促生长作用,具有广阔的应用前景。
附图说明
图1为本发明实施例1中干酪乳杆菌CICC 23185诱变致死率曲线;
图2为本发明实施例1中优良突变株96孔板初筛发酵结果示意图;
图3为本发明实施例2中优良突变株摇瓶复筛发酵结果示意图;
图4为本发明实施例2中干酪乳杆菌MSG308 20L发酵罐发酵曲线图。
具体实施方式
下面结合附图和较佳实施例对本发明做进一步的说明,在本发明中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值;对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开;
以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到;
以下实施例中的定量试验,均设置三次重复实验,结果取平均值;
下述实施例中苯乳酸的检测:参考《苯乳酸的快速检测研究》、《苯乳酸的微生物合成及分离》等文献,使用日立高效液相色谱仪,紫外吸收检测器,分析柱C18柱,柱温30℃,流速1mL/min,进样量10μL;流动相:A:0.5%的磷酸(V/V),B:0.5%的磷酸乙腈溶液(V/V),检测波长210nm。洗脱条件:0~12min,A:B=80:20,12~15min,A:B=0:100,15~16min,A:B=80:20进行检测;
γ-氨基丁酸的检测:参照中华人民共和国轻工行业标准QB/T4587-2013γ-氨基丁酸检测方法进行检测。
实施例1干酪乳杆菌及其诱变筛选
一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌MSG308,经由源于中国工业微生物菌种保藏管理中心的干酪乳杆菌CICC 23185经常压室温等离子体诱变筛选,进而得到所述干酪乳杆菌MSG308;
其中,发酵联产苯乳酸和γ-氨基丁酸的干酪乳杆菌MSG308的诱变筛选方法,包括如下步骤:
S1、诱变预处理:将出发菌干酪乳杆菌CICC 23185的甘油菌100μL接种至5mL MRS液体培养基培养,在37℃下静置培养至菌液OD600值为1.0,在8000rpm的转速下离心处理10min,弃上清液收集菌体,用含5%(v/v)甘油的生理盐水洗涤菌体两遍,并重悬制成分散均匀的菌悬液,调整细菌的终浓度为100~110CFU·mL-1;
S2、常压室温等离子体诱变:取20μL经过步骤S1制得的菌悬液均匀涂于无菌载片上,干燥后用镊子将载片转移至载物台,采用高纯氦气作为等离子体的工作气体,设置电源功率50W,照射距离4mm,等离子体的温度为26℃,气流量为10L/min,处理菌物载片,进行常压室温等离子体诱变处理,照射时间分别取0(对照)、5、10、15、20、25、30、35、40、45、50、55s;处理后将载片转移到1.5mL的EP管中,在振荡器上再生培养60min,再用含5%(v/v)甘油的生理盐水稀释10倍,形成新的菌悬液;
其中,将菌悬液涂布于MRS固体培养基平板后置于37℃培养箱内培养,培养48h,观察菌株生长情况,统计菌落数,绘制致死率曲线(如图1所示),选择致死率为75%的处理时间;
S3、诱变后培养:样品诱变处理完毕后,取经过步骤S2获得的新的菌悬液200μL涂布于MRS固体培养基,37℃静置培养48h分离单克隆;
S4、初筛:采用96孔板对诱变分离的突变株进行初筛,挑取单克隆到含1mL初筛液体培养基的96孔板培养,培养条件37℃,100rpm,培养72h,离心收集上清液,检测上清液中苯乳酸和γ-氨基丁酸含量,筛选苯乳酸和γ-氨基丁酸含量高的菌种,共筛选的到三株高产突变株作为初筛菌株,分别命名MSG308、MSG637、MSG1024,并进行甘油保藏,三株菌株的苯乳酸和γ-氨基丁酸产量如图2所示;其中,初筛液体培养基包含如下质量百分数成分:葡萄糖0.3%,牛大骨蛋白胨1%,酵母粉1%,七水硫酸镁0.02%,一水硫酸锰0.005%,吐温-80 0.1%,苯丙氨酸0.5%,谷氨酸6%,其余为水,pH调至6.5;
S5、复筛:将经过步骤S4初筛获取的优良突变株和对照菌的甘油菌分别按5%接种量,接种至MRS固体培养基上培养,再取1.25mL甘油菌液接种至25mL复筛种子培养基培养,摇管装液量50%(v/v),培养温度为37℃,静置培养时间48h;其中,复筛种子培养基包含如下质量百分数成分:葡萄糖0.2%,牛大骨蛋白胨0.5%,酵母粉0.5%,七水硫酸镁0.01%,一水硫酸锰0.002%,吐温-800.1%,其余为水,pH调至6.5;复筛种子培养基中的种液按15%接种量取18.75mL种子培养液接种至125mL复筛发酵培养基培养,250mL摇瓶装液量125mL,发酵温度37℃,静置发酵72h,然后离心收集上清液;其中,复筛发酵培养基包含如下质量百分数成分:葡萄糖0.3%,牛大骨蛋白胨2%,酵母粉1.5%,七水硫酸镁0.03%,一水硫酸锰0.01%,吐温-80 0.2%,苯丙氨酸1%,谷氨酸6%,其余为水,pH调至6.5;参见图3,检测上清液苯乳酸和γ-氨基丁酸的含量,筛选出高产菌株干酪乳杆菌MSG308,该菌经72h摇瓶发酵,发酵液中苯乳酸达到6.23g/L,与对照菌相比提高623倍,γ-氨基丁酸浓度达到16.3g/L,与对照菌相比提高10.18倍;
上述诱变筛选方法中,MRS液体培养基包含如下质量百分数成分:酪蛋白胨1%,牛肉膏1%,酵母粉0.5%,葡萄糖0.5%,乙酸钠0.5%,柠檬酸二铵0.2%,吐温80 0.1%,磷酸氢二钾0.2%,七水硫酸镁0.02%,一水硫酸锰0.005%,其余为水,pH调至6.5;
MRS固体培养基含如下质量百分数成分:酪蛋白胨1%,牛肉膏1%,酵母粉0.5%,葡萄糖0.5%,乙酸钠0.5%,柠檬酸二铵0.2%,吐温-80 0.1%,磷酸氢二钾0.2%,七水硫酸镁0.02%,一水硫酸锰0.005%,琼脂2%,其余为水,pH调至6.5;
最后,将筛选得到的干酪乳杆菌MSG308进行传代培养以考察其遗传稳定性,每2天传代一次,传代10代,每隔一代进行摇瓶发酵测定菌株生物量、苯乳酸和γ-氨基丁酸的含量,结果表明干酪乳杆菌突变株MSG308传代过程中菌株生物量、苯乳酸和γ-氨基丁酸均无明显变化,具有良好的遗传稳定性;将获得遗传稳定的、能够同时高效积累苯乳酸和γ-氨基丁酸的干酪乳杆菌MSG308于2022年5月15日保藏于中国典型培养物保藏中心(简称CCTCC;地址:中国武汉,武汉大学;邮编:430072),保藏编号为CCTCC NO:M 2022616。
实施例2干酪乳杆菌在发酵联产苯乳酸和γ-氨基丁酸中的应用
发酵联产苯乳酸和γ-氨基丁酸的方法,采用本发明提供的干酪乳杆菌MSG308,包括如下步骤:
A1、菌种活化:取50μL干酪乳杆菌MSG308甘油菌,接种至含MRS固体培养基的试管斜面,培养箱中于37℃培养48h进行菌种活化,再用无菌生理盐水洗下菌体细胞,制备菌株细胞悬液,为活化后的菌株悬液;
A2、种子培养:取75mL经过步骤A1制得的活化后的菌株悬液按5%的接种量转接于含1.5L种子培养基的5L摇瓶,于37℃进行静置培养48h,制备种子液,进一步提高菌株的数量,强化菌株活力;
A3、干酪乳杆菌MSG308补料反馈发酵:采用20L全自动发酵罐(装液量为10L)进行干酪乳杆菌MSG308补料反馈发酵;取经过步骤A2培养好的1.5L种子培养液按15%的接种量无菌接种于10L发酵罐内液体培养基中,在发酵培养起始阶段,通气比为0.15(V/V·m),罐压为0.02Mpa,搅拌控制为150rpm,温度控制在37℃,初始葡萄糖的浓度控制在20g/L,通过流加60%的葡萄糖控制发酵体系中的残糖(以葡萄糖计)为0.2-0.5g/L;在发酵罐内OD600达到20后,通过流加苯丙氨酸和谷氨酸控制发酵体系中的苯丙氨酸浓度保持在0.01-0.1g/L和谷氨酸的浓度保持在1-5g/L;发酵罐培养全过程控制溶解氧在5-10%,pH为6.0-6.5,发酵周期控制在72h,8000rpm离心10min,收集发酵上清液;
其中,发酵罐内液体培养基包括如下按质量百分比计的组份:葡萄糖0.5%,牛大骨蛋白胨2%,酵母粉2%,七水硫酸镁0.03%,一水硫酸锰0.01%,吐温-800.2%,谷氨酸5%,苯丙氨酸1%,其余为水;
最终,测定发酵上清液中苯乳酸和γ-氨基丁酸含量,将经过步骤A3获得的发酵液上清进行苯乳酸和γ-氨基丁酸检测,参见图4,检测结果如下:干酪乳杆菌MSG308经72h补料反馈发酵,发酵液中苯乳酸的浓度最高可达15.75g/L,发酵液中γ-氨基丁酸的浓度最高可达180.45g/L,极具工业化运用价值。
实施例3干酪乳杆菌MSG308在饲料添加剂上的应用
将本发明提供的干酪乳杆菌作为发酵菌株,将发酵菌株种子液接种至固态发酵培养基中,并于固态发酵池中通过固态发酵制备富含苯乳酸和γ-氨基丁酸的饲料添加剂,具体步骤如下:
(1)菌种活化:取50μL干酪乳杆菌MSG308甘油菌接种含MRS固体培养基的试管斜面,培养箱中于37℃培养48h,再用5mL无菌生理盐水洗下菌体细胞,制备菌株细胞悬液;
(2)种子培养:取活化好的菌株细胞悬液75mL按5%的接种量接到含1.5L种子培养基的5L摇瓶,于37℃进行静置培养48h,制备种子液;
(3)固态发酵制备复含苯乳酸和γ-氨基丁酸的干酪乳杆菌饲料添加剂:取黄豆饼粉60kg,麸皮30kg,玉米淀粉7.5kg,菜籽粕3kg,谷氨酸15kg,苯丙氨酸3kg,酵母提取物0.9kg,硫酸铵0.6kg,氯化钠0.3kg,其余为水,配置300kg,调节pH至6.5,搅拌均匀,配制成固态发酵培养基,置入发酵池;取经过步骤(2)获得的干酪乳杆菌种子液以占所述基质总重量5%的接种量,接种于固态发酵培养基中,固态发酵条件如下:发酵初始pH值为6.5,发酵过程温度为30℃,发酵时间为72h,发酵结束,于40℃在风干机中进行风干,制得饲料添加剂,将该添加剂装袋密封,保存在通风、阴凉、干燥处;
其中,采用MRS固体培养基倾注平板计数法计数在发酵过程中的干酪乳杆菌数量,干酪乳杆菌数量达到7.6×109cfu/g,取4g饲料添加剂于50mL离心管中,加入40mL去离子水搅拌混匀静置1h后10000rpm离心5min取上清,检测苯乳酸和γ-氨基丁酸含量,检测结果如下:干酪乳杆菌MSG308经72h固态发酵,发酵饲料中苯乳酸的浓度可达5.36g/kg,γ-氨基丁酸的浓度最高可达30g/kg。
实施例4干酪乳杆菌MSG308发酵饲料添加剂的饲喂效果
将60头体重为25kg(±0.5kg)肉猪随机分成两个组,一个试验组,一个对照组,各30头猪,分别分成6个重复组,每个重复组5头猪,将根据实施例3所制发酵饲料添加剂按重量百分比5%的比例添加添加到猪全价饲料中(玉米55%、豆粕23%、麸皮17.5%、鱼粉0.5%),用于饲喂试验组肉猪;将市售Z824型猪用预混料按重量百分比5%的比例添加到猪全价饲料中,用于饲喂对照组肉猪,连续饲喂30天;按猪场正常的养殖方式进行投料、加水、加药、防疫等饲养管理,对比干酪乳杆菌MSG308发酵饲料添加剂和市售Z824型猪用预混料的饲养效果,测定日均采食量、日增重和料重比,试验结果见表1;
表1干酪乳杆菌MSG308发酵饲料添加剂喂养结果
测定项目 | 日均采食量(g) | 日增重(g) | 料重比 |
对照组 | 1308.4±9.5 | 812.5±3.4 | 1.61 |
试验组 | 1342.6±10.2 | 854.3±6.2 | 1.57 |
由表1数据可知,试验组的日均采食量和日增重均高于对照组,试验组料重比降低;该结果说明,以本发明菌株干酪乳杆菌MSG308作为发酵菌株,按照本发明所述固态发酵方法制备饲料添加剂应用到猪养殖生产中,可提高肉猪的日均采食量和日增重,降低料重比,具有促生长作用。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书内容所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
Claims (8)
1.一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌,其特征在于:保藏编号为CCTCC M2022616的干酪乳杆菌MSG308。
2.如权利要求1所述的干酪乳杆菌在联产苯乳酸和γ-氨基丁酸中的应用。
3.一种联产苯乳酸和γ-氨基丁酸的方法,其特征在于:采用如权利要求1所述的干酪乳杆菌MSG308依次经过活化培养、种子培养、发酵罐补料反馈培养高效联产苯乳酸和γ-氨基丁酸。
4.如权利要求3所述的一种联产苯乳酸和γ-氨基丁酸的方法,其特征在于:所述发酵罐内液体培养基pH为6.0~6.5,包括如下按质量百分比计的组份:葡萄糖0.5%,牛大骨蛋白胨2%,酵母粉2%,七水硫酸镁0.03%,一水硫酸锰0.01%,吐温-80 0.2%,谷氨酸5%,苯丙氨酸1%,其余为水。
5.如权利要求3所述的一种联产苯乳酸和γ-氨基丁酸的方法,其特征在于:所述发酵罐培养周期划分为两个阶段:
(1)在发酵培养起始阶段,初始葡萄糖的浓度控制在20g/L,通过流加60%的葡萄糖控制发酵体系中的残糖(以葡萄糖计)为0.2-0.5g/L;
(2)在发酵罐内OD600达到20后,通过流加苯丙氨酸和谷氨酸控制发酵体系中的苯丙氨酸浓度保持在0.01-0.1g/L和谷氨酸的浓度保持在1-5g/L;
其中,所述发酵罐培养全过程控制溶解氧在5-10%,pH为6.0-6.5。
6.如权利要求1所述的一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌在饲料中的应用。
7.一种饲料添加剂的制备方法,其特征在于:以权利要求1所述的干酪乳杆菌作为发酵菌株,将发酵菌株种子液接种至固态发酵培养基中,并于固态发酵池中通过固态发酵制备富含苯乳酸和γ-氨基丁酸的饲料添加剂。
8.如权利要求7所述的一种饲料添加剂的制备方法,其特征在于:所述固态发酵培养基包括如下按质量百分比计的组份:黄豆饼粉20%,麸皮10%,玉米淀粉2.5%,菜籽粕1%,谷氨酸5%,苯丙氨酸1%,酵母提取物0.3%,硫酸铵0.2%,氯化钠1%,其余为水;所述固态发酵培养基的pH为6.0~6.5。
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