CN112538450B - 高产风味耐酸乳杆菌在食品生产中的应用 - Google Patents
高产风味耐酸乳杆菌在食品生产中的应用 Download PDFInfo
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Abstract
本发明公开了高产风味耐酸乳杆菌在食品生产中的应用,属于微生物领域、生物工程领域、食品领域。本发明的耐酸乳杆菌CGMCC NO.21135,耐高温和耐酸性能好,能适应多种食品生产环境;可在乙醇浓度为18%(v/v)正常生长;在发酵过程中产4‑乙烯基愈创木酚3254.82μg/L,有很强的产风味能力;可利用多种碳源,更好地提高底物利用率;本发明的耐酸乳杆菌基因组小,不具有生物胺合成的代谢通路和生物胺合成相关酶的编码基因,也不具有硝酸盐还原酶的编码基因,具有不产生物胺和亚硝酸盐的特征,安全性高。本发明的耐酸乳杆菌可广泛用于发酵食品,包括各种酒精产品、酱油、醋类产品、发酵茶、发酵蔬菜、发酵饮料、酸奶、干酪、豆豉、乳腐、发酵米面食品等。
Description
技术领域
本发明涉及高产风味耐酸乳杆菌在食品生产中的应用,属于微生物领域、生物工程领域、食品领域。
背景技术
传统发酵食品与人民生活息息相关,包括白酒、黄酒、酱油、啤酒、葡萄酒、食醋、发酵茶、传统发酵蔬菜、发酵饮料、酒精饮品、酸奶、干酪、果醋、酒酿、豆豉、乳腐、发酵米面食品等。乳酸菌是发酵食品中普遍存在的微生物,对食品发酵过程以及最终产品品质非常重要。但乳酸菌种类差异大,如乳杆菌是乳酸菌中最大的属,其不同种甚至不同株都有很大差异,因此获得优良性能的乳酸菌株,并应用于发酵食品中,对于保障与提升食品品质具有重要作用。
现有生产用乳酸菌主要是贡献乳酸,其它特性如风味特征、耐受性、安全性等则不够突出。传统发酵食品用优良性能乳酸菌菌株需要满足以下要求:第一,食品发酵过程对微生物而言属于微生物胁迫环境,如白酒发酵为高温、高酸与高乙醇环境等,这要求乳酸菌具有耐受以上胁迫环境的能力;第二,发酵食品安全性是食品品质的重要目标,这要求微生物满足食品用菌株安全特征;第三,发酵食品具有独特风味特征与要求,这要求微生物具有独特的风味代谢活性;第四,很多食品发酵是以谷物为主要原料,谷物原料中淀粉是主要基质,获得能够利用底物淀粉的菌株能够有效提高食品发酵资源利用率。因此,获得兼具有多种优良性能的乳酸菌菌株,才能真正有效应用于食品发酵生产,从而提升食品品质,并有助于保障食品安全。
发明内容
[技术问题]
本发明的技术问题为:传统生产用乳酸菌主要是贡献乳酸,其它特性如风味特征、耐受性、安全性等则不够突出。
[技术方案]
本发明提供了一株具有耐高温、耐酸、耐乙醇、安全、高产风味等特性的耐酸乳杆菌Lactobacillus acetotolerans JZ 16,其保藏编号为:CGMCC NO.21135,分类名称为耐酸乳杆菌Lactobacillus acetotolerans,保藏日期为2020年12月9日,保藏单位名称为中国普通微生物菌种保藏管理中心。
本发明还提供了含有上述耐酸乳杆菌CGMCC NO.21135的组合物。
在本发明的一种实施方式中,所述组合物是酒曲、酒醅、固态菌剂或液态菌剂。
在本发明的一种实施方式中,所述组合物是耐酸乳杆菌制剂。
在本发明的一种实施方式中,所述组合物含有CGMCC NO.21135菌体的活细胞、冷冻干燥得到的CGMCC NO.21135干菌体、固定化的CGMCC NO.21135细胞,或者以其他任何形式存在的CGMCC NO.21135菌株。
在本发明的一种实施方式中,所述组合物中还含有任何可以应用于食品或者食品制备的任意种属的菌株,比如地衣芽孢杆菌、酿酒酵母、枯草芽孢杆菌等等。
在本发明的一种实施方式中,所述组合物中还含有益生菌;所述益生菌包含罗伊氏乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌、约氏乳杆菌、格氏乳杆菌、干酪乳杆菌、副干酪乳杆菌、植物乳杆菌、发酵乳杆菌、卷曲乳杆菌、唾液乳杆菌、清酒乳杆菌、德氏乳杆菌、瑞士乳杆菌、假小链双歧杆菌、长双歧杆菌长亚种、长双歧杆菌婴儿亚种、青春双歧杆菌、短双歧杆菌、两歧双歧杆菌、动物双歧杆菌动物亚种以及动物双歧杆菌乳亚种中的一种或多种。
在本发明的一种实施方式中,所述微生物菌剂中还含有任意能用于食品的载体。
本发明还提供了上述耐酸乳杆菌CGMCC NO.21135在食品生产中的应用。
在本发明的一种实施方式中,所述食品是白酒、黄酒、酱油、啤酒、葡萄酒、食醋、发酵茶、传统发酵蔬菜、发酵饮料、酒精饮品、酸奶、干酪、果醋、酒酿、豆豉、乳腐、发酵米面食品中的任意一种或多种。
可选地,所述应用是耐酸乳杆菌CGMCC NO.21135至少解决如下一个问题:提升食品风味品质、提升食品安全品质、提高底物利用率、提高食品安全性、降低食品中生物胺/亚硝酸盐含量等。
可选地,所述食品是白酒;所述白酒的酿造方法中,包括将上述耐酸乳杆菌CGMCCNO.21135或含有耐酸乳杆菌CGMCC NO.21135的组合物以液体或固体培养物的形式接种于白酒大曲、堆积醅或窖池发酵的酒醅中。
可选地,所述白酒的酿造方法中,是在酒醅中额外接种含有CGMCC NO.21135组合物至含量为CGMCC NO.21135的终含量为103~107CFU/g酒醅。
可选地,所述食品为发酵蔬菜;所述发酵蔬菜的制备方法包括:将蔬菜清洗、榨汁、护色、加水加糖、杀菌,接种含有耐酸乳杆菌CGMCC NO.21135的菌剂进行发酵。
可选地,所述发酵蔬菜中的菌剂,还含有植物乳杆菌。可选地,所述发酵蔬菜可以是白菜、卷心菜、甜菜、萝卜、黄瓜、芹菜、青番茄、辣椒、青豆、菜豆等。可选地,所述发酵蔬菜的制备过程中,含包括加入纤维素酶、果胶酶等中的一种或者两种对榨汁后的蔬菜汁进行酶解处理。
可选地,所述食品为果醋。可选地,所述果醋的制备方法包括:挑选水果、清洗、榨汁、调配、预杀菌得到水果汁;然后依次接种果酒酵母进行酒精发酵、接种醋酸菌进行醋酸发酵、接种含有耐酸乳杆菌CGMCC NO.21135的乳酸菌菌剂进行乳酸发酵,得到水果发酵液,经过滤、杀菌、灌装得到产品。
可选地,所述食品是白酒、黄酒、酱油、啤酒、葡萄酒、食醋、发酵茶、传统发酵蔬菜、发酵饮料、酒精饮品、酸奶、干酪、果醋、酒酿、豆豉、乳腐、发酵米面食品等时,所述食品的制备方法中包括将耐酸乳杆菌CGMCC NO.21135直接作为附加物,或者替代原发酵体系中的微生物,或者部分替代原发酵体系中的微生物,添加到食品的发酵过程中。可选地,本领域技术人员可以结合常识对耐酸乳杆菌CGMCC NO.21135的添加方式或者发酵过程进行常规的调整。
本发明还提供了培养上述耐酸乳杆菌CGMCC NO.21135的方法,将所述耐酸乳杆菌于10-50℃,pH为2.5-7的液体培养基中厌氧培养若干天。
[有益效果]
本发明的Lactobacillus acetotolerans JZ 16耐受性好,能适应多种食品生产环境,在10-50℃温度范围内均可正常生长,具有良好耐高温能力;在pH值为2.5-7环境中能正常生长,具有良好的耐酸能力;可在乙醇浓度为18%(v/v)的环境中正常生长,具有很好的乙醇耐受性;在发酵过程中4-乙烯基愈创木酚产量可达3254.82μg/L,并具有很强的产风味能力;
本发明的Lactobacillus acetotolerans JZ 16具有利用多种碳源利用相关功能基因,例如编码寡聚1,6-葡萄糖苷酶、果聚糖酶等糖酶的基因,可利用多种碳源,能够更好地提高底物利用率;
本发明的Lactobacillus acetotolerans JZ 16基因组小于NCBI数据库中现有的Lactobacillus acetotolerans基因组,不具有生物胺合成的代谢通路和生物胺合成相关酶的编码基因,也不具有硝酸盐还原酶的编码基因,具有不产生物胺和亚硝酸盐的特征,安全性高,使用该乳杆菌用于白酒生产,可形成风味物质,并能保证所生产白酒的品质和安全。
生物材料保藏
耐酸乳杆菌,分类名称为耐酸乳杆菌Lactobacillus acetotolerans,保藏日期为2020年12月9日,保藏编号为CGMCC NO.21135,保藏单位为中国普通微生物菌种保藏管理中心,地址是北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
附图说明
图1:Lactobacillus acetotolerans JZ 16乳杆菌显微镜检图;
图2:Lactobacillus acetotolerans JZ 16乳杆菌在MRS培养基上的菌落形态;
图3:Lactobacillus acetotolerans JZ 16乳杆菌扩增凝胶电泳图;
图4:Lactobacillus acetotolerans JZ 16系统发育进化树;
图5:Lactobacillus acetotolerans JZ 16在18%乙醇浓度下(v/v)生长情况;
图6:Lactobacillus acetotolerans JZ 16全基因组图谱,其中,a:Chr1,DNA;b:Plas1,质粒,a,b的外圈是基因组序列位置坐标,由外到里,分别是编码基因、基因功能注释结果(包含COG(KOG),KEGG,GO数据库的注释结果信息)、ncRNA、基因组GC含量等。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明。
实施例1:高产风味Lactobacillus acetotolerans的筛选及鉴定
2019年,在江苏省无锡市从中国芝麻香型白酒酒醅中筛选到一株具有潜在的高产风味特性的菌株。具体筛选方法:
培养基的配制:胰蛋白胨15.0g/L、牛肉浸膏10.0g/L、酵母提取物6.0g/L、葡萄糖25.0g/L、无水山梨醇油酸酯1.5mL/L、K2HPO4 5.0g/L、三水合乙酸钠10.0g/L、柠檬酸三铵5.0g/L、MgSO4·7H2O 0.5g/L、MnSO4·4H2O 0.1g/L、琼脂25.0g/L调节pH为6.8±0.3,添加1000mL蒸馏水配制成主溶液,121℃灭菌15min;溴甲酚绿0.02g/mL、D-天冬氨酸二甲酯盐酸盐0.02g/mL、甲瓦龙酸内脂0.015g/mL、维生素B11 0.015g/mL和制霉素0.06g/mL,添加二甲基亚砜配制成副溶液,使用0.2μm无菌滤膜过滤灭菌;使用时,主溶液冷却至60℃,添加5mL副溶液混匀使用。
样品前处理:称取10g芝麻香型白酒酒醅于已灭菌装有100mL无菌PBS缓冲液(pH7.4,含0.5g/L D-天冬氨酸二甲酯盐酸盐)及3g玻璃珠的250mL三角瓶中,然后置于37℃、200r/min摇床振荡混匀30min,静置5min获得菌悬液。
微生物培养:取10-3、10-4和10-5 3个样品菌悬液稀释度涂布于上述MRS培养基平板中于37℃厌氧箱中培养84h,挑选挑取平板上周围培养基由蓝色变为黄色的单菌落并编号,使用特异性引物进行菌落PCR。
所述特异性引物对的核苷酸序列为LaF1:5′-AACATCCCCAGAGCGTCAAG-3′(SEQ IDNO.1);LaR1:5′-GGCACTACCCCAAGCATCTT-3′(SEQ ID NO.2)。PCR反应体系为25μL:PrimeSTAR12.5μL,上下游引物各0.5μL,菌液1mL。PCR的反应程序:预变性94℃4min,变性98℃10s,退火55℃1min,延伸72℃2min,共30个循环,72℃10min。琼脂糖凝胶电泳:2%的琼脂糖凝胶,2μL染液,4μL PCR产物。通过凝胶成像仪观察引物的特异性,选取在151bp附近具有明显的目的条带的菌株。进一步地,对菌株的产风味情况进行初筛。
获得一株菌株,其在MRS培养基中可保持良好的生长活性,厌氧条件下生长较快。该菌为革兰氏阳性菌,显微镜检形态为细长杆状(图1),在MRS固态培养基上所形成表面光滑的白色圆形小菌落(图2)。对该潜在高产风味菌株用1492R(5’-GGTTACCTTGTTACGACTT-3’)(SEQ ID NO.3)、27F(5’-AGAGTTTGATCCTGGCTCAG-3’)(SEQ ID NO.4)细菌通用引物对菌液进行PCR,得到片段约1500bp,电泳结果如图3所示。随后进行测序比对,与Lactobacillusacetotolerans LA749相似度为99.93%,确定筛选所得菌株在分类学上属于Lactobacillus acetotolerans(图4)。将其命名为Lactobacillus acetotolerans JZ 16,于2020年12月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.21135。
实施例2:菌株耐高温能力
将Lactobacillus acetotolerans JZ 16菌株,接种于MRS液体培养基中,37℃静置培养48h。按1%接种量添加至温度耐受性培养基。
MRS液体培养基的配方:
每1L培养基,含有葡萄糖20g,蛋白胨10g,酵母粉5g,牛肉膏10g,半胱氨酸盐酸盐0.02g,磷酸氢二钾2g,柠檬酸氢二铵2g,乙酸钠5g,硫酸锰0.25g,硫酸镁0.58g,吐温801mL,其余为水;
温度耐受性测试:将接种后的MRS培养基分别置于10,20,25,37,40和50℃培养箱中厌氧培养7d,结果显示本发明Lactobacillus acetotolerans JZ 16菌株在10-50℃温度范围内均可正常生长,具有良好耐高温能力。
实施例3:菌株耐酸能力
将Lactobacillus acetotolerans JZ 16菌株,接种于MRS液体培养基中,37℃静置培养48h。按1%接种量添加至酸度耐受性培养基。
酸度耐受性培养基:MRS培养基,用0.1M HCl调节培养基pH,使其成pH分别为2.5、3、4、5、6、7,置于37℃培养箱中厌氧培养7d,结果显示本发明Lactobacillusacetotolerans JZ 16菌株可在pH值为2.5-7环境中正常生长,具有良好的耐酸能力。
实施例4:菌株耐乙醇特征
将Lactobacillus acetotolerans JZ 16菌株,接种于MRS液体培养基中,37℃静置培养48h。按1%接种量添加至乙醇耐受度培养基。
乙醇耐受性培养基:MRS培养基,加入乙醇,使其成乙醇浓度(v/v)分别为0、6%、12%、18%,于37℃进行静置培养7d。结果显示本发明Lactobacillus acetotolerans JZ16菌株能够在乙醇浓度(v/v)6%-18%范围内正常生长,具有良好的乙醇耐受能力(图5)。
实施例5:菌株安全特征
采用SDS或STE的方法对Lactobacillus acetotolerans JZ 16的基因组DNA进行提取,之后利用琼脂糖凝胶电泳检测DNA的纯度和完整性,利用Qubit进行定量。采用SMRTbell TM Template kit(version 1.0)试剂盒构建10K SMRT Bell文库,将经电泳检测合格的DNA样品用Covaris g-TUBE打断成构建文库所需大小的目的片段,经DNA损伤修复及末端修复,使用DNA黏合酶将发卡型接头连接在DNA片段两端,并使用AMpure PB磁珠对DNA片段进行纯化,使用BluePipin片段筛选特定大小的片段,使用AMpure PB磁珠对SMRT Bell文库进行浓度筛选,随后修复DNA损伤,再次使用AMpure PB磁珠对SMRT Bell文库纯化,将构建好的文库经Qubit浓度定量,并利用Agilent 2100检测插入片段大小,最后用PacBio平台进行测序。
针对Lactobacillus acetotolerans JZ 16(即CGMCC NO.21135)的组装基因组序列,结合编码基因的预测结果,使用Circos软件对Lactobacillus acetotolerans JZ 16基因组进行展示,全基因组图谱如图6所示。
表1耐酸乳杆菌基本基因组比较信息
(L.acetotolerans NBRC 13120:Japanese sake;L.acetotoleransLA749:fermentedvinegar broth;
L.acetotoleransCN247:spoiled beer;L.acetotoleransLJ49:Chinesevinegar)
Lactobacillus acetotolerans JZ 16(即CGMCC NO.21135)基因组组装产生181个contigs,N50值为41242,组装基因组的大小为1.50Mb,GC%为36.58%。通过比较发现Lactobacillus acetotolerans JZ 16基因组大小小于NCBI数据库中现有的Lactobacillus acetotolerans基因组,共预测1700个基因,平均长度为830bp,占全基因组的88.44%。五组数据的GC%相近,而Lactobacillus acetotolerans JZ 16基因数目较多(表1)。在KEGG注释结果中查询生物胺和亚硝酸盐合成相关途径及所需酶(表2),结果显示Lactobacillus acetotolerans JZ 16并不具有生物胺合成的代谢通路,无生物胺合成相关酶的编码基因。Lactobacillus acetotolerans JZ 16不具有硝酸盐还原酶EC1.7.1.1,EC 1.7.1.2,EC 1.7.1.3,EC 1.7.5.1,EC 1.7.7.2,EC 1.9.6.1的编码基因。因此,Lactobacillus acetotolerans JZ 16具有不产生物胺和亚硝酸盐的特征,可以安全使用。
表2生物胺、亚硝酸盐合成相关途径及酶
实施例6:利用菌株代谢产风味
种子培养基:取20mL试管,装入15mL MRS培养基,并接种筛选获得的菌株Lactobacillus acetotolerans JZ 16,自然pH,37℃,静置厌氧培养2d。
发酵培养基:将培养好的种子培养液接种在装有15mL高粱汁培养基的20mL试管中,自然pH,接种量为10%,30℃,静置发酵7d。
运用顶空固相微萃取技术(HS-SPME)和气相色谱-质谱(GC-MS)方法对挥发性产物进行分析,取5mL样品,放入装有3g NaCl的顶空进样瓶中,加入10μL浓度为42.60mg/L的4-甲基-2-戊醇为内标。将顶空瓶于50℃恒温萃取45min,萃取完后进行GC-MS分析。
Lactobacillus acetotolerans JZ 16在发酵7d后,代谢产生的风味物质种类及含量如表3所示。
表3 Lactobacillus acetotolerans JZ 16产风味物质
各主要风味物质中,酚醚类物质为最多,能够生成4-乙烯基愈创木酚3254.82μg/L,该物质呈发酵香气,略带甜味,微带酚的气息,是决定酒类、酱油、茶叶、咖啡、干酪等食品品位及质量的一个主要香味成分。本菌株生成的酯类也为最主要的风味物质,其中,己酸乙酯含量最高为8.10μg/L,苯乙酸乙酯4.51μg/L、丁二酸二乙酯4.61μg/L等。本菌株产酸能力也较强,可生成己酸295.07μg/L,辛酸222.63μg/L,乙酸204.47μg/L,庚酸72.54μg/L,壬酸68.43μg/L,这些酸既是呈香物质又是呈味物质,与其他呈香呈味物质共同组成白酒所特有的芳香。本菌株可产生己醇、辛醇、戊醇、庚醇、苯乙醇等,其中苯乙醇29μg/L,己醇20.8μg/L,苯乙醇呈甜香气,似玫瑰气味,气味持久,微甜,带涩,为白酒中重要芳香物质。本专利中Lactobacillus acetotolerans JZ 16菌株具有高产风味的能力。
实施例7:菌株多碳源利用能力
全基因组注释结果表明Lactobacillus acetotolerans JZ 16具有利用多种碳源利用相关功能基因。具有编码寡聚1,6-葡萄糖苷酶,果聚糖酶等糖酶,可利用多种碳源,可以代谢环糊精、麦芽糖、糊精、纤维二糖、异麦芽糖、蔗糖、甘露糖、乳糖等多种碳源。将上述底物作为单一碳源培养,Lactobacillus acetotolerans JZ 16可正常生长。表面该菌株可利用多种碳源,能够更好的提高底物利用率。
实施例8:含Lactobacillus acetotolerans JZ 16的组合物的制备
利用菌株Lactobacillus acetotolerans JZ 16制备组合物—液体菌剂。
液体菌剂的制作方法是将Lactobacillus acetotolerans JZ 16接种于液体培养基中,于37℃静置培养48h,得到液体种子液。
液体培养基的配方是下列配方之一:
配方A:每1L培养基,含有葡萄糖20g,蛋白胨10g,酵母粉5g,牛肉膏10g,半胱氨酸盐酸盐0.02g,磷酸氢二钾2g,柠檬酸氢二铵2g,乙酸钠5g,硫酸锰0.25g,硫酸镁0.58g,吐温80 1mL,其余为水;
配方B:以白酒酿造所用的原料如高粱、大麦、小麦、豌豆、麸皮等为培养基成分,将原料粉碎后,按原料与水的比例为1:1-1:4w/v的比例混合,蒸煮1-5h,冷却后加入糖化酶10-50单位/g原料,于40-80℃保持2-10h,过滤,离心所得滤液调节糖度为10-15°Bx、pH为4-6。
实施例9:含Lactobacillus acetotolerans JZ 16的组合物的制备
利用菌株Lactobacillus acetotolerans JZ 16制备组合物—复合菌剂。
将Lactobacillus acetotolerans JZ 16接种于液体培养基中,于37℃静置培养48h,得到液体种子液。液体培养基的配方:每1L培养基,含有葡萄糖20g,蛋白胨10g,酵母粉5g,牛肉膏10g,半胱氨酸盐酸盐0.02g,磷酸氢二钾2g,柠檬酸氢二铵2g,乙酸钠5g,硫酸锰0.25g,硫酸镁0.58g,吐温80 1mL,其余为水。
采用MRS培养基培养益生菌,得到益生菌种子液。所述益生菌包含罗伊氏乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌、约氏乳杆菌、格氏乳杆菌、干酪乳杆菌、副干酪乳杆菌、植物乳杆菌、发酵乳杆菌、卷曲乳杆菌、唾液乳杆菌、清酒乳杆菌、德氏乳杆菌、瑞士乳杆菌、假小链双歧杆菌、长双歧杆菌长亚种、长双歧杆菌婴儿亚种、青春双歧杆菌、短双歧杆菌、两歧双歧杆菌、动物双歧杆菌动物亚种以及动物双歧杆菌乳亚种中的一种或多种。
将Lactobacillus acetotolerans JZ 16液体种子液与益生菌种子液混合,得到液态复合菌剂。
进一步地,可以将液态的复合菌剂离心,重悬,得到浓缩的液态的复合菌剂。进一步地,可以在液态的复合菌剂中加入保护剂。
进一步地,经常规喷雾干燥等方法对液态的复合菌剂进行干燥得到固态的复合菌剂。在干燥过程中,可以加入保护剂。
实施例10:Lactobacillus acetotolerans JZ 16在白酒生产中的应用
在白酒发酵过程中的醩醅中添加实施例8制备的液体菌剂,并对发酵结束时酒醅进行检测。结果表明添加液体菌剂的风味物质种类和总量明显增加,酯类和酸类物质含量增加,4-乙烯基愈创木酚含量更高,说明添加Lactobacillus acetotolerans JZ 16菌株可明显改善白酒风味。
实施例11:Lactobacillus acetotolerans JZ 16在发酵蔬菜生产中的应用
在传统发酵蔬菜的高酸度环境中耐酸乳杆菌可以正常生长,与多种挥发性风味物质相关,添加菌剂的风味物质种类和总量明显增加,改善了发酵蔬菜的风味。耐酸乳杆菌对碳源多利用的多样性使得蔬菜的利用度提高,增加了蔬菜的营养价值。可通过产酸抑制有害微生物生长。同时Lactobacillus acetotolerans JZ 16不具有产生生物胺和亚硝酸盐的途径,可避免由生物胺和亚硝酸盐带来的安全问题,保证了发酵蔬菜的品质。
实施例12:Lactobacillus acetotolerans JZ 16在果醋生产中的应用
以制备苹果醋为例:
(1)选果:挑选苹果;
(2)清洗:清洗苹果,沥干水分;
(3)榨汁:对苹果进行榨汁;果浆经离心分离去除果渣,再经纱布过滤制得苹果汁;
(4)调配:调整山苹果汁糖度12%、pH 3.5~4.5;
(5)预杀菌:将苹果汁置于80℃水浴中保温杀菌49min;
(6)酒精发酵:活化果酒酵母,将种子液按4%接种量接入调配好的山楂汁,搅拌并充分混合均匀,温度28℃下有氧发酵8h后无氧发酵48h;
(7)醋酸发酵:活化醋酸菌,将种子液按8%接种量接入酒精发酵液,搅拌并充分混合均匀;温度28℃下有氧发酵12h;
(8)乳酸发酵:活化Lactobacillus acetotolerans JZ 16,将种子液按4%接种量接入调配好的上一步制备的醋酸发酵液(糖度12%、pH 5),搅拌并充分混合均匀;38℃下无氧发酵12h;
(9)过滤、杀菌、灌装:将发酵完成的发酵液经过滤、杀菌、无菌灌装,得到苹果醋。
本发明的保护范围并不仅局限于上述实施例,凡是在本发明构思的精神和原则之内,本领域的专业人员能够做出的任何修改、等同替换和改进等均应包含在本发明的保护范围之内。
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<110> 江南大学
宿迁市江南大学产业技术研究院
<120> 高产风味耐酸乳杆菌在食品生产中的应用
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Claims (14)
1.耐酸乳杆菌,其特征在于,所述耐酸乳杆菌于2020年12月9日保藏于中国普通微生物菌种保藏管理中心,保藏编号为:CGMCC NO.21135。
2.含有权利要求1所述耐酸乳杆菌的组合物。
3.根据权利要求2所述的组合物,其特征在于,所述组合物是酒曲或酒醅。
4.根据权利要求2所述的组合物,其特征在于,所述组合物是固态菌剂或液态菌剂。
5.根据权利要求2所述的组合物,其特征在于,所述组合物中还含有任何能够应用于食品或者食品制备的任意种属的菌株。
6.权利要求1所述耐酸乳杆菌在食品生产中的应用。
7.根据权利要求6所述的应用,其特征在于,所述食品是白酒、黄酒、酱油、啤酒、葡萄酒、食醋、发酵茶、传统发酵蔬菜、酸奶、干酪、酒酿、豆豉、乳腐、发酵米面食品中的任意一种或多种。
8.根据权利要求6所述的应用,其特征在于,所述食品是果醋。
9.根据权利要求6所述的应用,其特征在于,所述食品是发酵饮料。
10.根据权利要求6所述的应用,其特征在于,所述食品是酒精饮品。
11.根据权利要求6或7所述的应用,其特征在于,所述食品是白酒;所述白酒的酿造方法中,包括将上述耐酸乳杆菌CGMCC NO.21135或含有耐酸乳杆菌CGMCC NO.21135的组合物以液体或固体培养物的形式接种于白酒大曲、堆积醅或窖池发酵的酒醅中。
12.根据权利要求7所述的应用,其特征在于,所述食品的制备方法中包括将耐酸乳杆菌CGMCC NO.21135直接作为附加物,或者替代原发酵体系中的微生物,或者部分替代原发酵体系中的微生物,添加到食品的发酵过程中。
13.一种微生物复合菌剂,其特征在于,所述复合菌剂中,含有权利要求1的耐酸乳杆菌,以及罗伊氏乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌、约氏乳杆菌、格氏乳杆菌、干酪乳杆菌、副干酪乳杆菌、植物乳杆菌、发酵乳杆菌、卷曲乳杆菌、唾液乳杆菌、清酒乳杆菌、德氏乳杆菌、瑞士乳杆菌、假小链双歧杆菌、长双歧杆菌长亚种、长双歧杆菌婴儿亚种、青春双歧杆菌、短双歧杆菌、两歧双歧杆菌、动物双歧杆菌动物亚种以及动物双歧杆菌乳亚种中的一种或多种。
14.权利要求1所述的耐酸乳杆菌在改善食品生产中的应用,其特征在于,所述改善食品至少包括如下任意一种改善:
(1)提升食品风味品质;
(2)提升食品安全品质;
(3)提高底物利用率;
(4)提高食品安全性;
(5)降低食品中生物胺含量;
(6)降低食品中亚硝酸盐含量。
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