CN106867935B - 白萝卜鲜酿酵素及其制备和发酵菌株 - Google Patents
白萝卜鲜酿酵素及其制备和发酵菌株 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
本发明公开了一种白萝卜鲜酿酵素及其制备和发酵菌株,本发明从自然发酵的白萝卜酵素出发,以发酵效率与产品风味为主要指标,筛选白萝卜酵素专用发酵菌株——肠膜明串珠菌(Leuconostoc mesenteroides)YZ18,由中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为CGMCC No.13332,保藏时间为2016年11月22日。利用该菌种接种发酵生产白萝卜鲜酿酵素,有益菌能够快速增殖并保持优势发酵,有效抑制杂菌污染与有害物质的产生,所得的酵素口感酸甜适宜,气味酸香,风味得到极大改善。
Description
技术领域
本发明属于食品加工技术领域,涉及以白萝卜为原料,生产乳酸型鲜酿酵素的制备方法,尤其涉及一株能够通过发酵改善产品风味的肠膜明串珠菌菌株。
背景技术
白萝卜又名莱菔、罗服,为十字花科萝卜属植物的新鲜根茎,是我国主栽的蔬菜品种之一,含有碳水化合物、蛋白质及氨基酸、膳食纤维等营养成分,钙、钠、磷、钾等矿物质及维A、维C的含量也很丰富。中医认为其味甘平辛,归肺脾经,具有下气消食、除痰润肺、和中止咳、利尿通便等功效,本草纲目称之为“蔬中最有利者”。现代医学证明,白萝卜所含酶系对致癌物质亚硝胺具有分解作用;其干扰素诱导物质对癌细胞及肿瘤具有抑制作用;所含菜菔硫烷对肺癌、结肠癌、食道癌等有很好的防治作用,在癌症的起始、发展和增生阶段均发挥作用,对增强机体免疫力、防癌、抗癌有重要意义。白萝卜是家庭常用的健康蔬菜与药膳原料,主要以煮菜粥、烹小炒、煲汤、制作泡菜为主,因其特殊的刺激气味而较少生食。
酵素最原始的定义为酶,是生物本身自然生成的具有生物催化功能的一类大分子物质。近期广受关注的植物酵素通常是指果蔬为原料,经自然发酵或者接种发酵后获得的一种包含微生物菌体及生物酶在内的复合物质体系,既保留了原料的部分营养物质,微生物发酵还带来丰富的次生代谢产物、维生素、矿物质等益生成分,具有特殊的发酵香与令人愉悦的果香,在健康、长寿、美容、养颜以及减肥等方面表现出来的独特功效已经逐渐被人们所认知。酵素在日本、中国台湾等国家、地区比较流行,目前中国大陆地区还较为少见。现有酵素产品质量良莠不齐、价格昂贵,且经历了加热、浓缩等处理过程,活性物质部分损失。而家庭自制酵素通常是将果蔬切块再与蔗糖、水按一定比例混合,密封放置几周甚至几个月,依靠果蔬表面携带的微生物进行发酵,最终得到黄色或褐色的粘稠液体。其发酵条件取决于制作人的微生物意识,稍有不慎有害微生物即快速繁殖,产生生物毒素、亚硝酸盐、甲醇等有害代谢产物;另外民间为追求口感、避免杂菌污染,一般采用层层码料方式,加入大量的蔗糖(30%以上),成品中仍然含有较高的糖度,无形中增加了人体的健康负担。
乳酸菌是一类可发酵糖类产生大量乳酸的革兰氏阳性菌的总称,具有提高营养物质利用率、预防和治疗多种疾病、调节机体免疫机能等功效,是食品、医药领域应用最广泛的益生菌之一。其作用机制一是靠菌体本身及其活性代谢物对有害物质具有直接作用,二是乳酸菌通过调节肠道菌群及提高免疫活性等方式起间接作用。以白萝卜为原料研发乳酸菌发酵型酵素,微生物的温和转化有利于保留原料中维生素、矿物质、硫苷等营养物质,菌体产生的酶类将原料中大分子物质降解为更易吸收的小分子物质,大量增殖的菌体及其产生的活性短链脂肪酸、抗菌物质、多糖、功能性酶类、维生素、挥发性成分等益生代谢产物及风味物质也保留在最终产品中,从而极大的提高产品营养价值,增进产品的益生功效,并有效的改善生萝卜的辛辣口感与风味,是集原料、代谢产物、菌株功效于一身的优秀健康食品,具有极大的开发前景。
目前还未见白萝卜酵素的相关报道,其发酵菌株的选择、基质复配、发酵工艺都不明确,而发明人采用现有商品菌株发酵所得产品的口感与风味皆不尽人意。
发明内容
本发明针对上述问题,从自然发酵的白萝卜酵素出发,以发酵效率与产品风味为主要指标,筛选白萝卜酵素专用发酵菌株,并对其进行生理生化与基因鉴定。
本发明的另一目的是明确接种比例、发酵温度与时间等关键工艺参数,建立与该新菌株相配套的白萝卜鲜酿酵素的制备方法。
本发明所涉及的物质含量及比例,无如特殊说明,均以质量计。
为了达到上述的目的,本发明采用了以下的技术方案:
用于乳酸型白萝卜鲜酿酵素的发酵菌株筛选、生物学特性研究及鉴定:
1、菌株筛选
筛选源制备:挑选无腐烂、无损伤、无虫害、无空心的新鲜大白萝卜,清洗、晾干、去皮、刨丝,添加15%蔗糖,混合均匀后28℃培养72h。
分离纯化:无菌条件下吸取上述自然发酵液25mL与225mL的无菌生理盐水混合均匀,然后依次进行10倍梯度稀释,分别取10-6、10-7、10-8稀释液涂布于(MRS+CaCO3)培养基上,28℃厌氧培养48h,取菌落形成单位在30‐100之间的培养皿,按50%比例挑取具有溶钙圈及典型乳酸菌特征的单菌落进行平板划线并保存备用。
筛选:对所得纯菌株进行菌落形态观察、革兰氏染色、菌体形态镜检,获得具有典型乳酸菌形态、革兰氏染色阳性且发酵性能良好、传代稳定的菌株,按2%接种量分别接种到萝卜汁培养基中,28℃厌氧发酵,每12h进行pH值测定,48h后进行感官评定,最终选择产酸性能优良、感官评分最高的YZ18为白萝卜鲜酿酵素生产菌种。
培养基:MRS固体培养基补水加热,添加0.5%的CaCO3混匀融化,121℃灭菌15min,放凉至55℃以下,轻轻摇晃使CaCO3在培养基中扩散均匀,倾倒培养皿备用。
萝卜汁培养基:新鲜白萝卜洗净去皮后刨丝,加入15%蔗糖混合均匀静置4h,以200目滤布挤压出汁,于70℃灭菌30min备用
2、YZ18菌株的生物学特征鉴定:
形态学特征:YZ18菌株在MRS固体培养基上生长状况良好,28℃厌氧培养48h后形成圆形菌落,直径在0.8-1.2mm之间,并随培养时间的延长而增大。菌落乳白色,半透明状,表面湿润、边缘整齐、中央凸起。镜检为革兰氏阳性球菌,成双球状、四球状或成短链状排列。
生理生化性质:过氧化氢酶阴性,发酵蔗糖、木糖、麦芽糖、甘露糖、海藻糖等产酸,不利用乳糖与半乳糖等,将反应结果输入数据库,经自动判读与明串珠菌相似性达99%。
基因序列分析:YZ18的16S rDNA序列与肠膜明串珠菌属(Leuconostocmesenteroides subsp.)的相似度达97%。
鉴定结果:将菌株YZ18的形态学特征对照伯杰氏细菌鉴定学手册,结合该菌株的理化特征及其16S rDNA序列分析结果,将该菌株鉴定为肠膜明串珠菌属(Leuconostocmesenteroides subsp.),将其命名为L.mesenteroides.YZ18。
一种肠膜明串珠菌(Leuconostoc mesenteroides)YZ18,由中国微生物菌种保藏管理委员会普通微生物中心(中国北京市朝阳区北辰西路1号院3号)保藏,保藏号为CGMCCNo.13332,保藏时间为2016年11月22日。
如上所述的肠膜明串珠菌(Leuconostoc mesenteroides)YZ18在制备白萝卜鲜酿酵素的应用。
一种乳酸型白萝卜鲜酿酵素,该酵素采用如上所述的肠膜明串珠菌(Leuconostocmesenteroides)YZ18为发酵菌种,经容器处理、基质配制、乳酸发酵、过滤冷藏等工艺获得。
应用如上所述的肠膜明串珠菌(Leuconostoc mesenteroides)YZ18制备乳酸型白萝卜鲜酿酵素的方法,其特征在于包括以下步骤:
(1)容器处理;
(2)基质配制:选取无腐烂、无损伤、无虫害、无空心的新鲜大白萝卜,清洗、晾干、去皮、刨丝,添加5%-30%蔗糖混合均匀,15℃-28℃静置2-6h后取出,以200目滤布挤压取汁;
(3)乳酸发酵:将上述挤出汁置入发酵容器中,装液量为65%-85%,添加0.5%-5%如权利要求1所述的肠膜明串珠菌(Leuconostoc mesenteroides)YZ18,0%-5%的植物乳杆菌,22℃-30℃发酵38-96h;
(4)过滤冷藏:无菌条件下将上述发酵液过800目滤布,所得滤液装入无菌容器,装液量90%,密封好置入0.5-15℃避光冷藏;
上述步骤中,物质含量比例为质量比例。
作为优选,所述容器处理步骤包括:生产过程中所涉及的器皿洗净、晾干,金属用具、玻璃容器高压灭菌,塑料容器及滤布浸泡75%酒精溶液后晾干,紫外照射30min。
本发明由于采用了以上的技术方案,具有下述有益效果:
(1)获得专用发酵菌种:从自然发酵的白萝卜酵素分离得到肠膜明串珠菌L.mesenteroides YZ18,该菌株可以单独发酵,也可以与其他乳酸菌混合发酵,改变了白萝卜酵素饮料无专用菌种的现状。
(2)发酵过程快速可控:利用本发明技术接种发酵生产白萝卜鲜酿酵素,有益菌能够快速增殖并保持优势发酵,有效抑制杂菌污染与有害物质的产生,所得的酵素口感酸甜适宜,气味酸香。
(3)益生作用增强:经过接种发酵,原料pH为由6下降为3.6左右,总酸由0.45g/kg增加至3.63g/kg。乳酸菌活菌数由104CFU/mL达到108CFU/mL,其抑制腐败菌及调节肠道菌群的作用增强。
(4)风味改善:经过该菌株的发酵,产品中的挥发性酯类发生明显变化,由原来的异硫氰酸酯类为主(刺鼻冲味的主要来源),转变为具有水果香型的有机酸酯类为主,如乙酸异戊酯、己酸乙酯、辛酸乙酯等,风味得到极大改善。
具体实施方式
下面实施例是对本发明的进一步说明,并非对本发明作任何形式上的限制,凡是依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
所涉及的物质含量及比例,无如特殊说明,均以质量计。
所采用原料对品种无特殊要求,无腐烂、无损伤、无虫害、无空心的新鲜白萝卜即可,一天内完成原料处理与接种;所采用蔗糖等辅料均为食品级。
实施例1:
应用肠膜明串珠菌(Leuconostoc mesenteroides)YZ18制备乳酸型白萝卜鲜酿酵素的方法,包括以下步骤:
(1)容器处理:实验过程中所用到器皿清洗干净,金属用具、玻璃容器进行高压灭菌,塑料容器及滤布浸泡75%酒精溶液后晾干,紫外照射杀菌30min。
(2)基质配制:挑选无腐烂、无损伤、无虫害、无空心的新鲜白萝卜,清洗、晾干、去皮、刨丝,添加5%蔗糖混合均匀,15℃静置6h后取出,通过200目滤布挤压取汁。
(3)乳酸发酵:将上述挤出汁置入发酵容器中,装液量为65%,添加0.5%L.mesenteroides YZ18,22℃发酵96h。
(4)过滤冷藏:无菌条件下将上述发酵液过800目滤布,所得滤液装入无菌容器,装液量90%,密封好置入15℃避光冷藏,一周内饮用完毕。
实施例2:
本例,按步骤(1)进行容器清洗与灭菌,步骤(2)中按相应的要求挑选新鲜白萝卜,按程序进行清洗、晾干、去皮、刨丝,添加30%蔗糖,28℃静置2h,经滤布挤压取汁;步骤(3)发酵液装液量85%,添加5%L.mesenteroides YZ18,5%植物乳杆菌,30℃发酵38h;步骤(4)中将步骤(3)过滤所得发酵液,装入无菌容器,密封好置入15℃避光冷藏备饮。其他与实施例1相同。
实施例3:
本例,按步骤(1)进行容器清洗与灭菌,步骤(2)中按要求挑选新鲜原料进行处理,添加10%蔗糖,20℃静置5h,经滤布挤压取汁;步骤(3)发酵液装液量70%,添加1%L.mesenteroides YZ18,1%植物乳杆菌,25℃发酵50h;步骤(4)中将所得发酵液过滤、分装、密封,置于5℃避光冷藏备饮。其他与实施例1相同。
实施例4:
本例,按步骤(1)进行容器清洗与灭菌,步骤(2)中按要求挑选新鲜原料进行处理,添加15%蔗糖,25℃静置4h,经滤布挤压取汁;步骤(3)发酵液装液量75%,添加2%L.mesenteroides YZ18,2%植物乳杆菌,25℃发酵48h;步骤(4)中将所得发酵液过滤、分装、密封,置于10℃避光冷藏备饮。其他与实施例1相同。
实施例5:
本例,按步骤(1)进行容器清洗与灭菌,步骤(2)中按要求挑选新鲜原料进行处理,添加20%蔗糖,27℃静置3h,经滤布挤压取汁;步骤(3)发酵液装液量80%,添加2.5%L.mesenteroides YZ18,2%植物乳杆菌,25℃发酵46h;步骤(4)中将所得发酵液过滤、分装、密封,置于5℃避光冷藏备饮。其他与实施例1相同。
经对上述实施例的白萝卜挤出液和白萝卜鲜酿酵素检测,经过接种发酵,原料pH由6下降为3.6左右,总酸由0.45g/kg增加至3.6g/kg左右。乳酸菌活菌数由104CFU/mL达到108CFU/mL,其抑制腐败菌及调节肠道菌群的作用增强。经过该菌株的发酵,产品中的挥发性酯类发生明显变化,由原来具有刺激气味的异硫氰酸戊酯、异硫氰酸己酯等异硫氰酸酯类为主,转变为具有水果香型的有机酸酯类为主,如乙酸异戊酯、己酸乙酯、辛酸乙酯等,风味得到极大改善。利用本发明技术接种发酵生产白萝卜鲜酿酵素,有益菌能够快速增殖并保持优势发酵,有效抑制杂菌污染与有害物质的产生,所得的酵素口感酸甜适宜,气味酸香。本发明从自然发酵的白萝卜酵素分离得到肠膜明串珠菌L.mesenteroides YZ18,该菌株可以单独发酵,也可以与其他乳酸菌混合发酵,改变了白萝卜酵素饮料无专用菌种的现状。
上述实施例所用植物乳杆菌为本实验室自行筛选于竹笋表面的L.PlantarumSZS001(由中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为CGMCCNo.11936,保藏时间为2015年12月25日),也可以是其他植物乳杆菌菌株。
其他需要强调的是:以上仅是本发明的部分实施例而已,并非对本发明作任何形式上的限制,凡是依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (5)
1.一种肠膜明串珠菌(Leuconostoc mesenteroides)YZ18,由中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为CGMCC No.13332,保藏时间为2016年11月22日。
2.如权利要求1所述的肠膜明串珠菌(Leuconostoc mesenteroides)YZ18在制备白萝卜鲜酿酵素中的应用。
3.一种乳酸型白萝卜鲜酿酵素,该酵素采用如权利要求1所述的肠膜明串珠菌(Leuconostoc mesenteroides)YZ18为发酵菌种。
4.应用如权利要求1所述的肠膜明串珠菌(Leuconostoc mesenteroides)YZ18制备乳酸型白萝卜鲜酿酵素的方法,其特征在于包括以下步骤:
(1)容器处理;
(2)基质配制:选取无腐烂、无损伤、无虫害、无空心的新鲜大白萝卜,清洗、晾干、去皮、刨丝,添加5%-30%蔗糖混合均匀,15℃-28℃静置2-6h后取出,以200目滤布挤压取汁;
(3)乳酸发酵:将上述挤出汁置入发酵容器中,装液量为65%-85%,添加0.5%-5%如权利要求1所述的肠膜明串珠菌(Leuconostoc mesenteroides)YZ18,0%-5%的植物乳杆菌,22℃-30℃发酵38-96h;
(4)过滤冷藏:无菌条件下将上述发酵液过800目滤布,所得滤液装入无菌容器,装液量90%,密封好置入0.5-15℃避光冷藏;
上述步骤中,物质含量比例为质量比例。
5.根据权利要求4所述的方法,其特征在于,所述容器处理步骤包括:生产过程中所涉及的器皿洗净、晾干,金属用具、玻璃容器高压灭菌,塑料容器及滤布浸泡75%酒精溶液后晾干,紫外照射30min。
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