CN113801800A - 一株酿酒酵母及其应用 - Google Patents
一株酿酒酵母及其应用 Download PDFInfo
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- CN113801800A CN113801800A CN202111119074.3A CN202111119074A CN113801800A CN 113801800 A CN113801800 A CN 113801800A CN 202111119074 A CN202111119074 A CN 202111119074A CN 113801800 A CN113801800 A CN 113801800A
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Abstract
本发明提供了一株酿酒酵母,所述菌株为酿酒酵母SF1(Saccharomyces cerevisiae SF1),于2020年7月16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.20379。本发明的酿酒酵母SF1(Saccharomyces cerevisiae SF1)对豆腐加工的豆清水、豆渣样品有发酵作用,能以豆清水为原料发酵生产豆清发酵饮料,还能以豆渣为原料发酵生产发酵豆渣,为豆清发酵饮料、发酵豆渣的生产及开发提供了新的发酵菌源。
Description
技术领域
本发明属于微生物技术领域,具体涉及一株酿酒酵母及其应用。所述菌株为酿酒酵母SF1(Saccharomyces cerevisiae SF1),该菌株能应用于制备豆清发酵饮料、发酵豆渣。
背景技术
大豆是我国第四大粮食作物,豆制品加工是大豆最为常见的食用方法。在豆制品加工过程中,会产生大量的副产物,我国每年因豆制品加工产生的豆清水约为2100-3500万吨,产生的豆渣约为200万吨。豆清水营养丰富,包括可溶性蛋白质、可溶性糖、维生素、脂类、微量元素、异黄酮及多糖等多种有效成分,但由于豆清水豆腥味重、口感差,且缺乏加工利用技术,而直接被企业以废水的方式排放。豆渣中含有50%-55%的膳食纤维和18%-23%的蛋白质,同时还富含人体多种维生素、矿物质、异黄酮等多种物质,但由于豆渣的口感差、豆腥味重、储存难、含有多种抗性因子,且蛋白分子量大不易消化,因此豆渣的利用率较低。
为解决上述问题,本申请人致力于筛选出能以豆清水或豆渣为原料制备豆清发酵饮料或发酵豆渣的菌株,为豆清水或豆渣的开发利用、豆清饮料及发酵豆渣的生产提供新的发酵菌源。
发明内容
本发明的目的在于提供一株酿酒酵母及其应用。所述菌株为酿酒酵母SF1(Saccharomyces cerevisiae SF1),于2020年7月16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,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCCNO.20379。酿酒酵母SF1(Saccharomyces cerevisiae SF1)能够以豆清水为主要原料生产豆清发酵饮料,还能够以豆渣为原料发酵生产发酵豆渣,为豆清水及豆渣的开发利用以及豆清发酵饮料及发酵豆渣的生产提供了新的发酵菌源。
本发明是通过以下技术方案解决上述技术问题的:
取南平农家传统家酿保存米曲饼10g,向其中加入250ml长汀传统酸浆豆腐生产工艺生产获得的豆清水,28℃驯化发酵,以7天为一个周期,连续更替数次至豆清水无豆腥味,并出现清香味,再将驯化的豆清发酵液接种至灭菌的豆渣中,固态发酵驯化3天,连续接种驯化至发酵物大分子蛋白显著下降,将经过驯化的发酵混合料稀释至10-4、10-5,各取0.1ml上述浓度梯度的稀释液涂布于酵母固体培养基的平板上,28℃倒置培养3天,挑选出菌落形态相似的疑似酵母菌的单菌落4株,在酵母固体平板上划线纯化培养3次,同时将各单菌落于豆清水培养基中28℃静置培养72h,获得豆清发酵培养液,测定分析其酒精含量,品尝其风味口感;将豆清发酵培养液分别接种于豆渣中发酵,最终挑选出一株豆清发酵风味独特,并能降解豆渣中大分子蛋白的菌种SF1,经通过对菌株进行形态学观察、18S rDNA基因序列测定及***发育树同源性分析,最终鉴定为酿酒酵母(Saccharomyces cerevisiae)。
进一步地,酿酒酵母SF1(Saccharomyces cerevisiae SF1)具有以下特征:
a. 菌落圆,在酵母培养基上呈乳白色、不透明;菌体椭圆形,宽2-4μm,长4-6μm;
b. 可利用葡萄糖、半乳糖、麦芽糖、蔗糖和果糖进行发酵产酸产气,不能利用乳糖、淀粉、木糖、***糖和海藻糖;
c. 可在pH值为2.0的条件下存活6h;在pH值为3.0的条件下,培养2h和6h, 落数可生长至原值的130.34%和225.64%。
上述酿酒酵母SF1(Saccharomyces cerevisiae SF1)可应用于制备豆清发酵饮料和发酵豆渣。
酿酒酵母SF1(Saccharomyces cerevisiae SF1)能以豆清水为原料,在6h短时间内即可发酵豆清水并去除豆腥味,同时提高豆清水中的蛋白含量至未发酵时的121.74%,生产出味道独特且口感适合的不含酒精的豆清发酵饮料,获得的豆清发酵饮料含有异黄酮(m/v)0.92%,氨基酸合计254.9 mg/100 ml,蛋白质0.3 %, 多糖3.01 mg/ml。
酿酒酵母SF1(Saccharomyces cerevisiae SF1)还可以以豆渣为原料进行发酵,将豆渣中分子量大于25kDa的大分子蛋白物质降解为小于25kDa的小分子蛋白物质,同时提高豆渣中的总蛋白质含量至未发酵时的114.05%,显著提高豆渣中的小分子肽蛋白质含量,发酵出的豆渣可用于制备发酵豆渣食品或发酵豆渣饲料,应用于豆渣的发酵处理及利用,并可作为饲料原料应用于养殖业。
本发明的有益效果在于:提供酿酒酵母SF1(Saccharomyces cerevisiae SF1),该菌株可利用豆清为原料在短时间内发酵生产出豆清发酵饮料,还能以豆渣为原料生产发酵豆渣,为豆清发酵饮料开发、发酵豆渣的生产及开发提供新的发酵菌源。
附图说明
图1为SF1菌的菌落形态图。
图2为SF1菌的扫描电镜图。
图3为目的片段扩增结果。
图4为SF1菌的***发育树。
图5为SF2菌的最适发酵pH。
图6为酿酒酵母SF1对豆渣和豆清水发酵的PAGE图。泳道M为蛋白Marker;泳道Z-CK为豆渣(未发酵);泳道Z1为豆渣(经SF1发酵3天);泳道Z2为豆渣(加入糖蜜后发酵产物);泳道S-CK为豆清水(未发酵);泳道S1为豆清发酵饮料(未过滤);泳道S2为豆清发酵饮料(过滤处理)。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是下述的实例仅仅是本发明其中的例子而已,并不代表本发明所限定的权利保护范围,本发明的权利保护范围以权利要求书为准。
本发明酿酒酵母SF1(Saccharomyces cerevisiae SF1)是从南平农家传统家酿保存米曲饼中分离获得的。
实施例1 菌的驯化筛选分离
取南平农家传统家酿保存米曲饼10g,向其中加入250ml长汀传统酸浆豆腐生产工艺生产获得的豆清水,在28℃下驯化发酵,以7天为一个周期,连续更替3次至豆清水无豆腥味,再将驯化的豆清发酵液根据10%(v/w)接种量接种至灭菌的豆渣中,固态28℃发酵驯化3天,连续接种驯化至发酵物大分子蛋白显著下降,将经过驯化的发酵混合料稀释至10-4,10-5,各取稀释液0.1ml涂布于酵母固体培养基的平板上,28℃倒置培养24h,挑选出菌落形态最多最相似的疑似酵母菌的单菌落,在酵母固体平板上划线纯化培养3次,获得4株单菌落,分别于200mL豆清水培养基中28℃静置培养7h,获得豆清发酵液,测定其酒精含量,品尝其风味口感;并将豆清发酵培养液根据10%(v/w)的接种量分别接种于灭菌的豆渣中发酵。发酵结果如表1所示,最终挑选出一株豆清发酵风味独特,并能降解豆渣中大分子蛋白的菌种1,保存并命名为SF1。通过对菌株进行形态学观察、及18S rDNA基因序列测定及***发育树同源性分析,最终鉴定为酿酒酵母(Saccharomyces cerevisiae)。
其中各培养基的配方如下:
(1)酵母培养基(1L):蛋白胨5.0g、麦芽提取物 3.0 g、酵母提取物 3.0g、葡萄糖3.0g溶于蒸馏水并定容到1000 mL,调节pH为6.2,分装后121 ℃高压灭菌 20 min。(注:固体培养基在此基础上添加琼脂粉 20.0g/L)
(2)豆清水培养基:传统酸浆豆腐加工工艺获得豆清水,根据(w/v)15:100的比例加入蔗糖, 115℃高压灭菌 20 min。
(3)灭菌的豆渣:传统酸浆豆腐加工工艺获得豆渣,115℃高压灭菌15 min。
表1 各种菌发酵豆清饮料情况分析
实施例2 菌种的鉴定
2.1 SF1菌的形态观察
SF1菌株的主要形态特征如下:菌落圆,在酵母培养基上呈乳白色、不透明(图1);菌体椭圆形,宽2-4 μm,长4-6 μm(图2)。
2.2 SF1菌的碳源利用分析
配置培养基(每1升培养基中含有蛋白胨10g,牛肉粉3g,氯化钠5g,溴甲酚紫0.04%,将培养基按10ml/管分装至试管中,分别加入1wt%的葡萄糖、半乳糖、麦芽糖、蔗糖、乳糖、淀粉、果糖、木糖、***糖、海藻糖,倒扣放入德汉氏小管,每种糖各3支,121 ℃灭菌20 min),根据1%(v/v)的接种量接种酿酒酵母SF1菌液(SF1菌液浓度为2.0×108 cfu/mL),放置30℃下发酵,产酸则培养基变成橙黄色(培养基原先为深紫色),产气则德汉氏小管中有气体,结果见表2,可知SF1可利用葡萄糖、半乳糖、麦芽糖、蔗糖、果糖进行发酵产酸产气,不能利用乳糖、淀粉、木糖、***糖、海藻糖。
表2 SF1对糖类的利用情况
注:“+”为实验阳性,“-”为实验阴性。
2.3 SF1菌的分子生物学鉴定
①细菌基因组DNA的提取:采用TIANGEN公司的酵母菌基因组DNA提取试剂盒提取。
②18S rDNA序列的PCR扩增:扩增18S rDNA基因序列,所用引物为:
Au2-F(SEQ ID NO.1):5’-TTTCGATGGTAGGATAGDGG-3’;
Au4-R(SEQ ID NO.2):5’-RTCTCACTAAGCCATTC-3’。
PCR反应体系:2*Mix 12.5μL,引物及DNA各1μL,ddH20 9.5μL。
PCR扩增程序:93℃预变性4min;然后94℃变性30s,50℃退火60s,72℃延伸90s,共35个循环;最后72℃充分延伸10min,4℃保存。
③PCR产物检测及测序分析:取5μL的PCR产物,在加入EB的1.0%的琼脂糖中凝胶电泳分离检验,扩增得到18S rDNA目的片段长为1257bp(图3)。将含有目标片段的产物委托铂尚生物测序公司完成测序,得到的实际有效核苷酸序列为18S rDNA其核苷酸序列见SEQ IDNO.3,大小为1257bp。
④***发育分析:在NCBI数据中对各18S rDNA序列进行Blast比对分析,得到序列与Saccharomyces cerevisiae的Per.Ident为99.92%,Query Cover为100 %,采用MEGA 4.1中的Maximum Parsimony进行发育树的构建分析,最终确定SF1与Saccharomyces cerevisiae (NR132207.1)在同一发育分支上(结果见图4),结合SF1菌的形态观察、细菌生理生化试验以及DNA***发育树中的同源性分析,确定SF1菌为酿酒酵母(Saccharomyces cerevisiae)菌种。
实施例3 酿酒酵母SF1(Saccharomyces cerevisiae SF1)在不同pH值条件下的生长状况
将酵母发酵培养基分别调整pH至1.5至3.5后,根据1%(v/v)的接种量接种酿酒酵母SF1菌液(SF1菌液浓度为2.0×108 cfu/mL),于30℃、150 r/min条件下分别培养2h、6h,然后取1ml菌悬液进行菌落计数,结果见表3,可知菌SF1可在pH值为2.0的条件下存活6h;在pH值为3.0的条件下可实现生长繁殖,在pH值为3.0的酵母培养基上培养2h时菌落数生长至原值的130.34%,培养6h时生长至原值的225.64%。说明SF1菌可在pH值为3.0的酸性条件下实现正增长进一步地,进行SF1最适发酵pH的测定,确定其最适发酵pH为6.0-6.5之间(图5)。
表3 菌SF1在酸性条件下的生长状况
实施例4 酿酒酵母SF1(Saccharomyces cerevisiae SF1)不同发酵时间内生产豆清发酵饮料分析
取1mL酿酒酵母SF1菌液(SF1菌液浓度为2.0×108 cfu/mL)接种于200 mL豆清水培养基中,于不同发酵时间取发酵样品,进行菌落数、pH、总糖、蛋白浓度、酒精度及口感分析检测,结果如表4,可知,经过SF1发酵6小时后,豆清水中的豆腥味完全消失,呈现极好的口感,此时豆清水:pH值为5.1;多糖3.00mg/mL;无酒精度;蛋白含量为0.298mg/mL,较未发酵(0h)增加了21.74%。基于此,选取6h为最佳发酵时间,进行豆清发酵饮料的生产。
表4 不同发酵时间内SF1对豆清水发酵情况分析
实施例5 酿酒酵母SF1(Saccharomyces cerevisiae SF1)在豆清发酵饮料中的应用
对豆清发酵饮料中的氨基酸含量,异黄酮及蛋白质进行分析,具体为:取1mL酿酒酵母SF1菌液(SF1菌液浓度为2.0×108 cfu/mL)接种于200ml豆清水培养基中,28℃下静止发酵培养6h,制成豆清发酵饮料,其参数如表5所示:经酿酒酵母SF1发酵后,可获得去除豆腥味的并产生淡淡香味的豆清发酵饮料,该饮料含有(m/v)0.92%异黄酮,总氨基酸254.9mg/100ml,蛋白质0.3%,多糖含量3.01mg/ml,pH值5.1。将发酵液过滤去除固体物质后,获得的液体饮料由于含有较高的大豆多糖可保持该发酵液长期处于稳定状态而不发生明显的沉淀现象,因此该类饮料无需再加稳定剂等物质。将发酵前后的豆清饮料进行同比例浓缩后,进行PAGE蛋白检测(图6),发现与未发酵的豆清水(S-CK)相比,发酵后的豆清饮料中蛋白质的含量明显增加,同时将发酵后的豆清饮料进行过滤处理,过滤前后的豆清饮料的蛋白含量未有明显变化,说明增多的蛋白质多以可溶性蛋白的形式存在。
表5 豆清发酵饮料产品参数检测
实施例6 酿酒酵母SF1(Saccharomyces cerevisiae SF1)发酵生产发酵豆渣
将酿酒酵母SF1进行发酵豆渣的生产。将豆渣进行115℃、15min灭菌处理,按照5%(v:w)的比例接种酿酒酵母SF1菌液(SF1菌液浓度为2.0×108 cfu/mL),28℃下恒温发酵3天。与此同时,另取豆渣按照质量比加入10%的糖蜜进行115℃、15min灭菌处理,按照5%(v:w)的比例接种酿酒酵母SF1菌液(SF1菌液浓度为2.0×108 cfu/mL),28℃下恒温发酵3天。将发酵豆渣进行PAGE电泳进行蛋白大小的检测分析(图6),经过SF1发酵后,豆渣中大于25kDa的大分子蛋白基本被降解为25kDa以下的小分子蛋白,并且发酵豆渣中的小分子多肽显著增加,与加入糖蜜等碳源相比,不加碳源可以保持更多的氨基酸,说明SF1可在不加碳源等其他物质的基础上显著将豆渣中的大分子蛋白降解为小分子量多肽,表明该菌可对豆渣进行发酵生产出富含小分子多肽物质。进一步地,采用凯氏定氮法GB/T 6432-2018进行豆渣发酵前后中总蛋白质的检测分析,结果见表6,经SF1发酵72h,豆渣中的总蛋白质由发酵前的23.525 %增加到26.831%,发酵豆渣中蛋白质增加了14.05%。采用SF1获得的发酵豆渣由于蛋白分子量低于25kDa,蛋白质含量显著增加,非常适合作为优质原料应用于食品、畜禽、水产的养殖中。
表6 发酵豆渣总蛋白质含量分析
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caaagtcttt gggttctggg gggagtatgg tcgcaaggct gaaacttaaa ggaattgacg 780
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caggtccaga cacaataagg attgacagat tgagagctct ttcttgattt tgtgggtggt 900
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Claims (3)
1.一株酿酒酵母,其特征在于:所述菌株为酿酒酵母SF1(Saccharomyces cerevisiaeSF1),于2020年7月16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,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC NO.20379。
2.如权利要求1所述的酿酒酵母在制备豆清发酵饮料中的应用。
3.如权利要求1所述的酿酒酵母在制备发酵豆渣中的应用。
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