CN116555059A - 一种酿酒酵母Saccharomyces cerevisiaeJXBK7-1及其应用 - Google Patents
一种酿酒酵母Saccharomyces cerevisiaeJXBK7-1及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种酿酒酵母Saccharomyces cerevisiae JXBK7‑1及其应用。本发明选择的酿酒酵母命名为Saccharomyces cerevisiaeJXBK7‑1,保藏于广东省微生物菌种保藏中心,保藏号GDMCC.No62908。本发明利用菌株GDMCC.No62908进行发酵,产生的多糖分泌到细胞外,聚合形成不规则颗粒状,易于收集,得率可达50%以上。且无需对酵母菌进行破壁、水解、分离等过程,可直接得到酵母多糖。具有酵母多糖得率高、操作过程简单等优点。
Description
技术领域
本发明属于生物技术领域,具体涉及一种酿酒酵母SaccharomycescerevisiaeJXBK7-1及其应用。
背景技术
酵母多糖(yeast polysaccharide,YPS)是一种从酵母细胞的细胞壁中提取出来的水溶性多糖,其主要活性成分为β-葡聚糖和甘露寡糖。葡聚糖(β-glucan)层靠近细胞膜,位于细胞壁的内层,是细胞壁结构的主要成分,约占细胞壁干重的30%~34%。甘露聚糖(MOS)位于细胞壁的外侧,约占细胞壁干重的30%。甘露聚糖和葡聚糖之间是蛋白质。
酵母多糖具有特殊的生物学功能,可参与细胞的多种生命调节,激活免疫细胞,抗菌、抗病毒、抗肿瘤,提高机体免疫功能,是一种有效的机体免疫促进调节剂,酵母菌的细胞壁中含有大量的多糖,常规酵母多糖生产方法是酵母经自溶和外源酶催化水解、分离、喷雾干燥而成,工艺复杂,耗能高,污染大。
目前,酵母多糖的提取方法主要有热水提取法,酸碱法,酶解法,超声波法等,其中,热水提取法是一种简单易行、成本低廉的方法,但提取效率较低;酸碱法提取效率较高,但操作复杂,易造成环境污染;酶解法提取效率高,但酶解剂价格昂贵;超声波法提取效率高,但设备成本高。因此,选择合适的提取方法需要综合考虑多种因素。
中国专利CN104610460A公开了一种从酵母细胞壁中提取多糖的方法,该方法是以培养的新鲜酵母或工业废酵母为原料,悬浮于含有锂盐和表面活性剂的提取液中,在水浴中提取多糖,然而,其最终粘红酵母的多糖提取率仅为17.3%,得率较低,造成大量原料浪费。
中国专利CN112760346A公开了一种高免疫活性酵母细胞壁多糖的提取方法。该提取方法主要包括以下步骤:S1、啤酒酵母预处理S2、冻融法破壁:将预处理的啤酒酵母泥置于-20℃下冷冻1~2h,再置于80℃水中骤然升温使其融化,后4000r/min离心5~10min,如此反复3次,得沉淀物;S3、酶解:将S2所得的沉淀物与蒸馏水按1:(10~15)的体积混合,用盐酸调pH值为6~8,加入蛋白酶、多糖酶、复配促进剂,进行超声波处理,将混合物5000r/min离心5~10min,收集上清液;S4、洗涤干燥:上清液用1~2倍体积的质量浓度为60%~85%乙醇沉淀,沉淀物丙酮洗涤两次,真空干燥至恒重即得酵母细胞壁多糖干品。但是该方法提取的酵母多糖得率最高仅为26.24%。
综上可知,现有技术皆是先要收集酵母菌体,破壁,再从破碎的细胞壁中提取酵母多糖,普遍具有酵母多糖得率低,筛选成本高,污染大,容易造成大量材料浪费等问题。
发明内容
针对现有技术普遍存在的技术问题,本发明提供了一株酿酒酵母SaccharomycescerevisiaeJXBK7-1及其应用。本发明的酿酒酵母产胞外多糖量高,利用其生产酵母多糖,无需破壁工艺,生产成本低,得率达50%以上,且环境污染低,有效提高了材料利用率。
为了达到上述技术效果,本发明采用的方案具体如下:
一种酿酒酵母Saccharomyces cerevisiaeJXBK7-1,保藏于广东省微生物菌种保藏中心,保藏日期为2022年12月6日,保藏编号为GDMCC.NO:62908。
本发明还提供了一种酿酒酵母Saccharomyces cerevisiaeJXBK7-1在制备酵母多糖中的应用。
优选地,所述酿酒酵母Saccharomyces cerevisiaeJXBK7-1制备酵母多糖的具体过程为:
S1、将酵母菌种GDMCC.No62908用麦芽汁培养基活化,获得活化后的菌种;
S2、将步骤S1所得活化后的菌种加入液体培养基中,培养5-10天,获得发酵培养液;
S3、收集步骤S2中发酵培养液中0.2-0.5cm大小不规则的透明颗粒,进一步检测,即得。
优选地,步骤S2所述液体培养基由如下重量百分数的组分制成:蔗糖1-5%,麦芽糖1-5%,棉子糖1-5%,蜂蜜0.1-2%,荔枝汁1-3%,硫酸铵0.1-3%,硫酸镁0.1-0.5%,磷酸二氢钾0.1-0.5%,加水至100%。
发明人在对发酵的研究过程中,发现在液体培养基中,添加适量蜂蜜及荔枝汁,可以为酵母菌提供足够的营养物质,加速产糖效率,提高了酵母多糖的得率。
优选地,所述培养的具体过程为:于液体培养基中,15~30℃静止培养两天,然后从第三天开始转到8-12℃培养,每天缓慢搅拌,每隔8小时搅拌一次。
在实际处理过程中,于15~30℃下静止培养一段时间后,有助于酿酒酵母Saccharomyces cerevisiaeJXBK7-1繁殖,增加菌种产量,而在第三天开始,进行搅拌,将发酵物与培养基组分充分混合均匀,可以进一步丰富培养基组分,有助于酿酒酵母Saccharomyces cerevisiaeJXBK7-1发酵,加速产糖,而经过多次筛选,发明人发现在8~12℃下,有助于颗粒物的形成,有利于其分泌到细胞外,便于收集。
优选地,所述缓慢搅拌的具体过程为:于60~100rpm转速下搅拌1~2min。
优选地,步骤S3所述收集透明颗粒的具体过程为:培养5-10天后发酵培养液中会形成0.2-0.5cm大小不规则透明颗粒,用孔径小于0.2cm滤网将颗粒过滤出来的。
本发明从蜂蜜发酵饮料中分离获得酵母菌,经鉴定与酿酒酵母Saccharomycescerevisiae的同源性达99.43%,命名为Saccharomyces cerevisiae JXBK7-1。发明人进一步利用筛选出来的菌株进行发酵,产生的多糖分泌到细胞外,聚合形成不规则颗粒状,易于收集,得率可达50%以上。
与现有技术相比,本发明具有如下优势:首次筛选出酿酒酵母Saccharomycescerevisiae JXBK7-1,并将其用于酵母多糖的提取,本发明酿酒酵母Saccharomycescerevisiae JXBK7-1产生的多糖,可以直接分泌到细胞外,聚合形成不规则颗粒状,无需破壁工艺,易于收集,大大简化了酵母多糖的提取步骤,节约了生产成本,有助于酵母多糖的大规模应用。
附图说明
图1为酿酒酵母Saccharomyces cerevisiae JXBK7-1的菌落形态图;
图2为酿酒酵母Saccharomyces cerevisiae JXBK7-1的显微结构图;
图3为酿酒酵母Saccharomyces cerevisiae JXBK7-1形成的假菌丝形态图。
具体实施方式
下面结合具体实施例对本发明作进一步解释,但是应当注意的是,以下实施例仅用以解释本发明,而不能用来限制本发明,所有与本发明相同或相近的技术方案均在本发明的保护范围之内。本实施例中未注明具体技术或条件者,按照本领域常规技术方法和仪器说明书内容进行操作;所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
其中,所述荔枝汁为荔枝浓缩汁,可购自陕西世泽天承生物科技有限公司,货号为LZNSZ01;所述自制蜂蜜发酵饮料的制备过程为:蜂蜜(龙眼蜜)9%+荔枝汁1%+凉开水90%,放置5℃冰箱冷藏一个月。
实施例1酿酒酵母Saccharomyces cerevisiae JXBK7-1的分离、鉴定和保藏1.酵母菌Saccharomyces cerevisiae JXBK7-1的分离:酵母菌Saccharomyces cerevisiaeJXBK7-8菌株是从蜂蜜发酵饮料中分离获得。分离过程如下:称取3g自制蜂蜜发酵饮料底部颗粒,用无菌研钵轻轻研磨后溶于97mL无菌生理盐水中,于25℃,200r/min的条件下震荡30min,用无菌水依次稀释样品为10-2~10-6,吸取不同浓度的样品100μL,分别涂布于麦芽汁培养基(广东环凯微生物科技有限公司,021120,下同)上,每个浓度梯度重复3次,置于25℃培养箱中倒置培养观察,培养3天左右,挑取形态、颜色不同的菌落划线,重复此步骤5-8次,获得纯的菌株。
2.酵母菌Saccharomyces cerevisiae JXBK7-1菌株的鉴定:根据菌落颜色、形态、等对菌株进行表观鉴定。如图1-3所示,其中,图1为菌落形态图,图2为显微结构图。酵母菌Saccharomyces cerevisiae JXBK7-1在麦芽汁培养基上,28℃培养3天,菌落为乳酪色,呈圆型,微凸起,边缘整齐,在显微镜下观察,营养细胞圆形、卵圆形或椭圆形,以无性繁殖为主,有少量假菌丝产生(如图3所示),有子囊孢子,每个子囊孢子中含有1-4个圆形或卵圆形子囊孢子。经过26SrRNA测序分析,其序列如下:
AAATTTGCAATCTGGTACCTTCGGTGCCCGAGTTGTAATTTGGAGAGGGC
AACTTTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGGG
TGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTGTAAAGTGCCTTCGAAGA
GTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGC
TAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAA
AAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAG
GGCATTTGATCAGACATGGTGTTTTGTGCCCTCTGCTCCTTGTGGGTAGGGGA
ATCTCGCATTTCACTGGGCCAGCATCAGTTTTGGTGGCAGGATAAATCCATAG
GAATGTAGCTTGCCTCGGTAAGTATTATAGCCTGTGGGAATACTGCCAGCTGG
GACTGAGGACTGCGACGTAAGTCAAGGATGCTGGCATAGTGGTTATATGCCGT
CCGTCTT(SEQ ID NO.1);
本发明菌株与Saccharomyces cerevisiae(酿酒酵母)的同源性达99.43%。结合菌株的形态特征,将该菌株鉴定为酵母菌,命名为Saccharomyces cerevisiae JXBK7-1。
3.酵母菌Saccharomyces cerevisiae JXBK7-1菌株的保藏:2022年12月6号将该菌种保藏于广东省微生物菌种保藏中心,保藏号GDMCC.No62908,12月9日鉴定为菌株存活。
实施例2一种酿酒酵母提取多糖的方法
所述酿酒酵母Saccharomyces cerevisiae JXBK7-1制备酵母多糖的过程如下:
S1、将酵母菌种Saccharomyces cerevisiae JXBK7-1用麦芽汁培养基活化,获得活化后的菌种;
S2、按如下组分及其质量百分比配制液体培养基:蔗糖5%,麦芽糖3%,棉子糖2%,蜂蜜0.8%,荔枝汁3%,硫酸铵0.7%,硫酸镁0.2%,磷酸二氢钾0.1%,加水至100%;将步骤S1所得活化后的菌种加入液体培养基中,于22℃下静止培养2天,然后从第三天开始转到12℃培养每隔8小时缓慢搅拌一次,搅拌条件为于60rpm转速下搅拌2min,连续培养10天,获得发酵培养液;
S3、连续培养10天后,发酵液中会形成0.2-0.5cm大小不规则透明颗粒,用孔径小于0.2cm滤网将颗粒过滤出来,进一步检测,即得。酵母多糖提取率为52.93%。
实施例3一种酿酒酵母提取酵母多糖的方法
所述酿酒酵母Saccharomyces cerevisiae JXBK7-1制备酵母多糖的过程如下:
S1、将酵母菌种Saccharomyces cerevisiae JXBK7-1用麦芽汁培养基活化,获得活化后的菌种;
S2、按如下组分及其质量百分比配制液体培养基:蔗糖2%,麦芽糖4%,棉子糖1%,蜂蜜2%,荔枝汁2%,硫酸铵0.15%,硫酸镁0.1%,磷酸二氢钾0.5%,加水至100%;然后将步骤S1所得活化后的菌种加入配制好的液体培养基中,于15℃静止培养2天,然后第3天开始转到10℃培养每隔8小时搅拌一次,每次于80rpm转速下搅拌2min,连续培养6天,获得发酵培养液;
S3、连续培养6天后,发酵液中会形成0.2-0.5cm大小不规则的透明颗粒,用孔径小于0.2cm的滤网将颗粒过滤出来,进一步检测,即得。酵母多糖纯度为92.78%,酵母多糖提取率为51.25%。
实施例4一种酿酒酵母提取酵母多糖的方法
所述酿酒酵母Saccharomyces cerevisiae JXBK7-1制备酵母多糖的过程如下:S1、将酵母菌种Saccharomyces cerevisiae JXBK7-1用麦芽汁培养基活化,获得活化后的菌种;
S2、按如下组分及其质量百分比配制液体培养基:蔗糖2%,麦芽糖5%,棉子糖4%,蜂蜜0.5%,荔枝汁1%,硫酸铵3%,硫酸镁0.5%,磷酸二氢钾0.2%,加水至100%;然后将步骤S1所得活化后的菌种加入配制好的液体培养基中,于30℃下静止培养2天,然后从第3天开始转到8℃培养,每隔8小时搅拌一次,搅拌过程为:于100rpm转速下搅拌1min,连续培养8天,获得发酵培养液;
S3、连续培养8天后,发酵液中会形成0.2-0.5cm大小不规则的透明颗粒,用孔径小于0.2cm的滤网将颗粒过滤出来,进一步检测,即得。酵母多糖纯度为93.01%,酵母多糖提取率为55.78%。
对比例1一种酿酒酵母提取酵母多糖的方法
所述酿酒酵母Saccharomyces cerevisiae JXBK7-1提取酵母多糖的过程与实施例4类似,与实施例4的区别在于,对比例1步骤S2中采用常规的LB液体培养基培养。最终,酵母多糖的纯度为91.18%,酵母多糖提取率为27.45%。
对比例2一种酿酒酵母提取酵母多糖的方法
所述酿酒酵母Saccharomyces cerevisiae JXBK7-1提取酵母多糖的过程与实施例4类似,与实施例4的区别在于,对比例2中在配制好的液体培养基中培养时,于30℃下连续静止培养8天。最终,酵母多糖的纯度为87.55%,酵母多糖提取率为32.17%。
最后,需要说明的是,上述实施例仅例示性说明本发明的原理、性能及功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (7)
1.一种酿酒酵母Saccharomyces cerevisiaeJXBK7-1,其特征在于,所述酿酒酵母Saccharomyces cerevisiaeJXBK7-1保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC.NO:62908。
2.一种如权利要求1所述的酿酒酵母Saccharomyces cerevisiaeJXBK7-1在制备酵母多糖中的应用。
3.如权利要求2所述的应用,其特征在于,所述制备酵母多糖的具体过程为:
S1、将酿酒酵母Saccharomyces cerevisiaeJXBK7-1用麦芽汁培养基活化,获得活化后的菌种;
S2、将步骤S1所得活化后的菌种加入液体培养基中,培养5-10天,获得发酵培养液;
S3、收集步骤S2中发酵培养液中0.2-0.5cm大小不规则的透明颗粒,即得。
4.如权利要求3所述的应用,其特征在于,步骤S2所述液体培养基由如下重量百分数的组分制成:蔗糖1-5%,麦芽糖1-5%,棉子糖1-5%,蜂蜜0.1-5%,荔枝汁0.1-3%,硫酸铵0.1-3%,硫酸镁0.1-0.5%,磷酸二氢钾0.1-0.5%,加水至100%。
5.如权利要求3所述的应用,其特征在于,所述培养的具体过程为:于液体培养基中,15~30℃静止培养两天,然后从第三天开始转到8-12℃培养,每天缓慢搅拌,每隔8小时搅拌一次。
6.如权利要求5所述的应用,其特征在于,所述缓慢搅拌的具体过程为:于60~100rpm转速下搅拌1~2min。
7.如权利要求3所述的应用,其特征在于,步骤S3所述收集透明颗粒的具体过程为:培养5-10天后发酵培养液中会形成0.2-0.5cm大小不规则透明颗粒,用孔径小于0.2cm的滤网将颗粒过滤出来。
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