CN114854609A - 一株能增加红枣乳酸菌饮料发酵香气的葡萄汁有孢汉逊酵母及其应用 - Google Patents
一株能增加红枣乳酸菌饮料发酵香气的葡萄汁有孢汉逊酵母及其应用 Download PDFInfo
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- CN114854609A CN114854609A CN202210523252.7A CN202210523252A CN114854609A CN 114854609 A CN114854609 A CN 114854609A CN 202210523252 A CN202210523252 A CN 202210523252A CN 114854609 A CN114854609 A CN 114854609A
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- hansenula polymorpha
- red date
- fermentation
- lactobacillus
- juice
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Abstract
本发明公开了一株能增加红枣乳酸菌饮料发酵香气的葡萄汁有孢汉逊酵母及其应用,属于生物工程技术领域。该葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)MY‑00于2021年6月28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.22784。该葡萄汁有孢汉逊酵母能够高产乙酸乙酯并且发酵过程中不产酒精,与植物乳杆菌CGMCC No.24097混菌发酵红枣汁促进果香酯类物质的发生,增加了红枣乳酸菌饮料中的温带水果香气,使其发酵后的风味更为丰富,并且还提升了红枣乳酸菌饮料中植物乳杆菌的含量,增强了益生效果。同时,本发明提供的葡萄汁有孢汉逊酵母有望添加到其他食品饮料的生产中以丰富风味物质增加香气并促进其他益生菌的生长。
Description
技术领域
本发明涉及一株能增加红枣乳酸菌饮料发酵香气的葡萄汁有孢汉逊酵母及其应用,属于生物工程技术领域。
背景技术
我国目前果酒生产行业中,大多数选用商业酵母进行发酵,其优点是发酵快、成品酒稳定性较高,但菌株专用性程度低也造成了成品酒在香气复杂性和风格独特性方面有所欠缺。非酿酒酵母广泛存在于浆果的果皮表面、果园土壤及酿造环境中,虽然不能单独完成发酵,但是可以以糖、醛、有机酸、盐类等为底物,在酯酶催化下合成酯类,在果酒呈香方面具有酿酒酵母所不具有的优势,因此其对于成品酒的风味具有至关重要的影响。
葡萄汁有孢汉逊酵母在分类学上属于真核生物原界,真菌界,子囊菌门,酵母亚门,酵母纲,酵母目,酵母科,有孢汉逊酵母属。葡萄汁有孢汉逊酵母通常应用在与酿酒酵母共同发酵果酒领域,其发酵可以促进一些果香酯类物质的发生,在葡萄酒中可以增加温带水果香气,并且在玫瑰香葡萄酒增香方面可以增加玫瑰香葡萄特征香气的含量,例如2-苯乙醇、单萜醇等。但同时,大多数葡萄汁有孢汉逊酵母在果汁体系下的生长能力较弱,不能独自完成发酵,同时葡萄汁有孢汉逊酵母在发酵的同时大多会产酒精,其也会抑制自身的生长。
许多传统发酵食品的制作过程都是自然发酵并不是单一菌种发酵,而是混合微生物共同发酵的结果,多种的微生物群落此消彼长,协同完成整个发酵过程,整个过程中代谢路径较为复杂,最终形成了所加工成品的独特风味。葡萄汁有孢汉逊酵母菌株具有混合发酵可调节风味物质的种类和含量的能力,且不产硫化氢,因此筛选一株低产酒精,果汁中发酵能力良好且高产香的葡萄汁有孢汉逊酵母,在发酵食品领域具有很大的应用潜能。
发明内容
本发明提供了一种增加发酵香气的方法,并将其应用于红枣乳酸菌饮料发酵中,以增加其独特的风味。
本发明的第一个目的是提供一株葡萄汁有孢汉逊酵母菌株(Hanseniasporauvarum)MY-00,已于2021年06月28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.22784,保藏地址为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。
本发明的第二个目的是提供含有所述葡萄汁有孢汉逊酵母的微生物菌剂。
在一种实施方式中,所述葡萄汁有孢汉逊酵母不低于106cfu/mL或106cfu/g。
在一种实施方式中,所述微生物菌剂还含有植物乳杆菌,所述植物乳杆菌已于2021年12月13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.24097。
在一种实施方式中,所述植物乳杆菌不低于108cfu/mL或108cfu/g。
本发明的第三个目的是提供一种制备红枣乳酸菌饮料的方法,其特征在于,以红枣汁为原料,利用权利要求1所述葡萄汁有孢汉逊酵母或所述微生物菌剂和植物乳杆菌发酵生产红枣乳酸菌饮料,或者利用含有所述植物乳杆菌的微生物菌剂发酵生产红枣乳酸菌饮料。
在一种实施方式中,所述方法步骤为:
(1)将菌浓为1×106~6×106CFU/mL的所述葡萄汁有孢汉逊酵母与菌浓为1×108~6×108CFU/mL的所述植物乳杆菌等比例混合得到混合菌液;
(2)将混合菌液按照发酵总体积的5%~10%添加至红枣汁中;
(3)在35~40℃下主发酵2~3天。
在一种实施方式中,所述红枣汁的含糖量为140g/L,可滴定酸含量为2g/L。
优选地,将混合菌液按照发酵总体积的10%添加至红枣汁中,在37℃下主发酵2天。
本发明的第四个目的是提供所述制备红枣乳酸菌饮料的方法制备得到的红枣乳酸菌饮料。
在一种实施方式中,所述红枣乳酸菌饮料中含有所述的葡萄汁有孢汉逊酵母。
本发明的第五个目的是提供一种食品,所述食品含有所述的植物乳杆菌或所述的微生物菌剂。
在一种实施方式中,所述食品为使用所述植物乳杆菌、或所述的微生物菌剂制备得到的发酵乳、发酵饮料或果酒。
本发明的第六个目的是提供所述植物乳杆菌或所述的微生物菌剂在制备发酵产品中的应用。
在一种实施方式中,所述发酵产品包括发酵乳、发酵饮料或果酒等发酵食品。
在一种实施方式中,所述发酵产品为红枣乳酸菌饮料。
有益效果:
本发明的葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)MY-00与其他非酿酒酵母相比能够高产乙酸乙酯并且发酵过程中不产酒精,有助于延长其自身在发酵过程中的寿命,从而使得香气成分更加丰富持久。将葡萄汁有孢汉逊酵母MY-00与植物乳酸菌进行混合发酵生产红枣汁饮料,与植物乳杆菌CGMCC No.24097混菌发酵可以促进果香酯类物质的发生,与植物乳杆菌CGMCC No.24097单独发酵相比,混菌发酵产物分析发现乙酸乙酯相对含量提高了9倍,乙酸异戊酯相对含量提高了7倍,己酸乙酯相对含量提高了4倍,辛酸乙酯相对含量提高了4倍。增加了红枣乳酸菌饮料中的温带水果香气,使红枣汁发酵后的风味更为丰富。并且能够提升发酵后红枣乳酸菌饮料中的植物乳杆菌含量,提升了植物乳杆菌的益生效果。
生物材料保藏
本发明提供的葡萄汁有孢汉逊酵母MY-00,分类命名为葡萄汁有孢汉逊酵母Hanseniaspora uvarum,已于2021年06月28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.22784,保藏地址为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。
本发明提供的植物乳杆菌MY-12,分类命名为植物乳杆菌Lactobacillusplantarum,已于2021年12月13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.24097,保藏地址为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为乙酸乙酯初筛平板图。
图2为TTC一级复筛平板图。
图3为杜氏小管产气情况图。
图4为菌株葡萄汁有孢汉逊酵母MY-00号系统发育树图。
图5为在枣汁中MY-12单菌发酵和MY-12+MY-00混菌发酵生长曲线图。
图6为红枣汁两种发酵方式的感官评定结果图。
具体实施方式
WL培养基(g/L):酵母浸粉:4.0g,酪胨:5.0g,葡萄糖:50.0g,磷酸二氢钾:0.55g,氯化钾:0.425g,氯化钙:0.125g,硫酸镁:0.125g,氯化铁:0.0025g,硫酸锰:0.0025g,溴甲酚绿:0.022g,pH5.5±0.2(25℃)。
YPD液体培养基(g/L):葡萄糖20.0g,蛋白胨20.0g,酵母粉10.0g。
YPD培养基平板(g/L):葡萄糖20.0g,蛋白胨20.0g,酵母粉10.0g,琼脂粉20.0g。
TTC底层培养基(g/L):葡萄糖50.5,蛋白胨10.0,酵母浸膏7.5,酸性磷酸钾5.0,硫酸镁2.0,柠檬酸1.35,氨苄青霉素1.0,调整pH在4.0以上,琼脂30.0。
TTC上层培养基(g/L):葡萄糖0.5,琼脂15.0;灭菌后冷却后加入TTC 0.5。
扩增培养基:葡萄糖10.0%,蛋白胨5.0%,K2HPO4 1.0%,MgSO4 0.5%,pH 7.2。
发酵培养基:蛋白胨2.0%,酵母浸粉1.0%,硝酸铵(NH4NO3)0.3%,磷酸二氢钾(KH2PO4)0.4%,葡萄糖2.0%,蒸馏水配制,121℃灭菌25min。在温度降到60~70℃左右加入0.1%p-NPG。
50%糖水(g/L):白砂糖500g。
实施例1:葡萄汁有孢汉逊酵母菌株的筛选与鉴定
(1)菌株初筛
六种水果挤碎室温自然发酵,稀释涂布于含氨苄青霉素YPD平板培养48h后,共得酵母菌株200株。初筛培养基为碳源仅含乙酸乙酯的培养基,乙酸乙酯水解产酸导致培养基PH改变从而引起溴甲酚紫颜色变化,由于酯化过程为可逆反应,因此采用此方法进行高产乙酸乙酯菌株的初筛。为便于判断颜色变化将分离出的酵母菌每八株菌划线至一个乙酸乙酯平板,于28℃培养48h后共有50株菌株产生变色圈,将其编号为1-50。
(2)菌株的一级复筛
氯化三苯基四氮,也称红四氮唑。原理在于TTC为一种氧化还原反应指示剂,TTC可以与活细胞内的脱氢酶(尤其是线粒体内的琥珀酸脱氢酶)发生反应而被还原为红色化合物,酵母体内的呼吸酶活力与酵母产酒精能力有密切关系,产酒精能力较强的酵母呈现深红色,弱者呈浅红色。将于斜面保藏的菌株活化后分区划线至TTC底层平板,每八株菌划线至一个平板。将TTC底层平板倒置于28℃培养箱培养2~3d,倒入TTC上层平板,于避光环境培养2~3h后观察菌落颜色,共有34株菌菌落没有呈红色,初步判定其为产酒精能力弱的菌株,保藏后进行二级筛选。
(3)菌株的二级复筛
发酵能力强弱与菌株的产气量多少成正比,采用杜氏小管进行二级复筛。将上述一级复筛所得34株菌株活化后进行杜氏小管复筛,所用红枣汁的糖含量为140g/L,可滴定酸2g/L,,每支试管装液量为10mL,接种量为枣汁体积的5%,接种后于25℃静置培养,分别在24h,48h时观察各菌株的产气速度和产气量。24h时共有8株产气充满小管,48h时共有17株产气充满小管。产气实验观察结束后,继续发酵5d,待发酵结束后初步进行香气感官品评,杜氏小管产气量如下图3所示。综合以上筛选选出17株菌株进行三级筛选。
(4)菌株的三级复筛
对实施例1步骤(3)中初步筛选到的17株菌株在桃汁和红枣汁中分别发酵产生的香气进行了闻香实验,部分结果如下表所示。
表1菌株在桃汁和红枣汁发酵中的香气特点
(5)菌株的生物学鉴定
挑取斜面保藏的菌株于10ml YPD培养基中,28℃,150rpm培养24h。取适量培养液离心1min,弃上清,采用酵母DNA提取试剂盒提取菌体DNA,用酵母18s通用引物扩增基因组DNA,PCR产物用2%琼脂糖凝胶电泳检测,并进行测序。采用BLAST方式将测定的18srDNA序列与GenBank中酵母菌的序列进行比对,结果如图4所示,确定15号菌株为葡萄汁有孢汉逊酵母,并重新命名为葡萄汁有孢汉逊酵母MY-00。
实施例2:混菌发酵在红枣乳酸菌饮料中的应用
菌浓为106左右的葡萄汁有孢汉逊酵母MY-00与菌浓为108左右的植物乳杆菌MY-12菌液不同接种比例、接种先后顺序混种发酵,总接种量为10%(v/v)。
步骤1:挑取菌株MY-00接种于10mL YPD液体培养基,28℃培养20h培养成种子培养液。挑取菌株MY-12接种于10mL MRS液体培养基,37℃培养16h培养成种子培养液。
步骤2:调整葡萄汁有孢汉逊酵母MY-00和植物乳杆菌MY-12两菌种浓度分别为5×106和5×108CFU/mL,MY-00与MY-12按体积接种比例3:1、1:1或1:3;接种时间为同时接种、MY-00先接种1d(将MY-00到发酵体系中,然后发酵1天后再接种MY-12)或MY-00后接种1d(将MY-12到发酵体系中,然后发酵1天后再接种MY-00)。其中空白对照组为MY-12单独发酵。种子培养液以10%(v/v)接种总量接种于装有200mL红枣汁250mL三角瓶中,调整含糖量至140g/L,可滴定酸2g/L。控制发酵温度为37℃,使得主发酵2d,发酵结束后测定各指标。
步骤3:进行糖酸比的测定。
糖酸比失重测定方式:接菌之后先测定每个发酵三角瓶的初始糖酸比,之后每隔12h再测定一次总酸和总糖,直到糖酸比保持不变则意味着发酵完成。
表2酵母菌混种发酵实验结果
根据表2显示,葡萄汁有孢汉逊酵母MY-00与植物乳杆菌MY-12的最适发酵方式为按1:1同时接种,该发酵方式下最终产品的糖酸比最接近20,接近饮料最优感官糖酸比和符合对产品的需求。
步骤4:生长曲线与感官评定。
对红枣汁分别单独接种5×107CFU/mL MY-12菌株和同时接种5×105CFU/mL葡萄汁有孢汉逊酵母MY-00与5×107CFU/mL的植物乳杆菌MY-12的发酵产物进行不同发酵时间的活菌数检测与可滴定酸检测,单一菌种发酵与乳酸菌葡萄汁有孢汉逊酵母共培养发酵生长曲线如图5所示,MY-12单一乳酸菌发酵红枣汁在12h后活菌数保持在9.5log CFU/mL左右,而通过添加葡萄汁有孢汉逊酵母MY-00后发现发酵体系中,发酵结束时间会提前,且乳酸菌的活菌数达到了10.5log CFU/mL。由此可以得到,葡萄汁有孢汉逊酵母MY-00可通过混菌发酵达到提升益生菌活性同时提高其活菌数的效果。
对以上发酵48h的红枣发酵产物进行感官评定,结果如图6所示。由图可知添加了葡萄汁有孢汉逊酵母MY-00发酵在香气方面改善明显。对比单菌发酵,混菌发酵结果的水果味与花香明显,使红枣汁发酵特征与典型性得到增强。同时,添加了MY-00使得酸感减弱,单宁感增强,涩味的添加让口感也更加丰富,颜色更加浑浊,但是总体接受度会得到提升。
步骤5:发酵产物成分分析。
分别对以上单独接种MY-12菌株和同时接种葡萄汁有孢汉逊酵母MY-00与植物乳杆菌MY-12的发酵产物进行分析,分析方法采用GC-MS技术检测红枣乳酸菌饮料中的风味物质。
由MY-12菌株单独发酵红枣乳酸菌饮料的风味物质分析以及MY-00与MY-12混菌发酵红枣乳酸菌饮料的风味物质分析可知,混菌发酵新增了一些风味物质,这些物质对增加红枣乳酸菌饮料香气的贡献很大,结果如下表所示,比如相对含量0.02%的乙酸异丁酯(成熟水果香味)相对含量0.03%的丙酸乙酯(菠萝香味)、相对含量0.02%的反-2-庚烯醛(青草香气)、相对含量0.11%的乙酸苏合香酯(强烈的绿叶以及栀子花香)、相对含量0.03%的1,3-二叔丁基苯(芳香气味)和相对含量0.02%的十二醛(松叶油和橙油的强烈香气);同时,乙酸乙酯相对含量提高了9.28倍,乙酸异戊酯相对含量提高了6.53倍,己酸乙酯相对含量提高了4.63倍,辛酸乙酯相对含量提高了4.68倍。这些气味使得红枣乳酸菌饮料的风味层次增多,构建了红枣乳酸菌饮料特征风味,对于增加饮料香气的平衡性、复杂性和典型性具有重要的作用。
表3发酵产物挥发性物质分析
实施例3:含有葡萄汁有孢汉逊酵母MY-00的微生物菌剂的制备
(1)挑取葡萄汁有孢汉逊酵母MY-00的单菌落接种于10mL YPD液体培养基中,28℃下活化2至3代,待葡萄汁有孢汉逊酵母MY-00达到107cfu/mL以上活菌数时,5000~10000rpm下离心10~20min,去除上清液后,在无菌环境下依次加入缓冲液和冻干保护剂,待细胞浓度不低于105cfu/mL时,真空冷冻干燥处理得到固体菌剂。
(2)挑取葡萄汁有孢汉逊酵母MY-00的单菌落接种于10mL YPD液体培养基中,28℃下培养2至3代;挑取植物乳杆菌MY-12的单菌落接种于10mL MRS液体培养基,37℃培养2至3代;调节葡萄汁有孢汉逊酵母MY-00为107cfu/mL以上和植物乳杆菌MY-12的菌浓不低于109cfu/mL时,将两者等比例混合,5000~10000rpm下离心10~20min,去除上清液后,在无菌环境下依次加入缓冲液和冻干保护剂,待细胞浓度不低于106cfu/mL时,真空冷冻干燥处理得到固体菌剂。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一株葡萄汁有孢汉逊酵母(Hanseniaspora uvarum),已于2021年06月28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.22784。
2.含有权利要求1所述的葡萄汁有孢汉逊酵母的微生物菌剂。
3.根据权利要求2所述的微生物菌剂,其特征在于,所述葡萄汁有孢汉逊酵母不低于106cfu/mL或106cfu/g。
4.根据权利要求2或3所述的微生物菌剂,其特征在于,还含有植物乳杆菌,所述植物乳杆菌已于2021年12月13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.24097。
5.一种制备红枣乳酸菌饮料的方法,其特征在于,以红枣汁为原料,利用权利要求1所述葡萄汁有孢汉逊酵母或权利要求2~3任一所述微生物菌剂和植物乳杆菌发酵生产红枣乳酸菌饮料,或者利用权利要求4所述微生物菌剂发酵生产红枣乳酸菌饮料。
6.根据权利要求5所述的方法,其特征在于,所述方法步骤为:
(1)将菌浓为1×106~6×106CFU/mL的所述葡萄汁有孢汉逊酵母与菌浓为1×108~6×108CFU/mL的所述植物乳杆菌等比例混合得到混合菌液;
(2)将混合菌液按照发酵总体积的5%~10%添加至红枣汁中;
(3)在35~40℃下主发酵2~3天。
7.根据权利要求6所述的方法,其特征在于,将菌浓为5×106CFU/mL的所述葡萄汁有孢汉逊酵母与菌浓为5×108CFU/mL的所述植物乳杆菌等比例混合得到混合菌液。
8.根据权利要求7所述的方法,其特征在于,在37℃下主发酵2天。
9.利用权利要求5~8任一所述方法制备得到的红枣乳酸菌饮料。
10.权利要求1所述植物乳杆菌或权利要求2或3所述的微生物菌剂在制备发酵产品中的应用。
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