CN117448183A - 马克斯克鲁维酵母菌km-20及其在樱桃番茄发酵果汁中的应用 - Google Patents
马克斯克鲁维酵母菌km-20及其在樱桃番茄发酵果汁中的应用 Download PDFInfo
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- CN117448183A CN117448183A CN202311525578.4A CN202311525578A CN117448183A CN 117448183 A CN117448183 A CN 117448183A CN 202311525578 A CN202311525578 A CN 202311525578A CN 117448183 A CN117448183 A CN 117448183A
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Abstract
本发明属于微生物技术领域,具体涉及马克斯克鲁维酵母菌KM‑20及其在樱桃番茄发酵果汁中的应用。本发明公开了一株适用于发酵樱桃番茄汁的酵母菌KM‑20,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.27835。该菌分离于宁夏产区葡萄酒自然发酵过程中。该菌可改善乳酸菌单一发酵果蔬汁香气不足、生青味重的缺点,如樱桃番茄汁,在与植物乳杆菌HY‑1(保藏编号为CGMCC No.23619)混合接种樱桃番茄汁并发酵48h后生成大量有机酸,同时抗氧化活性和香气物质增加,生青味减少,使樱桃番茄发酵果汁的品质得到很大提升。
Description
技术领域
本发明属于微生物技术领域,涉及马克斯克鲁维酵母菌KM-20及其在樱桃番茄发酵果汁中的应用。
背景技术
樱桃番茄(Lycopersicon esculentum var.cerasiforme A.Gray),是一年生草本植物,属茄科番茄属。具有生津止渴、健胃消食、清热解毒、凉血平肝,补血养养血和增进食欲的功效。黄丽华曾对樱桃番茄果实营养成分进行分析,内约含有13种维生素以及17种矿物质,其中维生素C质量分数最高可达66mg/g,约是普通番茄的1.7倍。成熟的圣女果中富含葡萄糖、果糖;柠檬酸、苹果酸,还有少量醋酸、乳酸和富马酸。此外,樱桃番茄还含有番茄红素、多酚类、黄酮类等功能成分,其中抗坏血酸、番茄红素和还原型谷胱甘肽的含量均高于普通番茄,具有更高的抗氧化活性。
近年来,随着人们健康意识的提高,绿色、天然的果蔬制品越来越受到消费者的青睐,发酵果蔬汁就以其独特的品质优势脱颖而出。随着研究深入,学者发现与单一菌种发酵果蔬汁相比,多种菌种协同发酵可以使发酵果蔬汁风味更加丰富,营养更加均衡。目前,混合发酵菌剂逐渐受到研究人员的关注。混菌发酵果蔬汁借鉴“果蔬酵素”的发酵方式,以一种或多种果蔬(果蔬汁)为原料,利用多菌种发酵,如:乳酸菌、酵母菌、醋酸菌等,生产富含多种营养物质并具有多种功能性的饮料。果蔬汁经多菌种协同发酵,其风味、营养以及贮藏品质获得改善。发酵果蔬汁中的微生物,通过利用原料中的营养物质进行自身物质代谢,微生物与微生物、微生物与底物之间的相互作用以及多种复杂的代谢过程,促进多种生物活性物质的产生,如有机酸、蛋白酶、多酚类物质等,可以提升果蔬汁的风味、口感、营养价值和货架期等各方面性能。
发明内容
本发明的一个目的是提供一种马克斯克鲁维酵母KM-20,(Kluyveromycesmarxianus KM-20),该菌具有很强改善发酵果蔬汁香气的能力。
本发明的另一个目的是提供上述马克斯克鲁维酵母KM-20在混合发酵果蔬汁中的应用。
本发明提供一株马克斯克鲁维酵母KM-20,该菌已于2023年07月07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,地址:中国北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.27835。
本发明的马克斯克鲁维酵母KM-20(CGMCC No.27835)于宁夏产区葡萄酒自然发酵过程中分离获得,采用16S rDNA基因组测序法鉴定马克斯克鲁维酵母。
本发明的马克斯克鲁维酵母KM-20(CGMCC No.27835)的菌落特征为圆形、直径2~4mm、白色、透明、表面光滑,在固体培养基表面呈突起状态且边缘整齐平滑。
本发明的马克斯克鲁维酵母KM-20(CGMCC No.27835)在较高酸度环境下生长良好,在YPD液体培养基中生长24h即可达到最大OD600。
本发明还提供了菌株组合,包括所述的马克斯克鲁维酵母菌(Kluyveromycesmarxianus)KM-20和植物乳杆菌(Lactobacillus plantarum)HY-1,所述植物乳杆菌(Lactobacillus plantarum)HY-1的保藏编号为CGMCCNo.23619。
基于上述研究,本发明还提供了所述马克斯克鲁维酵母菌(Kluyveromycesmarxianus)KM-20或所述菌株组合在制备樱桃番茄发酵果汁中的应用。
在本发明的一些具体实施方案中,将活化后得到接种菌液离心处理并用无菌水反复洗涤,接种于经巴氏杀菌后迅速冰水冷却得到的待发酵樱桃番茄中,所述马克斯克鲁维酵母菌(Kluyveromyces marxianus)KM-20接种量为1×106CFU/mL,植物乳杆菌(Lactobacillus plantarum)HY-1接种量为1×107CFU/mL,在28℃恒温条件下静置发酵48h;
所述马克斯克鲁维酵母菌(Kluyveromyces marxianus)KM-20的活化:-80℃下50%甘油保藏的菌液在YPD琼脂培养基上划线,28℃静置培养48~72h得到单菌落,用接种环挑取单菌落于YPD液体培养基,28℃静置培养24h得到接种菌液。所述植物乳杆菌(Lactobacillus plantarum)HY-1的活化:-80℃下50%甘油保藏的菌液在MRS琼脂培养基上划线,37℃静置培养48~72h得到单菌落,用接种环挑取单菌落于MRS肉汤培养基,37℃静置培养24h得到接种菌液。
与现有技术相比,本发明的有益效果在于:
本发明的马克斯克鲁维酵母KM-20(CGMCC No.27835)具有较快的生长速度且产香气能力较强,可用于相关用途。本发明实施例具体提供了该菌与植物乳杆菌HY-1(保藏编号为CGMCC No.23619)混合发酵在樱桃番茄汁中的应用。
本发明的马克斯克鲁维酵母KM-20耐酸性较强、生长繁殖速度快、发酵的樱桃番茄汁抗氧化活性增强、挥发性香气化合物含量增多、生青味减少,感官品质得到很大改善。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1为本发明马克斯克鲁维酵母KM-20的菌落;
图2为本发明马克斯克鲁维酵母KM-20与植物乳杆菌HY-1混合发酵时,益生菌植物乳杆菌HY-1的活菌数;
图3为本发明马克斯克鲁维酵母KM-20与植物乳杆菌HY-1混合发酵时的pH;
图4为本发明马克斯克鲁维酵母KM-20与植物乳杆菌HY-1混合发酵后发酵樱桃番茄汁后的总酚含量;
图5为本发明马克斯克鲁维酵母KM-20与植物乳杆菌HY-1混合发酵樱桃番茄汁后的DPPH自由基清除能力;
图6为本发明马克斯克鲁维酵母KM-20与植物乳杆菌HY-1混合发酵樱桃番茄汁后的总抗氧化能力(TAOC);
图7为本发明马克斯克鲁维酵母KM-20与植物乳杆菌HY-1混合发酵樱桃番茄汁后的感官评价。
生物保藏说明
生物材料:KM-20,分类命名:马克斯克鲁维酵母(Kluyveromyces marxianus),于2023年07月07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(CGMCC),保藏中心地址为: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,保藏编号为CGMCCNo.27835;
生物材料:HY-1,分类命名:植物乳杆菌(Lactobacillus plantarum),于2021年10月18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(CGMCC),保藏中心地址为: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,保藏编号为CGMCC No.23619。
具体实施方式
下面结合附图和实施例对本发明进行进一步说明。
实施例1:马克斯克鲁维酵母KM-20的分离纯化及鉴定
宁夏贺兰山东麓产区提供的酿酒葡萄(品种为赤霞珠),该葡萄经自然发酵结束后,取酒样品稀释涂布于YPD琼脂培养基(添加100mg/L氯霉素),在28℃恒温培养箱中培养48~72h,挑取形态不同的菌落划线纯培养即得到分离菌株。酵母菌的鉴定采用16S rDNA基因组测序法,其中菌株基因组DNA提取方法为:挑取划线纯培养得到的单菌落在12,000r离心1min,弃去上清液,然后用酵母菌DNA基因组提取试剂盒按说明书进行菌株基因组DNA提取。16S rDNA基因PCR扩增方法为:将提取得到的菌株DNA利用正向引物NL1(GCATATCAATAAGCGGAGGAAAA)和反向引物NL4
(GGTCCGTGTTTCAAGACGG)对菌株DNA的16S rDNA基因区域在PCR扩增仪中扩增,PCR扩增体系及程序如表1所示。扩增产物用1%的琼脂糖凝胶电泳和凝胶成像仪进行检测,观察到约为1500bp的条带,说明PCR成功。然后送往上海生工进行单向测序。登陆NCBI网站运用BLAST程序将所得菌株16S rDNA基因序列在GenBank数据库中与已知细菌基因序列进行对比,所分离菌株与马克斯克鲁维酵母同源相似性大于99%,因此被确认为克思克鲁维酵母菌,命名为KM-20。马克斯克鲁维酵母菌KM-20的菌落特征如图1所示,菌落特征为圆形、直径2~4mm、白色、透明、表面光滑,在固体培养基表面呈突起状态且边缘整齐平滑。
鉴定成功的马克斯克鲁维酵母菌KM-20,挑取其划线纯培养得到的单菌落于YPD液体培养基中静置培养约24h至对数中期,以50%甘油作冷冻保护剂,培养液与50%甘油(1:1)混合加入保藏管,于-80℃保存。
将本发明获得的马克斯克鲁维酵母菌KM-20送交中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏日期为2023年07月07日,保藏编号为CGMCC No.27835。
表1PCR扩增体系及程序
实施例2:
本发明的马克斯克鲁维酵母菌KM-20可以有效改善乳酸菌单一发酵香气不足的缺点,且该酵母耐酸、生长速率快,果蔬汁的原料可以选自樱桃番茄、沙棘、柠檬、猕猴桃、青梅、山楂中的一种或多种。
本实施例以果蔬汁“樱桃番茄汁”为例。
1.樱桃番茄汁的制备:用自来水清洗樱桃番茄,剔除坏果、烂果,并去除番茄梗。沸水热烫处理然后用食品级榨汁机将樱桃番茄果肉压碎,用80目纱布过滤,然后立即加入0.08%的抗坏血酸以防止酶促褐变。将锥形瓶中的樱桃番茄汁80℃水浴加热15秒,使樱桃番茄汁中的多酚氧化酶失活。在室温(25℃)下静置冷却,然后储存在-20℃备用。
2.马克斯克鲁维酵母菌KM-20的活化:-80℃下50%甘油保藏的菌液在YPD琼脂培养基上划线,28℃静置培养48~72h得到单菌落,用接种环挑取单菌落于YPD液体培养基,28℃静置培养24h得到接种菌液。
3.植物乳杆菌HY-1的活化:-80℃下50%甘油保藏的菌液在MRS琼脂培养基上划线,37℃静置培养48~72h得到单菌落,用接种环挑取单菌落于MRS肉汤培养基,37℃静置培养24h得到接种菌液。
4.樱桃番茄汁的接种发酵:将解冻的樱桃番茄汁在80℃加热杀菌15分钟,并冷却到室温(25℃),然后将菌液加入樱桃番茄汁(500mL)中,设置三组平行。酵母菌KM-20接种量为1×106CFU/mL,植物乳杆菌HY-1接种量为1×107CFU/mL,乳酸菌、酵母菌菌液同时加入到樱桃番茄汁中,混匀后安装发酵栓,在30℃恒温条件下静置发酵48h。发酵结束后,在4℃冰箱中贮藏14天。
5.测定发酵樱桃番茄汁的理化指标:包括pH、可溶性固形物(Total solublesolids,TSS)、植物乳杆菌HY-1活菌数、还原糖、总酚、有机酸、葡萄糖、果糖和蔗糖,设置两组生物学重复。测定发酵樱桃番茄汁的颜色、抗氧化活性和挥发性香气,并进行感官品评。单独接种植物乳杆菌HY-1作为对照组,设置两组生物学重复。
(1)理化指标测定:利用MRS琼脂培养基(加入100mg/L放线菌酮)平板计数法(参考GB 4789.35-2016)测定植物乳杆菌HY-1活菌数以观察在酵母菌作用下植物乳杆菌HY-1的生长情况,植物乳杆菌HY-1的数量变化如图2所示,pH的测定使用pH计,取发酵0、24、48h的发酵果汁,单独接种植物乳杆菌HY-1作为对照组,设置两组生物学重复,pH变化如图3所示。PAL-1数显糖度计测定TSS值,如表2所示。以上指标测定结束后,将发酵液离心(12,000r×2min)后取上清液存放于-20℃冰箱中,待用。福林酚法测定发酵果汁中总酚的含量,如图4所示;DNS法测定总还原糖含量,如表2所示。
(2)主代谢产物测定:发酵液经0.22μm水相滤膜(PES)过滤后,进行葡萄糖、果糖及有机酸(柠檬酸、乳酸、酒石酸、琥珀酸、苹果酸及乙酸)氨基酸浓度的测定。使用安捷伦1200高效液相色谱仪,离子交换色谱柱为HPX-87H(Bio-Rad公司,美国)。使用5mmol/L的硫酸做流动相,并以0.6mL/min的流速进行等度洗脱。葡萄糖、果糖的测定使用示差折光检测器(安捷伦,美国),进样量为20μL,柱温45℃,测定时间为30min。每个样品设置两个技术平行。有机酸的测定采用紫外检测器(安捷伦,美国),进样量为20μL,柱温为60℃,测定时间为25min。每个样品设置两个技术平行。通过主发酵产物的出峰时间定性,根据主发酵产物标准品的标准曲线进行定量。
(3)樱桃番茄汁的抗氧化活性测定:使用DPPH(2,2-Diphenyl-1-picrylhydrazyl)自由基清除能力试剂盒和总抗氧化能力(T-AOC)检测试剂盒(FRAP法)测定(购自南京建成生物工程研究所),方法参考说明书。
(4)挥发性风味化合物测定:用Agilent 7890气相色谱(GC)和Agilent5975质谱(MS)联用仪(Agilent,美国)检测上述获得的发酵樱桃番茄果汁中各种挥发性香气物质种类和含量。具体条件为:毛细管柱HP-INNOWAX Polyethylene Glycol 60m×0.25mm×0.25μm(J&W scientific,美国)载气为高纯氦气,流速1mL/min;顶空固相微萃取自动进样,采用不分流模式,***气相色谱的进样口,温度250℃,热解析25min。柱温箱的升温程序是:40℃保持5min,然后以3℃/min的速度升温至200℃,保持2min。质谱口温度为280℃,离子源温度为230℃,电离方式EI,离子能量70ev。
(5)感官品评方法:15名具有相关经验和背景知识的教师和学生小组成员对樱桃番茄果汁的颜色、形态、香气、味道和甜度进行评价,评价结果如图7所示。
实验结果:
1.马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵樱桃番茄汁后的理化指标如表2所示。马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵(KM+HY)的樱桃番茄汁中活菌数、产酸能力、产多酚量和还原糖消耗量均高于植物乳杆菌HY-1(HY1)单独发酵的樱桃番茄汁。
表2马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵樱桃番茄汁后的理化指标
注:HY1为植物乳杆菌HY-1的缩写,KM+HY为马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵的缩写。小写字母(a~c)为表中的显著性差异(Duncan test,p<0.05)。
2.马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵樱桃番茄汁后的抗氧化活性如图5、6所示。发酵樱桃番茄汁的DPPH自由基清除能力和FRAP抗氧化活性较樱桃番茄原汁升高1.24倍和2.12倍,较植物乳杆菌HY-1单独发酵樱桃番茄果汁升高1.06倍和1.89倍,可能是混合发酵产生了更高含量的酚类化合物,而酚类化合物已被证明是良好的还原剂和自由基清除剂,主要与它们对自由基的氢原子转移或电子传递有关。马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵能够明显提高樱桃番茄汁的抗氧化活性。
3.马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵樱桃番茄汁后的挥发性化合物如表3所示。发酵樱桃番茄汁的总挥发性化合物是樱桃番茄原汁的2.90倍,是植物乳杆菌HY-1单独发酵的1.77倍。挥发性醇类、酯类、醛类、酮类和酸类分别是原汁的5.61倍、24.32倍、0.09倍、5.71倍和7.32倍,是植物乳杆菌HY-1单独发酵的12.64倍、7.53倍、1.06倍、1.14倍和1.42倍。混合发酵增加了植物乳杆菌HY-1单独发酵时不具备的己二酸二异丙酯和三甲基-1,3-戊二醇二异丁酯,且3-甲基-1-丁醇乙酸酯、L-乳酸甲酯、2-羟基丙酸乙酯、辛酸甲酯和辛酸乙酯的含量明显增多,这些酯类是樱桃番茄发酵果汁香气的主要贡献者,提供了层次丰富的果香、花香和酒香味。同时混合发酵樱桃番茄汁中己醇、壬醇、辛醇和苄醇等高级醇的含量增高,赋予发酵樱桃番茄汁花果香、油脂香和草木香等芳香味道。己醛、庚醛、苯甲醛、2-壬烯醛、1-环己烯-1-甲醛是发酵樱桃番茄汁的醛类特征香气,混合发酵使得其含量上升,赋予果汁更加浓郁的果香和青草香。戊酮、庚酮、壬酮等含量的增加则为发酵果汁提供了更加浓郁的甜香、果香等芳香味。另外发酵后丁香酚、呋喃、环己烯等物质,贡献了焦香、丁香花香、柑橘香和木制香料等复杂气味。同时发酵后甲苯、1,2,3-三甲基苯等具有刺激性气味的物质含量减少,这是混合发酵后生青味减弱的一个重要原因。总而言之,马克斯克鲁维酵母菌KM-20(CGMCC No.27835)与植物乳杆菌HY-1协同发酵,可以改善植物乳杆菌HY-1单独发酵时香气单一、生青味较重的情况,从而获得更加宜人的香气,更易被大众所接受。
表3植物乳杆菌HY-1发酵樱桃番茄汁后的挥发性化合物
注:结果为三个独立重复的平均值;标准误差均小于平均值的10%;ND:未检测到
4.马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵樱桃番茄汁后的感官评价如图7所示。马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵的樱桃番茄汁在香气、颜色、组织形态及口感较樱桃番茄原汁有所改善,大众接受度相对也更高。
综上所述,马克斯克鲁维酵母KM-20(CGMCC No.27835)具有良好的发酵潜力,可以作为与乳酸菌协同发酵的潜力菌种,且发酵后的樱桃番茄汁的品质得到提升。马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵改变了樱桃番茄在发酵过程中的产酸情况,产生了更多乳酸,减少了带有刺激性的其他有机酸含量。马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵后樱桃番茄汁的总酚含量增加、抗氧化活性增强,植物乳杆菌HY-1的活菌数增加,具有更高的营养价值和保健功能,更易被消费者和市场接受。马克斯克鲁维酵母菌KM-20与植物乳杆菌HY-1混合发酵发酵不仅保持了樱桃番茄汁的原有特征性酯类物质,同时苯、C6等生青味物质减少,还增加了大量的高级醇、醛、酮、酚、酯和其它萜烯类香气物质,使樱桃番茄发酵果汁增加了花果香以及其他宜人香气,整体香气更加浓郁,层次更丰富,掩盖了樱桃番茄原汁和植物乳杆菌HY-1单独发酵时的不良气味。感官评价结果也表明发酵樱桃番茄果汁的香气、组织形态、颜色和大众接受度均优于樱桃番茄原汁和植物乳杆菌HY-1单独发酵果汁。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种马克斯克鲁维酵母菌(Kluyveromyces marxianus)KM-20,其特征在于,其保藏编号为CGMCC No.27835。
2.菌株组合,其特征在于,包括如权利要求1所述的马克斯克鲁维酵母菌(Kluyveromyces marxianus)KM-20和植物乳杆菌(Lactobacillus plantaru m)HY-1,所述植物乳杆菌(Lactobacillus plantarum)HY-1的保藏编号为CGMCC No.23619。
3.如权利要求1所述的马克斯克鲁维酵母菌(Kluyveromyces marxianus)KM-20或如权利要求2所述的菌株组合在制备樱桃番茄发酵果汁中的应用。
4.根据权利要求3所述的应用,其特征在于,将活化后得到接种菌液离心处理并用无菌水反复洗涤,接种于经巴氏杀菌后迅速冰水冷却得到的待发酵樱桃番茄中,所述马克斯克鲁维酵母菌(Kluyveromyces marxianus)KM-20接种量为1×106CFU/mL,植物乳杆菌(Lactobacillus plantarum)HY-1接种量为1×107CFU/mL,在28℃恒温条件下静置发酵48h;
所述马克斯克鲁维酵母菌(Kluyveromyces marxianus)KM-20的活化:-80℃下50%甘油保藏的菌液在YPD琼脂培养基上划线,28℃静置培养48~72h得到单菌落,用接种环挑取单菌落于YPD液体培养基,28℃静置培养24h得到接种菌液;
所述植物乳杆菌(Lactobacillus plantarum)HY-1的活化:-80℃下50%甘油保藏的菌液在MRS琼脂培养基上划线,37℃静置培养48~72h得到单菌落,用接种环挑取单菌落于MRS肉汤培养基,37℃静置培养24h得到接种菌液。
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