CN110179031A - 酿酒酵母菌株以及起泡型苹果酵素饮料的生产方法 - Google Patents
酿酒酵母菌株以及起泡型苹果酵素饮料的生产方法 Download PDFInfo
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Abstract
本发明提供一株适宜苹果酵素饮料发酵、能够显著改善产品褐变的菌株以及相应的起泡型苹果酵素饮料的生产方法,本发明解决了苹果酵素生产过程中易发生褐变、生产工艺未标准化且以及产品安全隐患大的技术问题。
Description
技术领域
本发明属于食品加工技术领域;具体涉及一种酿酒酵母菌株,以及利用该酿酒酵母菌株结合二步混菌发酵法生产苹果酵素饮料的方法。
背景技术
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,是维持机体正常功能、消化食物、修复组织等生命活动的一种必需物质。随着认识的不断深入,目前所提及的酵素一般是指以果蔬为原料,通过自然发酵或者接种复合菌剂发酵而成、含有微生物及其代谢产物的物质体系。果蔬酵素能够保留果蔬原料的部分营养物质,微生物发酵还带来丰富的有益代谢产物,具有微生物的发酵香气与令人愉悦的果蔬香气。国内外相关研究表明,果蔬酵素具有消除自由基、抗肿瘤、抗衰老、提高机体免疫力、限制炎症、清除血管垃圾等功效,是发展前景较好的健康食品。
目前市场上流行的果蔬酵素产品一则价格较昂贵,二则发酵后经历了浓缩、过滤等多步处理过程,其活性物质部分损失;三则商品化的酵素产品因其独特特性,不宜进行高温杀菌,为保持适宜的货架期,需要添加部分防腐剂,消费者的认可度降低。目前自制果蔬酵素主要为自然发酵,依靠果蔬表面携带的微生物进行,制作的过程中稍有不慎,有害微生物就会快速繁殖,产生较多的生物毒素、亚硝酸盐、甲醇等有害代谢物,对健康造成威胁。筛选合适的原料与发酵菌株、建立精确的工艺、生产安全可靠的酵素产品是产业的发展需求。
中国是苹果生产和出口大国,栽培面积、总产量、人均占有量与出口量均居世界第一,其口感香甜,营养丰富,自古就有“一天一苹果,医生远离我”的谚语,现代研究更是明确了苹果全营养提取物的长寿功效,是生产益生菌发酵酵素的良好原料。褐变是苹果在加工或储存过程中颜色由诱人的金黄色逐渐转变为发暗的红棕色过程,主要包括酶促褐变和非酶褐变,一直是困扰苹果加工的重要问题,也是果蔬加工业普遍存在的关键性技术难题之一。酶促褐变是由于在打浆、取汁等过程中,酶与底物的细胞区域化被打破,果蔬中的多酚氧化酶(PPO)通过氧气介导催化酚类物质氧化从而引起变色,是主要的褐变形式。非酶褐变包括多酚氧化聚合反应、美拉德反应、焦糖化反应等,其中美拉德反应和多酚氧化聚合反应是苹果汁非酶褐变的主要形式,其还原糖和氨基酸都是果蔬汁本身所含的成分,因此较难控制。
果汁发生褐变时感官品质劣化,营养价值降低,商品价值也随之下降。从业人员做了许多研究,分别从选料、添加其他物质、去除部分酚类物质等方面来减轻褐变。加热灭酶是防止酶促褐变的一种传统方法,但果汁在打浆、取汁等工序中,已经发生了部分酶促褐变,因此加热并不能完全消除酶促褐变,且过分加热会造成营养物质的破坏,从而促进非酶褐变。随着公众健康意识的增强,添加剂的使用受到严格的限制,食品安全性已越来越受关注,利用生物方法抑制果蔬褐变,已经成为果蔬加工行业的研究热点。
发明内容
本发明的目的之一在于筛选出一株适宜苹果酵素饮料发酵、能够显著改善产品褐变的菌株,该菌株解决了苹果酵素生产过程中易褐变的问题。
基于本发明提供的菌株,本发明的另一个目的在于建立乳酸菌混菌与酵母菌混合发酵的酵素饮料生产方式,从而解决苹果酵素生产工艺未标准化以及产品安全隐患大的技术问题。具体地,提供一种采用二步混菌发酵生产起泡型苹果酵素饮料的方法。
本发明的再一个目的在于提供一种起泡型混菌发酵苹果酵素饮料。
为实现上述发明目的,本发明提供一种酿酒酵母菌株,该菌株的分类命名:酿酒酵母Saccharomyces cerevisiae,生物株:AP 68,命名为S.cerevisiae AP 68,于2018年6月4日保藏于“中国微生物菌种保藏管理委员会普通微生物保藏中心”,保藏号为CGMCCNo.15839。
本发明提供的一种采用二步混菌发酵生产起泡型苹果酵素饮料的方法,其中,该方法包括如下步骤:以苹果原汁为原料,以乳酸菌混菌对苹果原汁进行第一步发酵,后采用安琪葡萄酒酵母与上述所述的酿酒酵母菌株S.cerevisiae AP 68的混菌进行第二步发酵,过滤获得起泡型苹果酵素饮料。
在本发明一些实施方式中,所述乳酸菌混菌包括第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌。优选地,所述的第一种植物乳杆菌保藏号为CICC 20022;所述植物乳杆菌植物亚种保藏号为CICC 20242;所述干酪乳杆菌保藏号为CICC 20296;所述类干酪乳杆菌保藏号为CICC 20251;所述鼠李糖乳杆菌保藏号为CICC 6012;所述第二种植物乳杆菌保藏号为CGMCC No.11936。
在本发明一些优选地实施方式中,本发明提供的一种采用二步混菌发酵生产起泡型苹果酵素饮料的方法,包括如下步骤:
(1)果汁的制备与复配:选择无腐烂、无病虫害的苹果果实,清水洗净,沥水晾干,切块,热烫,凉开水冲洗降温,榨汁,过滤去除少量残留颗粒与固体得苹果果汁;以蔗糖调整可溶性固形物浓度至120~200g/L,调节pH值至4.2~6.0,升温至60~80℃保持15~40min,获得苹果原汁备用;
(2)乳酸菌菌株的活化与复配:将第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌分别接种于一级苹果汁改良MRS培养基中,26~35℃厌氧培养48~90h后,按接种量10~50mL/L分别接入二级苹果汁改良MRS培养基中,28~32℃厌氧培养60~72h;
其中,所述一级苹果汁改良MRS培养基为:苹果汁200~400mL/L,去离子水600~800mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温801mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5~7,121℃灭菌20min;
所述二级苹果汁改良MRS培养基为:苹果汁500~700mL/L,去离子水300~500mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温801mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为4~6,121℃灭菌20min;
(3)乳酸菌发酵:将步骤2)活化好的第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌按照重量比例1~3:1~3:1~3:0.5~2:0.5~2:0.5~1.5混合后以10~50mL/L的接种量接入步骤(1)苹果原汁中,进行液体深层发酵,控制发酵温度为26~35℃,发酵时间12~60h;
(4)酵母菌的活化与扩大:将安琪葡萄酒酵母与上述所述的酿酒酵母菌株S.cerevisiae AP68分别接种于一级苹果汁培养基,28~32℃培养60~72h;然后按接种量10~50mL/L分别接入二级苹果汁培养基中,20~30℃培养48~96h后作为种子培养基应用;
其中,所述一级苹果汁培养基为:苹果汁200~400mL/L,去离子水600~800mL/L,葡萄糖50~100g/L,酵母粉3~8g/L,pH值为4~6,121℃灭菌20min;
所述二级苹果汁培养基为:苹果果汁500~800mL/L,去离子水200~500mL/L,葡萄糖100~150g/L,酵母粉1~2g/L,pH值为4~6,121℃灭菌20min;
(5)酵母菌平静态发酵:将步骤(4)活化后的安琪葡萄酒酵母与所述的酿酒酵母菌株S.cerevisiae AP 68按重量比例2:1~1:3混合,以按0.2~5mL/L的接种量接入步骤(3)得到的果汁发酵液中,再次进行液体深层发酵,控制发酵温度为20~32℃,发酵时间12~60h,期间液面无剧烈气泡溢出,保持相对稳定的发酵状态,获得苹果酵素原液;
(6)降温及后续处理:去除苹果酵素的下层沉淀,上清液置于2~8℃、避光处保存2~5d后精滤获得起泡型苹果酵素饮料。
在本发明一些具体实施方式中,所述步骤(3)中活化后的第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌的混合体积比例为2:2:2:1.5:1.5:1。
在本发明一些具体实施方式中,所述步骤(5)中活化后的安琪葡萄酒酵母与S.cerevisiae AP 68按体积比例1:2混合。
在本发明一些具体实施方式中,所述步骤(6)获得的起泡型苹果酵素饮料经60~85℃灭菌25~33min后灌装、封口。
在本发明一些具体实施方式中,所述一级苹果汁改良MRS培养基为:苹果汁300mL/L,去离子水700mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温801mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.5,121℃灭菌20min;所述二级苹果汁改良MRS培养基为:苹果汁600mL/L,去离子水400mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温801mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.0,121℃灭菌20min。
在本发明一些具体实施方式中,所述一级苹果汁培养基为:苹果汁300mL/L,去离子水700mL/L,葡萄糖80g/L,酵母粉5g/L,pH值为5.5,121℃灭菌20min;所述二级苹果汁培养基为:苹果汁600mL/L,去离子水400mL/L,葡萄糖120g/L,酵母粉1.5g/L,pH值为5.0,121℃灭菌20min。
本发明以苹果原汁为原料,以乳酸菌混菌与酿酒酵母(安琪葡萄酒酵母与S.cerevisae AP68)进行二步发酵,经果汁的制备与复配、菌株的活化与扩大、乳酸菌发酵、酵母菌平静态发酵、降温储存、过滤、密封、杀菌工艺获得了起泡型苹果酵素饮料。
与现在技术相比,本发明具有以下有益效果:
(1)改善色泽,加速澄清:苹果汁营养丰富,口感好香气足,但极易发生褐变,在不加化学试剂的情况下,仅靠物理方法很难控制,即使经过热烫,榨汁后也会马上发生褐变,最终产品呈现暗淡的咖啡色,影响产品的感官品质与消费者的购买欲望。从自然发酵苹果原酒中筛选获得酿酒酵母S.cerevisiaeAP 68菌株,经其发酵后能够明显改良酵素饮料的色泽,从暗淡的咖啡色变为浅黄色,并加速了发酵液中大分子物质与固体颗粒的沉降,增加了上清液的澄清度,降低后续精滤的工作量,产品的感官品质大大增加。
(2)增加营养因子,增强健康功效:一般果蔬发酵汁以乳酸菌家族单菌株发酵为主,营养因子主要是短链脂肪酸;本发明选择乳酸菌混菌组合,不仅能够产生短链脂肪酸,还能产生多糖、氨基酸等营养物质,在此基础上,本发明进一步增加酵母发酵流程,能够赋予发酵液B族维生素、氨基酸、葡聚糖、甘露聚糖等营养因子,其健康功效进一步增强。
(3)在保证安全的前提下调整肠道菌群:对所得酵素饮料的急毒性、亚慢性毒性及肠道微生物影响进行了测试。小鼠的急性经口毒性试验结果表明,该酵素饮料属无毒物;大鼠的90d经口亚慢性毒性试验表明,苹果酵素饮料对大鼠无亚慢性全身毒性反应。90d饲喂过程中,苹果酵素饮料可以促进大鼠肠道有益菌(主要是乳杆菌)增殖,抑制病原菌生长。
(4)研发新品,增加发酵果蔬汁的种类:一般果蔬发酵汁以乳酸菌发酵为主,其口感以酸甜爽口为主,本发明以纯果汁为原料,在发酵第二阶段有控制的叠加酵母发酵,产品风味清香,口感酸甜略带刺激感和气泡的爽口感,适合年轻人的口感与消费观念,是一款特征鲜明的二步发酵酵素饮品,为果蔬发酵品家族增加了新的品种。
附图说明
图1为本发明提供的S.cerevisiae AP 68菌株的菌落与细胞形态;
图2为不同发酵情况下苹果酵素饮料的色泽变化示意图。
具体实施方式
本发明将就下面实施例来作进一步说明,但应了解的是,该等实施例仅是用来作为例示说明,而不应被解释为本发明的实施上的限制。
实施例1.酿酒酵母菌株的筛选与鉴定
酿酒酵母菌株的筛选:选择无新鲜无损害的苹果,以无菌刀具切块后,称取10g接入装有90mL无菌生理盐水、50颗无菌玻璃珠的三角瓶,振摇混合60min,然后以无菌生理盐水进行梯度稀释,分别取10~1、10-3及10-5的稀释液涂布于PDA培养基上,28℃微好氧培养48h;取菌落形成单位在10~50之间的培养皿,按50%比例挑取单菌落进行TTC显色实验;选择显色红色菌落进行划线分离,培养并镜检,选择菌体为圆形或椭圆形的纯菌落,接入苹果汁中进行发酵实验,研究其发酵性能、防褐变性能、传代稳定性等,选择发酵风味好、防褐变效果好、传代稳定性高的菌株,进行酒精转化率、二氧化硫耐受性试验,获得所得精酿鲜饮风味最佳,防褐变效果强,产品澄清度高等综合性能最佳的AP68菌株,作为起泡型苹果酵素饮料的酿造菌株。
酿酒酵母菌株的鉴定:本发明提供的酿酒酵母菌株鉴定:VITEK2COMPACT梅里埃全自动菌种鉴定仪的运行结果表明,本发明提供的菌株为酿酒酵母(S.cerevisae),置信区间为99%;18S rDNA序列分析的结果表明,AP 68与S.cerevisiae菌株的部分18S rDNA核苷酸序列同源性达到99%。该菌株的分类命名:酿酒酵母S.cerevisiae,生物株:AP 68,于2018年6月4日保藏于北京市朝阳区北辰西路,中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物保藏中心,保藏号为CGMCC No.15839。
本发明提供酿酒酵母菌株具有如下特征:耐受140mL/L的酒精,220×10-6mg/L的偏重亚硫酸氢钾,105×10-6mg/L的二氧化硫。在PDA培养基上生长良好,28℃静置培养42h形成明显菌落,菌落直径在1~5.0mm之间,规则圆形,乳白色,不透明,表面较湿润;镜检呈椭圆形,出芽,单个或者成对或成成链存在,可被美兰染色(参考图1)。
表1列出了S.cerevisiae AP 68菌株的生理生化性质。
表1
底物 | 结果 | 底物 | 结果 | 底物 | 结果 | 底物 | 结果 |
LysA | - | lMLTa | - | LeuA | + | ARG | + |
ERYa | - | GLYLa | - | TyrA | - | BNAG | - |
ARBa | - | AMYa | - | dGALa | + | GENa | - |
dGLUa | + | LACa | - | MAdGa | - | dCELa | - |
GGT | - | dMALa | + | dRAFa | + | NAGA1 | - |
dMNEa | + | dMELa | + | dMLZa | - | lSBEa | - |
lRHAa | - | XLTa | - | dSORa | - | SACa | + |
URE | - | AGLU | - | dTURa | + | dTREa | + |
NO3a | - | lARAa | - | dGATa | (﹣) | ESC | - |
lGLTa | - | dxyLa | - | LATa | + | ACEa | + |
CITa | - | GRTas | - | lPROa | - | 2Kga | - |
NAGa | - | dGNTa | - |
备注:“﹢”为阳性,“﹣”为阴性,“(﹢)”为弱阳性,“(﹣)”为弱阴性
实施例2.二步混菌发酵生产苹果酵素饮料
一种起泡型混菌发酵苹果酵素饮料的制备方法,包括如下步骤:
(1)果汁的制备与复配:选择无腐烂、无病虫害的新鲜苹果果实,清水洗净,沥水晾干,切块,90℃热烫1min,凉开水冲洗降温,榨汁后以200目无菌滤布去除少量残留颗粒与固体后得苹果果汁;以蔗糖调整可溶性固形物浓度至120~200g/L,调节pH值至4.2~6.0,升温至60~80℃保持20~40min,获得苹果原汁备用;
(2)乳酸菌菌株的活化与复配:将第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌分别接种于一级苹果汁改良MRS培养基,26~35℃厌氧培养48~90h,按50mL/L的接种量分别接入二级苹果汁改良MRS培养基中,28~32℃厌氧培养60~72h;
其中,所述第一种植物乳杆菌保藏号为CICC 20022;所述植物乳杆菌植物亚种保藏号为CICC20242;所述干酪乳杆菌保藏号为CICC 20296;所述类干酪乳杆菌保藏号为CICC20251;所述鼠李糖乳杆菌保藏号为CICC 6012,购于中国工业微生物菌种保藏管理中心;所述第二种植物乳杆菌为浙江省农业科学院的实验室保藏菌种,实验室编号为SZS001,来源于自然酸化水煮笋,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.11936;
所述一级苹果汁改良MRS培养基为:苹果汁300mL/L,去离子水700mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.5,121℃灭菌20min;
所述二级苹果汁改良MRS培养基为:苹果汁600mL/L,去离子水400mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.0,121℃灭菌20min。
(3)乳酸菌发酵:将上述活化好的乳酸菌按照2:2:2:1.5:1.5:1的重量比例混合作为种子,按10~50mL/L的接种量接入步骤(1)果汁原汁中,进行液体深层发酵,控制发酵温度为26~35℃,发酵时间为12~60h;
(4)酵母菌的活化与扩大:将安琪葡萄酒酵母与实施例1获得酿酒酵母菌株S.cerevisiae AP 68分别接种于一级苹果汁培养基,28℃培养48h,按接种量50mL/L接入二级苹果汁培养基中,25℃培养72h后作为种子培养基应用;
其中,所述一级苹果汁培养基为:苹果汁300mL/L,去离子水700mL/L,葡萄糖80g/L,酵母粉5g/L,pH值为5.5,121℃灭菌20min;
所述二级苹果汁培养基为:苹果汁600mL/L,去离子水400mL/L,葡萄糖120g/L,酵母粉1.5g/L,pH值为5.0,121℃灭菌20min;
(5)酵母菌平静态发酵:将步骤(4)活化扩大后的安琪葡萄酒酵母与S.cerevisiaeAP 68按照2:1的比例混合,以按0.2~5mL/L的接种量接入步骤(3)得到的果汁处理液中,进行液体深层发酵,控制发酵温度为20~32℃,厌氧发酵12~60h,期间发酵面无剧烈气泡溢出,保持相对稳定的发酵状态;
(6)降温及后续处理:将步骤(5)的苹果酵素去除下层沉淀,上清液置于2~8℃、避光处保存2~5d后精滤,家庭制备可置于冰箱中直接饮用,或者经60~85℃灭菌25~33min后灌装、封口,即成成品起泡型苹果酵素饮料。
实施例3.二步混菌发酵生产苹果酵素饮料
一种起泡型混菌发酵苹果酵素饮料的制备方法,包括如下步骤:
(1)果汁的制备与复配:选择无腐烂、无病虫害的新鲜苹果果实,清水洗净,沥水晾干,切块,90℃热烫1min,凉开水冲洗降温,榨汁后以200目无菌滤布去除少量残留颗粒与固体后得苹果果汁;以蔗糖调整可溶性固形物浓度至120~200g/L,调节pH值至4.2~6.0,升温至60~80℃保持20~40min,获得苹果原汁备用;
(2)乳酸菌菌株的活化与复配:将第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌分别接种于一级苹果汁改良MRS培养基,26~35℃厌氧培养48~90h,按50mL/L的接种量分别接入二级苹果汁改良MRS培养基中,28~32℃厌氧培养60~72h后;
其中,所述第一种植物乳杆菌保藏号为CICC 20022;所述植物乳杆菌植物亚种保藏号为CICC 20242;所述干酪乳杆菌保藏号为CICC 20296;所述类干酪乳杆菌保藏号为CICC 20251;所述鼠李糖乳杆菌保藏号为CICC 6012,购于中国工业微生物菌种保藏管理中心;所述第二种植物乳杆菌为浙江省农业科学院的实验室保藏菌种,实验室编号为SZS001,来源于自然酸化水煮笋,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.11936;
所述一级苹果汁改良MRS培养基为:苹果汁300mL/L,去离子水700mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.5,121℃灭菌20min;
所述二级苹果汁改良MRS培养基为:苹果汁600mL/L,去离子水400mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.0,121℃灭菌20min;
(3)乳酸菌发酵:将上述活化好的乳酸菌按照2:2:2:1.5:1.5:1的重量比例混合作为种子,按10~50mL/L的接种量接入步骤(1)所述果汁原汁中,进行液体深层发酵,控制发酵温度为26~35℃,发酵时间12~60h;
(4)酵母菌的活化与扩大:将安琪葡萄酒酵母与实施例1获得酿酒酵母菌株S.cerevisiae AP 68分别接种于一级苹果汁培养基,28℃培养48h,按接种量50mL/L接入二级苹果汁培养基中,25℃培养72h后作为种子培养基应用;
其中,所述一级苹果汁培养基为:苹果汁300mL/L,去离子水700mL/L,葡萄糖80g/L,酵母粉5g/L,pH值为5.5,121℃灭菌20min;
所述二级苹果汁培养基为:苹果汁600mL/L,去离子水400mL/L,葡萄糖120g/L,酵母粉1.5g/L,pH值为5.0,121℃灭菌20min;
(5)酵母菌平静态发酵:将步骤4)活化扩大后的安琪葡萄酒酵母与S.cerevisiaeAP 68按照1:2的比例混合,以按0.2~5mL/L的接种量接入步骤(3)得到的果汁处理液中,进行液体深层发酵,控制发酵温度为20~32℃,厌氧发酵12~60h,期间发酵面无剧烈气泡溢出,保持相对稳定的发酵状态;
(6)降温及后续处理:将步骤(5)的苹果酵素去除下层沉淀,上清液置于2~8℃、避光处保存2~5d后精滤,家庭制备可置于冰箱中直接饮用;或者经60~85℃灭菌25~33min后灌装、封口,即成成品起泡型苹果酵素饮料。
对比例1.乳酸混菌一步发酵生产苹果酵素饮料
一种发酵苹果酵素饮料的制备方法,包括如下步骤:
(1)果汁的制备与复配:选择无腐烂、无病虫害的新鲜苹果果实,清水洗净,沥水晾干,切块,90℃热烫1min,凉开水冲洗降温,榨汁后以200目无菌滤布去除少量残留颗粒与固体后得苹果果汁。以蔗糖调整可溶性固形物浓度至120~200g/L,调节pH值至4.2~6.0,升温至60~80℃保持20~40min,获得苹果原汁备用;
(2)乳酸菌菌株的活化与复配:将第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌分别接种于一级苹果汁改良MRS培养基,26~35℃厌氧培养48~90h,按50mL/L的接种量分别接入二级苹果汁改良MRS培养基中,28~32℃厌氧培养60~72h;
其中,所述第一种植物乳杆菌保藏号为CICC 20022;所述植物乳杆菌植物亚种保藏号为CICC 20242;所述干酪乳杆菌保藏号为CICC 20296;所述类干酪乳杆菌保藏号为CICC 20251;所述鼠李糖乳杆菌保藏号为CICC 6012,购于中国工业微生物菌种保藏管理中心;所述第二种植物乳杆菌为浙江省农业科学院的实验室保藏菌种,实验室编号为SZS001,来源于自然酸化水煮笋,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.11936;
所述一级苹果汁改良MRS培养基为:苹果汁300mL/L,去离子水700mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.5,121℃灭菌20min;
所述二级苹果汁改良MRS培养基为:苹果汁600mL/L,去离子水400mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.0,121℃灭菌20min;
(3)乳酸菌发酵:将上述活化好的乳酸菌按照2:2:2:1.5:1.5:1的重量比例混合作为种子,按10~50mL/L的接种量接入步骤(1)所述果汁中,进行液体深层发酵,控制发酵温度26~35℃之间,发酵时间24~120h得到苹果酵素;
(4)降温及后续处理:将步骤(3)的苹果酵素去除下层沉淀,上清液置于2~8℃、避光处保存2~5d后精滤,所得发酵酵素饮料置于冰箱冷藏,以备饮用,或者经60~85℃灭菌25~33min后灌装、封口,常温避光保存。
试验例1.苹果酵素饮料的色度试验
如图2所示,从左到右依次为对比例1、实施例2、实施例3制备得到的苹果酵素饮料,可以看出对比例1获得苹果酵素饮料呈现暗淡的咖啡色,而实施例2、3获得酵素饮料的色泽为浅黄色;此外,表2列出了对比例1、实施例2、实施例3制备所得苹果酵素饮料的色度值。
表2
从图2中可以看出从苹果中筛选获得酿酒酵母S.cerevisiaeAP 68菌株能够明显改良酵素的色泽,从暗淡的咖啡色变为浅黄色,并加速了发酵液中大分子物质与固体颗粒的沉降,增加了上清液的澄清度,降低后续精滤的工作量。表2的色差分析结果显示,三者的L值依次增加,说明其亮度增加,a值稍有下降,说明其红度下降,而b值显著降低,说明其黄度降低,L、a、b值的变化规律与实物颜色变化相符。
试验例2.苹果酵素饮料经口急性毒性实验
供试品:按实施例3制备得到的苹果酵素饮料。
实验方法:取20只ICR小鼠,雌雄各半,试验前实验动物禁食4~6h,不限制饮水。试验日当天染色、称重、分组,小鼠最大灌胃容量20mL/kg BW染毒,之后禁食3~4h。灌胃后连续观察14天。观察期内记录动物死亡数、死亡时间和中毒表现,每7天称量动物体重。
结果:给予供试品后,雌雄动物均未发现死亡,所有动物均未发现任何异常临床症状,所有动物体重及体重变化均正常。观察期结束后,对所有存活动物进行大体病理学检查,未发现异常组织和器官。
结论:在本次试验条件下,供试品小鼠急性经口LD50>5000mg/kg,按照小鼠急性经口毒性分级标准,供试品属无毒物。
试验例3.苹果酵素饮料经口亚慢性毒性实验
供试品:按实施例3制备得到的苹果酵素饮料。
分组:SD大鼠80只,雌雄各半,每只大鼠称重、标号、随机分组(高、中、低3个剂量组,1个空白对照组),每个试验组各20只大鼠(10只雄性,10只雌性)。
试验剂量:对照组按最大灌胃量20mL/kg体重以0.9%氯化钠注射液灌胃,其他组按低剂量组7mL/kg,中剂量组14mL/kg,高剂量组20mL/kg灌胃,不足20mL/kg的组以0.9%氯化钠注射液补足到20mL/kg,持续90天。
每天观察大鼠临床表现,终止日腹主动脉取血收集血液标本做临床病理学检测,解剖做大体标本观察及称取脏器湿重,脏器组织病理学检测。临床指标见表3。
表3
大体病理观察指标:试验终止日剖取脏器(心脏、肝脏、脾脏、胸腺、肾脏、肾上腺、睾丸、附睾、子宫、卵巢、脑等),称取湿重,计算器官/体重(‰)比值。并观察其大体病理(体表变化,脏器的位置、形态、大小、颜色、质地、有无分泌物或渗出物、淤血、水肿、出血、坏死等)情况。
血液学和生化指标:收集血液做临床病理学检测,具体见表4:
表4
血液学 | 生化检测 |
白细胞计数(WBC) | 白蛋白(ALB) |
血红蛋白浓度(HGB) | 球蛋白(GLB) |
红细胞计数(RBC) | 谷丙转氨酶(ALT) |
血小板计数(PLT) | 谷草转氨酶(AST) |
红细胞压积(HCT) | 白球比(A/G) |
凝血酶原时间(PT) | 氯化物(Cl) |
活化部分凝血活酶时间(APTT) | 肌酐(CREA) |
钙(Ca) | |
碱性磷酸酶(ALP) | |
谷酰转肽酶(GGT) | |
葡萄糖(GLU) | |
钾(K) | |
钠(Na) | |
总蛋白(TP) | |
甘油三酯(TG) |
结果:低剂量组、中剂量组、高剂量组及对照组大鼠的临床表现无异常,t检验分析试验实验组与对照组大鼠的体重变化均无显著性差异(P>0.05)。实验组及对照组大鼠的大体病理观察无异常,器官与体重比值、病理学数值等数据均在大鼠正常生物学范围之内,无显著性差异(P>0.05),未见明显组织病理学差异。
结论:试验样品对大鼠无亚慢性全身毒性反应。
试验例4.对大鼠肠道菌群的影响
分别在I期(30天)、II期(60天)、III期(90天)收集亚慢性毒性实验所饲喂大鼠的粪便,提取粪便肠道菌DNA后,基于同属细菌的特异性引物,采用RT-PCR技术测定乳杆菌属(Lactobacillus)、双歧杆菌属(Bifidobacterium)、肠杆菌属(Enterobacteriaceae)、肠球菌属(Enterococcus)的相对含量,结果见表5。
表5
结论:在灌胃苹果酵素饮料II期、III期,与空白对照组相比,中、低剂量组对肠道乳杆菌具有一定的促进作用,而对双歧杆菌的影响不明显。肠杆菌在灌胃苹果酵素饮料II期无差异,在灌胃III期中、低剂量组肠杆菌数量与空白组较接近,中、高剂量组肠杆菌数量增加;在灌胃III期,中、低剂量组肠球菌数量与空白组较接近。试验结果表明,灌胃中、低剂量苹果酵素饮料在一定程度上可以促进大鼠肠道有益菌增殖,抑制病原菌生长。
以上通过具体实施方式对本发明进行了详细描述,然而,本领域技术人员将会理解,可以在不偏离本发明的原理和精神的情况下对这些实施方式进行改变。
Claims (10)
1.一种酿酒酵母菌株,该菌株的分类命名:酿酒酵母Saccharomyces cerevisiae,于2018年6月4日保藏于“中国微生物菌种保藏管理委员会普通微生物保藏中心”,保藏号为CGMCC No.15839。
2.一种采用二步混菌发酵生产起泡型苹果酵素饮料的方法,该方法包括如下步骤:以苹果原汁为原料,以乳酸菌混菌对苹果原汁进行第一步发酵,后采用安琪葡萄酒酵母与权利要求1所述的酿酒酵母菌株的混菌进行第二步发酵,过滤获得起泡型苹果酵素饮料。
3.如权利要求2所述的方法,其特征在于,所述乳酸菌混菌包括第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌;优选地,所述的第一种植物乳杆菌保藏号为CICC 20022;所述植物乳杆菌植物亚种保藏号为CICC 20242;所述干酪乳杆菌保藏号为CICC 20296;所述类干酪乳杆菌保藏号为CICC20251;所述鼠李糖乳杆菌保藏号为CICC 6012;所述第二种植物乳杆菌保藏号为CGMCCNo.11936。
4.如权利要求2所述的方法,其特征在于,该方法包括如下步骤:
(1)果汁的制备与复配:选择无腐烂、无病虫害的苹果果实,清水洗净,沥水晾干,切块,热烫,凉开水冲洗降温,榨汁,过滤去除少量残留颗粒与固体得苹果果汁;以蔗糖调整可溶性固形物浓度至120~200g/L,调节pH值至4.2~6.0,升温至60~80℃保持15~40min,获得苹果原汁备用;
(2)乳酸菌菌株的活化与复配:将第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌分别接种于一级苹果汁改良MRS培养基中,26~35℃厌氧培养48~90h后,按接种量10~50mL/L分别接入二级苹果汁改良MRS培养基中,28~32℃厌氧培养60~72h;
其中,所述一级苹果汁改良MRS培养基为:苹果汁200~400mL/L,去离子水600~800mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5~7,121℃灭菌20min;
所述二级苹果汁改良MRS培养基为:苹果汁500~700mL/L,去离子水300~500mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为4~6,121℃灭菌20min;
(3)乳酸菌发酵:将步骤(2)活化好的第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌按照重量比例1~3:1~3:1~3:0.5~2:0.5~2:0.5~1.5混合后以10~50mL/L的接种量接入步骤(1)苹果原汁中,进行液体深层发酵,控制发酵温度为26~35℃,发酵时间12~60h;
(4)酵母菌的活化与扩大:将安琪葡萄酒酵母与所述的酿酒酵母菌株分别接种于一级苹果汁培养基,28~32℃培养60~72h;然后按接种量10~50mL/L分别接入二级苹果培养基中,20~30℃培养48~96h后作为种子培养基应用;
其中,所述一级苹果汁培养基为:苹果汁200~400mL/L,去离子水600~800mL/L,葡萄糖50~100g/L,酵母粉3~8g/L,pH值为4~6,121℃灭菌20min;
所述二级苹果汁培养基为:苹果果汁500~800mL/L,去离子水200~500mL/L,葡萄糖100~150g/L,酵母粉1~2g/L,pH值为4~6,121℃灭菌20min;
(5)酵母菌平静态发酵:将步骤(4)活化后的安琪葡萄酒酵母与所述的酿酒酵母菌株按重量比例2:1~1:3混合后,以0.2~5mL/L的接种量接入步骤(3)得到的果汁发酵液中,再次进行液体深层发酵,控制发酵温度为20~32℃,发酵时间12~60h,期间液面无剧烈气泡溢出,保持相对稳定的发酵状态,获得苹果酵素原液;
(6)降温及后续处理:去除苹果酵素的下层沉淀,上清液置于2~8℃、避光处保存2~5d后精滤,获得起泡型苹果酵素饮料。
5.如权利要求4所述的方法,其特征在于,所述步骤(3)中活化后的第一种植物乳杆菌、植物乳杆菌植物亚种、第二种植物乳杆菌、干酪乳杆菌、类干酪乳杆菌及鼠李糖乳杆菌的混合重量比例为2:2:2:1.5:1.5:1。
6.如权利要求4所述的方法,其特征在于,所述步骤(5)中活化后的安琪葡萄酒酵母与所述的酿酒酵母菌株按重量比例1:2混合。
7.如权利要求4所述的方法,其特征在于,所述步骤(6)获得的起泡型苹果酵素饮料经60~85℃灭菌25~33min后灌装、封口。
8.如权利要求4所述的方法,其特征在于,所述一级苹果汁改良MRS培养基为:苹果汁300mL/L,去离子水700mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.5,121℃灭菌20min;
所述二级苹果汁改良MRS培养基为:苹果汁600mL/L,去离子水400mL/L,蛋白胨10g/L,牛肉膏10g/L,酵母膏5g/L,柠檬酸氢二铵2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,pH值为5.0,121℃灭菌20min。
9.如权利要求4所述的方法,其特征在于,所述一级苹果汁培养基为:苹果汁300mL/L,去离子水700mL/L,葡萄糖80g/L,酵母粉5g/L,pH值为5.5,121℃灭菌20min;
所述二级苹果汁培养基为:苹果汁600mL/L,去离子水400mL/L,葡萄糖120g/L,酵母粉1.5g/L,pH值为5.0,121℃灭菌20min。
10.如权利要求2或3或4或5或6或7或8或9所述的方法制备得到的起泡型苹果酵素饮料。
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