CN104357353B - 一株植物乳杆菌在发酵果蔬汁制作中的应用 - Google Patents
一株植物乳杆菌在发酵果蔬汁制作中的应用 Download PDFInfo
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- C12N1/20—Bacteria; Culture media therefor
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Abstract
本发明公开了一株植物乳杆菌及其在发酵果蔬汁制作中的应用。本发明提供了一株植物乳杆菌Lactobacillus plantarum ZZU 208,其保藏号CGMCC NO.8990。本发明的实验证明了,从玉米青贮饲料中分离纯化到的乳酸菌ZZU 208对病原菌具有较好的抑菌活性。在发酵果蔬汁制作中能够有效抑制病原菌的生长,延长果蔬汁的保存期,改善果蔬汁的口感,增加乳酸菌及其代谢产物,有益于健康,在作为发酵果蔬汁制作方面具有潜在的应用价值。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一株植物乳杆菌及其在发酵果蔬汁制作中的应用。
背景技术
益生菌是指有益于人类生命健康的一类肠道生理细菌。益生菌发挥作用需要满足2个基本条件,一是活的微生物,二是通过摄入充足的数量,对宿主产生功能性健康益处。益生菌有很多种,乳酸菌是一类常见的益生菌,可以通过与有害菌竞争营养素来抑制有害菌生长,或通过释放抗菌性物质杀伤有害菌,也可以在肠道上皮细胞形成一层保护粘膜,阻止有害菌的入侵,还能够在肠道内产生酸性物质,抑制有害菌生长,以起到改善肠道微生态环境,保持健康的胃肠道功能。另外,乳酸菌还可以产生乳糖酶,缓解乳糖不耐受症,合成短链脂肪酸和维生素(B族、K族),加强营养物质的吸收。此外,乳酸菌还能够增强免疫功能。因此,乳酸菌的补充不仅可以改善胃口不好,吸收差的症状,还有益于体质弱,反复生病感染,过敏等症状的改善。目前市场上发酵饮品种类较少,且产品多集中于各种发酵乳制品。果蔬汁含有丰富的维生素、矿物质、果胶质等天然营养元素,服用果蔬汁可以降低癌症的发病率,同时还能有效防止动脉硬化、高血脂和冠心病等心血管疾病的发生。以果蔬汁为原料,接种乳酸菌进行发酵,进行果蔬汁发酵饮品的开发,有助于丰富风味发酵饮品的种类,能够满足在补充果蔬营养的同时,增加益生菌。
发明内容
本发明的一个目的是提供一株植物乳杆菌Lactobacillus plantarum ZZU 208。
本发明提供的植物乳杆菌Lactobacillus plantarum ZZU 208,其保藏号为CGMCCNO.8990。
上述的植物乳杆菌或其发酵产物或其菌悬液或其培养液在作为发酵果蔬汁添加剂或制备发酵果蔬汁添加剂中的应用。
上述的植物乳杆菌或其发酵产物或其菌悬液或其培养液在发酵果蔬汁中的应用。
上述应用中,所述果蔬汁按照包括如下的方法制备:将质量百分含量分别为25%西红柿、12.5%胡萝卜、25%苹果、7.5%的白糖和水混合匀浆后,灭菌,得到果蔬汁。
上述应用中,所述灭菌温度为90℃,所述灭菌的时间为5min。
本发明的另一个目的是提供一种发酵果蔬汁的方法。
本发明提供的方法,包括如下步骤:
1)、将质量百分含量分别为25%西红柿、12.5%胡萝卜、25%苹果、7.5%的白糖和水混合匀浆后,灭菌,得到果蔬汁;
2)将上述的植物乳杆菌接入所述果蔬汁中,发酵,得到发酵后果蔬汁。
上述方法中,所述植物乳杆菌是以植物乳杆菌种子液接入,所述植物乳杆菌种子液中菌含量大于等于108cfu/g。
所述植物乳杆菌种子液按照体积百分含量2.5%接入果蔬汁中,其植物乳杆菌种子液中菌含量大于等于108cfu/g。
上述植物乳杆菌种子液按照包括如下步骤的方法进行:将所述植物乳杆菌在MRS固体培养基30℃厌氧培养箱培养48h,收集菌体,将其重悬于水中;
上述方法中,所述发酵条件为30℃静置培养17h-23h。
本发明的第三个目的是提供一种发酵果蔬汁添加剂。
本发明提供的发酵果蔬汁添加剂,其活性成分为上述的植物乳杆菌或其发酵产物或其菌悬液或其培养液。
上述的植物乳杆菌在制备抑菌产品中的应用;
所述抑菌为抑制Salmonella enterica、抑制Escherichia coli或抑制Micrococcus luteus。
乳酸菌ZZU 208于2014年4月2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(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号),保藏编号为CGMCCNO.8990,分类命名为植物乳杆菌(Lactobacillus plantarum)。
本发明的实验证明了,菌株ZZU 208在混合果汁发酵中不仅能够有效补充乳酸菌、降低果蔬汁pH,而且能够进一步改善果蔬汁的气味、色泽及口感,在发酵果蔬汁饮料制作方面具有潜在的应用价值。
附图说明
图1为recA基因的PCR扩增产物电泳图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、植物乳杆菌的获得
1、分离纯化
从河南省郑州市取得的玉米(品种:郑超甜2号)青贮饲料中分离纯化到乳酸菌ZZU208,为同型发酵乳酸菌,具体如下:
称取10g玉米青贮饲料样品加入灭菌的90ml蒸馏水中,利用漩涡振荡器震荡30s,10倍梯度稀释,然后分别取10-1、10-3和10-5倍样品稀释液涂布在MRS固体培养基上,放置于厌氧培养箱培养48h,然后将其取出,依据菌落的大小、形态和颜色,挑取MRS上生长的单菌落,进行过氧化氢酶实验及革兰氏染色。将过氧化氢酶阴性及革兰氏染色阳性者暂定为乳酸菌,于MRS固体培养基上继续划线纯化2次。-80℃保存于10%二甲基亚砜的Nutrient肉汤培养基中,该菌株命名为ZZU 208,为同型发酵乳酸菌。
2、生理生化检测
乳酸菌ZZU 208在10℃-45℃条件下及pH3.5-9.0条件生长良好,能够耐受3.0%的盐浓度,部分理化特性如表1所示。该菌株能够利用的碳源如表2所示。
表1菌株ZZU 208的生理生化特性
表2菌株ZZU 208的碳源发酵试验结果
注:+代表反应为阳性;—代表反应为阴性;w代表反应为弱阳性
3、分子鉴定
1)16rRNA基因序列鉴定
将固体平板上培养48h的乳酸菌ZZU 208,采用菌落PCR扩增,进行16rRNA基因序列鉴定。引物选用细菌的16S rRNA基因扩增通用引物:27F(5’-AGAGTTTGA-TCCTGGCTCAG-3’)和1492R(5’-GGTTACCTTGTTACGACTT-3’)。PCR反应体系为50μl:Premix Taq25μl,27F和1492R(均为20μM)1μl,补充灭菌蒸馏水至50μl,直接将菌落加入反应体系,进行PCR扩增,得到PCR产物。
将PCR产物送去测序,结果为序列表中序列1所示。
2)recA基因检测
将固体平板上培养48h的乳酸菌ZZU 208,采用菌落PCR扩增,进行recA基因检测。引物选用基于recA基因的引物:paraF(5’-GTCACAGGCATTACGAAAAC-3’),pentF(5’-CAGTGGCGCGGTTGATATC-3’),planF(5’-CCGTTTATGCGGAACACCTA-3’)和pREV(5’-TCGGGATTACCA AACAT CAC-3’)。PCR扩增体系(40μl)包括Premix Taq20μl,引物paraF,pentF,和pREV(各0.25μM),引物planF0.12μM,补充灭菌蒸馏水至40μl,用10μl无菌枪头在单菌落挑取少许,加入反应体系,进行PCR反应。
结果如图1所示,1:LactobacilluscaseiJCM 16167T(阴性对照);2:L.paraplantarum JCM 12533T;3:L.pentosus JCM 1558T;4:L.plantarumsubsp.plantarum JCM 1149T;5:L.plantarum subsp.argentoratensisJCM 16169T;6:ZZU208;M:DNA Markers,可以看出,得到318bp的PCR产物。
经过上述鉴定,乳酸菌ZZU 208于2014年4月2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(简称CGMCC,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.8990,分类命名为植物乳杆菌(Lactobacillus plantarum)。
实施例2、乳酸菌ZZU 208在抑菌中的应用
乳酸菌ZZU 208接种到MRS液体培养基,30℃培养48h,得到的发酵液经10000rpm离心5min,收集上清液,将上清液采用牛津杯双层平板法进行抑菌活性检测,结果菌株ZZU208发酵上清液对革兰氏阳性及阴性指示菌都有较强的抑菌活性,如表3所示。
表3菌株ZZU 208发酵上清液的抑菌活性
注:抑菌活性以抑菌圈直径表示,牛津杯外径为7.80mm。+,抑菌圈直径:8.00-12.00mm;++,12.00-16.00mm;+++,16.00-20.00mm;++++,>20.00mm
其发酵上清液在排除酸和过氧化氢的作用后对革兰氏阳性及阴性指示菌仍然有较强的抑菌活性,然而在经过蛋白酶K或胰蛋白酶的作用后抑菌活性消失,结果如表4所示,表明该抑菌活性物质可能为蛋白类或肽类物质。具体方法为:用2mol/L NaOH调发酵上清液pH值至6.0,并用2mol/L HCL调未接种的MRS液体培养基pH值至6.0作为对照,按照过氧化氢酶、胰蛋白酶或蛋白酶K的终浓度分别为1.0mg/mL、100μg/mL及100μg/mL分别加入到发酵液中,37℃水浴2h,采用牛津杯双层平板法进行抑菌活性检测。
表4不同处理后菌株ZZU 208发酵上清液的抑菌活性
注:抑菌活性以抑菌圈直径表示,牛津杯外径为7.80mm。+,抑菌圈直径:8.00-12.00mm;++,12.00-16.00mm;+++,16.00-20.00mm;++++,>20.00mm;-,≤7.80mm
乳酸菌ZZU 208接种到MRS液体培养基,30℃培养48h,得到的发酵液经10000rpm离心5min,收集上清液,在不同的温度条件下处理不同时间,或用2mol/L NaOH或2mol/L HCL将上清液调至不同的pH值,应用采用牛津杯双层平板法进行抑菌活性检测,结果如表5所示,
表5温度和pH对菌株ZZU 208发酵上清液抑菌活性的影响
注:抑菌活性以抑菌圈直径表示,牛津杯外径为7.80mm。+,抑菌圈直径:8.00-12.00mm;++,12.00-16.00mm;+++,16.00-20.00mm;++++,>20.00mm;-,≤7.80mm
该发酵上清液中的抑菌活性物质在100℃保持60min具有较好的稳定性,在pH≤6的条件下具有抑菌活性,且随pH降低活性增强。
实施例3、ZZU 208在发酵果蔬汁制作中的应用
1、乳酸菌种子液的制备
乳酸菌ZZU 208接种到MRS固体培养基,于30℃厌氧培养箱培养48h,取出,将固体MRS培养基上生长的菌株ZZU 208无菌刮擦于无菌水中,使乳酸菌浓度≥108cfu/ml,备用。
2、乳酸菌在发酵果蔬汁制作中的应用
将质量百分含量分别为25%西红柿、12.5%胡萝卜及25%苹果切块、匀浆,加入质量百分含量为7.5%的白糖混匀,90℃灭菌5min,待温度降至30℃时,无菌取出部分未发酵果蔬汁作为0h对照,其余果蔬汁接种乳酸菌浓度108cfu/ml的ZZU 208种子液,接种量为体积百分含量2.5%,分别30℃静置发酵17h、23h无菌取出发酵果蔬汁。
应用酸度计测定pH;梯度稀释平板计数法统计乳酸菌活菌数;
由30人组成的试饮评价组对3种果蔬汁的气味、色泽及口感进行满意度评价,评价分为3个级别,分别是一般、好及很好。
未发酵果蔬汁、发酵17h及23h果蔬汁的pH、乳酸菌含量及对3种果蔬汁的试饮评价调查结果如表6所示,随发酵时间延长果蔬汁的pH下降,在发酵的17h及23h,pH分别降至3.6及3.2,乳酸菌数在发酵17h已超过108cfu/ml,随发酵时间延长,数量有所降低,发酵果蔬汁在放置一段时间后,乳酸菌活菌数有下降趋势,在4℃存放10d,乳酸菌活菌数均比存放前有所减少,但仍然高于108cfu/ml。30人对未发酵果蔬饮料、发酵17h及23h果蔬饮料的试饮评价结果表明,无论是气味、色泽,还是口感,发酵后果蔬汁的满意度都超过未发酵果蔬汁,因此接种乳酸菌发酵果蔬汁不仅增加果蔬汁中益生菌的数量,而且能够赋予果蔬汁更好的气味、色泽及口感。
综上所述,菌株ZZU 208在混合果汁发酵中不仅能够有效补充乳酸菌、降低果蔬汁pH,而且能够进一步改善果蔬汁的气味、色泽及口感,在发酵果蔬汁饮料制作方面具有潜在的应用价值。
表6为未发酵与乳酸菌发酵果蔬饮料的pH、乳酸菌含量及试饮评价
Claims (3)
1.植物乳杆菌(Lactobacillus plantarum)ZZU 208 CGMCC NO.8990或其菌悬液在发酵果蔬汁中的应用;
所述果蔬汁按照包括如下的方法制备:将质量百分含量分别为25%西红柿、12.5%胡萝卜、25%苹果、7.5%的白糖和水混合匀浆后,灭菌,得到果蔬汁;
所述灭菌温度为90℃,所述灭菌的时间为5min。
2.一种发酵果蔬汁的方法,包括如下步骤:
1)、将质量百分含量分别为25%西红柿、12.5%胡萝卜、25%苹果、7.5%的白糖和水混合匀浆后,灭菌,得到果蔬汁;
2)将植物乳杆菌(Lactobacillus plantarum)ZZU 208 CGMCC NO.8990接入所述果蔬汁中,发酵,得到发酵后果蔬汁;
所述植物乳杆菌是以植物乳杆菌种子液接入,所述植物乳杆菌种子液的菌含量大于等于108cfu/ml;
所述发酵条件为30℃静置培养17h-23h。
3.植物乳杆菌(Lactobacillus plantarum)ZZU 208 CGMCC NO.8990在制备抑菌产品中的应用;
所述抑菌为抑制肠道沙门氏菌(Salmonella enterica)、抑制大肠埃希氏菌(Escherichia coli)或抑制藤黄微球菌(Micrococcus luteus)。
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