CN109112083A - 一种高产辛酸的瑞士乳杆菌及其应用 - Google Patents
一种高产辛酸的瑞士乳杆菌及其应用 Download PDFInfo
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- A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
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- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
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Abstract
本发明公开了一种高产辛酸的瑞士乳杆菌及其应用,属于微生物学发酵领域。本发明的瑞士乳杆菌已于2018年1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,保藏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.15147。该菌株产酸快,10h辛酸产量达66.47μg/kg,具有主发酵剂应用潜力;且该菌株发酵脱脂乳时可高产特征风味物质,占所有挥发性风味物质总和的7.01%,赋予乳制品产品吸引人的清香风味。
Description
技术领域
本发明涉及一种高产辛酸的瑞士乳杆菌及其应用,属于微生物学发酵领域。
背景技术
酸类物质是发酵制品主要特色风味物质之一,可赋予发酵制品多样的特征风味,例如酸味、奶味、淡果香味等。其中辛酸是挥发性中链脂肪酸中的典型风味物质。
酸类物质存在于各种食品中,在发酵食品中更为常见,其中8种脂肪酸是呈味脂肪酸,即乙酸、丁酸、异丁酸、异戊酸、丙酸、辛酸、己酸、癸酸(Cheng H.Volatile flavorcompounds in yogurt:a review.[J].Critical Reviews in Food Science&Nutrition,2010,50(10):938-50.)。当前提倡健康饮食,通过低糖低盐低脂的饮食方式降低患病风险,例如发酵乳制品所用的原料从全脂乳改为脱脂乳,有研究表明(艾娜丝,仝令君,张晓梅,等.全脂乳与脱脂乳挥发性风味成分对比分析[J].食品研究与开发,2016,37(11):1-6.),脱脂乳未被检测出香气强度较高的呈香成分,而全脂乳中辛酸(清香味)、癸酸(油脂味)香气强度较高,这对乳制品风味的改善方向提出了要求。梁钻好等人研究发现(梁钻好.超高压处理对黄油物理特性及挥发性风味物质的影响[D].华南农业大学,2016.),在乳制品中,中短链脂肪酸的缺失会导致乳制品的香气不足,而目前增加中短链脂肪酸含量的主要方式是酶法水解,成本较高,且专一性太强,易造成其他风味的缺失。因此,筛选出能够直接发酵脱脂乳,并产生强度较高的挥发性风味物质的乳酸菌极其有必要,同时可简化发酵工艺,节约成本。
瑞士乳杆菌是一种常用附属发酵剂,广泛应用于奶酪中,具有很强的蛋白酶解能力,可产生丰富的风味物质和极具活性的多肽类物质,是对人体有益生作用的一种乳酸菌(Slattery L,O'Callaghan J,Fitzgerald G F,et al.Invited review:Lactobacillushelveticus--a thermophilic dairy starter related to gut bacteria.[J].Journalof Dairy Science,2010,93(10):4435-54.)。殷捷等人(殷捷,卢姗姗,张少辉,等.不同瑞士乳杆菌发酵牛乳产肽的比较研究[J].食品科学,2010,31(15):186-189.)研究了具有益生特性的不同株瑞士乳杆菌发酵脱脂乳时的产酸情况,结果显示产酸能力最强的瑞士乳杆菌菌株自15h开始剧烈产酸,且在相同发酵过程中该菌株的最大水解度也明显高于其他菌株,这对当前筛选具备益生特性的发酵乳酸菌提供了指导。张佳等人(张佳,马永昆,崔凤杰,等.乳酸菌发酵酸豆乳香气成分分析及评价[J].食品科学,2010,31(20):298-302.)于2010年就已研究发现嗜热链球菌:保加利亚乳杆菌=1:1的发酵豆乳的辛酸含量为1.54%,嗜热链球菌:瑞士乳杆菌=1:1的发酵豆乳的辛酸含量为1.68%,两者辛酸含量相近,风味相近,但后者由于乙酸含量较高导致刺激性酸味较强,说明该组别中的清香风味不足,不能掩盖刺激性酸味。武士美等人(武士美,靳汝霖,任为一,丹彤,孙天松,孟和毕力格.德氏乳杆菌保加利亚亚种发酵乳中挥发性风味物质的比较分析[J].中国乳品工业,2017,45(9):4-10)制备了四株保加利亚乳杆菌发酵的全脂酸奶,经42℃发酵至pH4.7终点后测定其挥发性风味物质,发现四株保加利亚乳杆菌发酵后的酸奶中的辛酸含量分别为:4.39%、1.54%、1.54%和0.64%,与20%左右的酮类化合物相比,含量偏低。
综上,在产辛酸的菌株中,辛酸产量普遍较低,其中包括瑞士乳杆菌,而瑞士乳杆菌是发酵乳制品中常用的一种乳酸菌,因此为挖掘具有高产辛酸特色的瑞士乳杆菌对于增强乳制品的特征风味同时不因风味过强而导致异味感具有重要的意义。
发明内容
本发明采集了中国四川阿坝藏区传统牦牛酸奶,并从中分离筛选出一株高产辛酸的瑞士乳杆菌菌株,以期作为发酵剂应用于乳制品发酵,以提升乳制品的清香风味。
本发明的第一个目的是提供一株高产辛酸的瑞士乳杆菌(Lactobacillushelveticus),已于2018年1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,保藏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.15147。
本发明提供的瑞士乳杆菌CGMCC No.15147基本特征如下:
1、该菌株在固体LBS培养基上呈白色,稍有光泽,边缘光滑,不透明,中等大小,凸起;
2、该菌株生长速度较快,LBS中16h进入稳定期,脱脂乳中8h进入稳定期,适合工业发酵;
3、该菌株产酸快,有利于凝乳和风味物质的产生;
4、该菌株产辛酸含量高,10h可产66.47μg/kg,相对含量为7.01%;
本发明的第二个目的是提供含有所述瑞士乳杆菌的菌剂。
在本发明的一种实施方式中,所述菌剂包括所述瑞士乳杆菌(Lactobacillushelveticus)和食品用载体。
在本发明的一种实施方式中,所述菌剂的形式包括是液体、固体、冻干制剂、粉末、凝胶或薄膜。
本发明的第三个目的是提供所述瑞士乳杆菌(Lactobacillus helveticus)在制备食品方面的应用。
在本发明的一种实施方式中,所述食品包括发酵食品。
在本发明的一种实施方式中,所述应用包括制备烘焙食品或乳制品。
本发明的第四个目的是提供一种提升乳制品风味的方法,所述方法是向乳制品生产过程中接种含有所述瑞士乳杆菌的微生物。
本发明还要求保护应用所述瑞士乳杆菌(Lactobacillus helveticus)制备的食品或饮料。
有益效果:1、本发明的瑞士乳杆菌属于常用附属发酵剂菌株,该发明菌株产酸快,10h辛酸产量达66.47μg/kg,具有主发酵剂应用潜力;
2、本发明的菌株发酵脱脂乳时可高产特征风味物质辛酸,占所有挥发性风味物质总和的7.01%,赋予乳制品产品吸引人的清香风味。
附图说明
图1:瑞士乳杆菌CGMCC No.15147分离源样品与市售样品的风味特征差异;
图2:瑞士乳杆菌CGMCC No.15147在LBS中24h内的生长情况;
图3:瑞士乳杆菌CGMCC No.15147在LBS中24h内的pH变化情况;
图4:瑞士乳杆菌CGMCC No.15147在脱脂乳中14h内的活菌数变化情况;
图5:瑞士乳杆菌CGMCC No.15147在脱脂乳中14h内的生长速率变化情况;
图6:瑞士乳杆菌CGMCC No.15147在脱脂乳中14h内的产酸能力。
生物材料保藏
一株瑞士乳杆菌(Lactobacillus helveticus),分类命名为瑞士乳杆菌Lactobacillus helveticus,已于2018年1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,保藏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.15147。
具体实施方式
实施例1:瑞士乳杆菌分离源样品与市售样品的风味特征差异评定
(1)准备样品
将-80℃冰箱中贮藏的西藏阿坝传统牦牛酸奶原样取出,置于冰上解冻,振荡混匀后取6g于气相瓶中,加入1g NaCl,并迅速拧紧气相瓶瓶盖,同时购买新鲜的市售酸奶,震荡混匀后取6g于气相瓶中,加入1g NaCl,并迅速拧紧气相瓶瓶盖,测定其挥发性风味,平行三次测定。
(2)GC-MS风味测定条件
气谱条件为:Rtx-WAX毛细管柱;柱子规格:30m×0.25mm×0.25mm,进口温度为250℃,分流比5,柱流速1mL/min,载气为氦气;程序升温:初始温度40℃,保持3min;10℃/min升温至90℃;5℃/min升温至200℃,保持5分钟。
质谱条件为:离子化方式EI,发射能量为70eV,发射电流为200μA,检测器电压为1.4kV,离子源温度250℃,接口温度230℃,四级杆温度:150℃,质荷比30-500。化合物检索结果与NIST和Varian2个标准谱库进行匹配,相似度达到80%以上确认为目的化合物。以2μl的0.05mg/ml癸酸乙酯为内标,计算各挥发性风味物质的含量。
本发明使用的样品编号、样品名称如表所示:
表1样品信息表
由图1可知,西藏传统发酵酸奶的挥发性风味特征多样,大部分传统酸奶与工业发酵的市售酸奶的差异显著。其中TD1-Y酸奶整体挥发性风味组成与市售酸奶相似,辛酸占比较高。
实施例2:瑞士乳杆菌的分离与鉴定
(1)制备合适的样品稀释梯度并培养
将-80℃冰箱中贮藏的西藏阿坝传统牦牛酸奶甘油样品取出,置于冰上解冻。震荡混匀后取0.5mL样品加入4.5mL无菌生理盐水中,完成一次10倍稀释,振荡混匀后再从该稀释液中取出0.5mL稀释液加入4.5mL无菌生理盐水中,完成第二次10倍稀释,以此类推,直至稀释到10-6,从每个梯度的稀释液中吸取100μL,均匀涂布于LBS固体培养基平板上,平行制备两块平板,倒置,放于37℃厌氧下培养36-48h,并及时观察。
(2)划线分离与纯化
将长出菌落的平板取出后,选取单菌落明显的梯度平板,挑取不同菌落形态的菌落,进行二次划线,直至纯化出所有单菌落。
(3)革兰氏染色和过氧化氢酶实验
挑取单菌落于载玻片上,经过涂片、干燥、固定、初染、水洗、媒染、水洗、脱色、复染、水洗、干燥后镜检,记录革兰氏染色结果;并挑取单菌落于载玻片上,加入3%过氧化氢溶液,观察有无气泡产生,并记录过氧化氢酶接触结果。
(4)菌种保藏
将纯化完成后的每种菌株的单菌落挑入5mL液体LBS培养基中,置于37℃厌氧下静置培养20-24h,吸取1mL菌液至保菌管中,6000rpm 3min离心,去上清,加入1mL30%无菌甘油溶液,重悬,置于-80℃保存。
(5)16S rDNA序列扩增
吸取1mL菌液6000rpm 3min离心,去上清,水洗两次,得菌泥,加入SDS裂解液破碎细胞壁,再加入酚-氯仿萃取核酸DNA,12000rpm 10min离心取上清,乙醇溶液水洗两次,12000rpm 5min离心去上清,烘干,加入50μL无菌水重悬,即得菌株DNA模板。以此为模板,进行PCR扩增,流程如下:
1)扩增体系25μL:
其中模板量为1μL,Taq酶Mix为12.5μL,27F为0.5μL,1492R为0.5μL,ddH2O为10.5μL。所用引物为27F:AGA GTT TGA TCC TGG CCT CA(核苷酸序列如SEQ ID NO:1)和1492R:GGT TAC CTT GTT ACG ACT T(核苷酸序列如SEQ ID NO:2)。扩增片段长度1500bp。
2)扩增条件:
预变性温度:105℃;
第一步变性:95℃7min;95℃30s;
第二步退火:55℃30s;
第三步延伸:72℃90s;
循环次数:Go to 95℃30s,33次循环;
第四步最后延伸:72℃5min;
第五步保持:12℃10min。
(6)琼脂糖凝胶电泳
称取80mL琼脂糖加入锥形瓶中,加入80mL1xTAE,微波间断式加热4min,至液体澄清透明,稍稍冷却,加入2‰核酸染料,摇匀,无气泡,倒入胶板槽中,冷却40min凝固后,置于电泳槽中,排气泡,依次加入PCR扩增产物,每个孔中加入4μL产物,120V 30min跑胶结束后取出,置于凝胶电泳成像仪中拍照保存,记录PCR成功样品的编号,将成功的PCR产物置于-20℃冰箱中保藏。
(7)菌株PCR产物送测与鉴定
将PCR成功的样品PCR送予华大基因进行检测,根据华大基因反馈的序列结果,结合NCBI菌株序列数据库进行BLAST检索,选取匹配度最高的菌株信息进行结果记录,该菌株的16S rDNA序列全长共1406bp(核苷酸序列如SEQ ID NO:3),经Genbank比对相似性达到99%,确定为瑞士乳杆菌。该编号TD1的瑞士乳杆菌,于2018年1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,保藏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.15147。
本发明使用的菌株编号、菌株名称以及分离源等菌株信息如表所示:
表2菌株信息表
实施例3:瑞士乳杆菌在LBS中24h内的生长曲线
从-80℃下保藏的菌株按2%的接种量接种到5mL液体LBS培养基中,37℃厌氧下培养16-18h,活化三代培养,取菌液按2%的接种量接种至LBS液体培养基中培养14h,从起点开始每隔2h取菌液1mL,振荡混匀,利用紫外分光光度计测定OD600吸光度,平行三次,并按时间绘制生长曲线。同时取出菌液100μL均匀涂布于固体LBS培养基上,倒置,37℃厌氧下培养36-48h,平板计数。由图2可知,瑞士乳杆菌CGMCC No.15147(即编号TD1的瑞士乳杆菌)在16h进入稳定期,市售菌株C1、C2均在18h进入稳定期,本发明提供的瑞士乳杆菌更早进入稳定期。
实施例4:瑞士乳杆菌在LBS中24h内的pH变化
从-80℃下保藏的菌株按2%的接种量接种到5mL液体LBS培养基中,37℃厌氧下培养16-18h,活化三代培养,取菌液按2%的接种量接种至LBS液体培养基中培养14h,从起点开始每隔2h取菌液,振荡混匀,利用pH计测定pH值,平行三次并记录。根据图3可知,在降低pH上,瑞士乳杆菌CGMCC No.15147在12h内可将pH从5.51降至4.89,菌株C1的pH从5.52降至5.47,菌株C2的pH从5.98降至4.57,因此本发明提供的瑞士乳杆菌在LBS中的pH比市售菌株C1降低更快,与市售菌株C2相当。
实施例5:瑞士乳杆菌在脱脂乳中14h内的活菌数变化
从-80℃下保藏的菌株按2%的接种量接种到5mL液体LBS培养基中,37℃厌氧下培养16-18h,活化三代培养,取菌液按2%的接种量接种至无菌脱脂乳中培养14h,从起点开始每隔2h取发酵液100μL均匀涂布于固体LBS培养基上,倒置,37℃厌氧下培养36-48h,平板计数。由图4、图5可知,本发明提供的瑞士乳杆菌在脱脂乳中的生长速度远远快于LBS中的生长速度,瑞士乳杆菌CGMCC No.15147 8h可进入稳定期,活菌量高于同样在脱脂乳中生长的市售保加利亚乳杆菌C1、C2,活菌量可达到5.6*108CFU/mL。
实施例6:瑞士乳杆菌在脱脂乳中14h内的产酸能力、凝乳能力
从-80℃下保藏的菌株按2%的接种量接种到5mL液体LBS培养基中,37℃厌氧下培养16-18h,活化三代培养,取菌液按2%的接种量接种至无菌脱脂乳中培养14h,从起点开始每隔2h取发酵液,振荡混匀,利用pH计测定pH值;同时取出10mL发酵液,加入20无菌水稀释混匀后,加入2-3滴2%酚酞,经0.1M NaOH滴定至变色且30min不褪色,平行三次,记录滴定体积并计算酸度值。
同时,每个时间点进行发酵乳的质地观察,可通过倾斜震荡以观察是否凝固,其中发酵乳在倾斜角大于45°时不具有流动性视为凝固,在倾斜角大于45°时具有流动性视为微凝。
由图6可知,与两株市售保加利亚乳杆菌相比,本发明提供的瑞士乳杆菌CGMCCNo.15147可更快发酵脱脂乳产酸,与表3提供的凝乳观察结果一致,这将有利于快速凝乳,产生活跃的酶系水解脱脂乳的成分形成芳香物质。
表3不同时间点凝乳情况
实施例7:瑞士乳杆菌在脱脂乳中10h内的辛酸生成能力
1)发酵乳的制备
从-80℃下保藏的菌株按2%的接种量接种到5mL液体LBS培养基中,37℃厌氧下培养16-18h,活化三代培养,取菌液按2%的接种量接种至无菌脱脂乳中培养14h,从起点开始每隔2h取发酵液6g,置于顶空气相瓶中,并加入1g NaCl迅速拧紧气相瓶瓶盖,测定其挥发性风味。
2)GC-MS风味测定条件
气谱条件、质谱条件均同实施例1,计算辛酸含量。
本发明所提供的菌株发酵脱脂乳产生的辛酸含量如下表所示,由表4、表5可知,瑞士乳杆菌CGMCC No.15147可在10h内产生66.47μg/kg的辛酸,占所有挥发性风味物质总和的7.01%,绝对值含量远高于市售菌株C1、C2。
表4不同时间点辛酸产量
表5不同时间点辛酸相对含量
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。
SEQUENCE LISTING
<110> 江南大学
<120> 一种高产辛酸的瑞士乳杆菌及其应用
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> 人工合成
<400> 1
agagtttgat cctggcctca 20
<210> 2
<211> 19
<212> DNA
<213> 人工合成
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 1406
<212> DNA
<213> Lactobacillus helveticus 16S rDNA
<400> 3
taggccaccg gctttgggca ttgcagactt ccatggtgtg acgggcggtg tgtacaaggc 60
ccgggaacgt attcaccgcg gcgttctgat ccgcgattac tagcgattcc agcttcgtgc 120
agtcgagttg cagactgcag tccgaactga gaacagcttt cagagattcg cttgccttcg 180
caggcttgct tctcgttgta ctgtccattg tagcacgtgt gtagcccagg tcataagggg 240
catgatgact tgacgtcatc cccaccttcc tccggtttat caccggcagt ctcattagag 300
tgcccaactt aatgctggca actaataaca agggttgcgc tcgttgcggg acttaaccca 360
acatctcacg acacgagctg acgacagcca tgcaccacct gtcttagcgt ccccgaaggg 420
aactcctaat ctcttaggat ggcactagat gtcaagacct ggtaaggttc ttcgcgttgc 480
ttcgaattaa accacatgct ccaccgcttg tgcgggcccc cgtcaattcc tttgagtttc 540
aaccttgcgg tcgtactccc caggcggagt gcttaatgcg ttagctgcag cactgagagg 600
cggaaacctc ccaacactta gcactcatcg tttacggcat ggactaccag ggtatctaat 660
cctgttcgct acccatgctt tcgagcctca gcgtcagttg cagaccagag agtcgccttc 720
gccactggtg ttcttccata tatctacgca ttccaccgct acacatggaa ttccactctc 780
ctcttctgca ctcaagaaaa acagtttccg atgcagttcc tcggttaagc cgagggcttt 840
cacatcagac ttattcttcc gcctgcgctc gctttacgcc caataaatcc ggacaacgct 900
tgccacctac gtattaccgc ggctgctggc acgtagttag ccgtgacttt ctggttgatt 960
accgtcaaat aaaggccagt tactacctct atccttcttc accaacaaca gagctttacg 1020
atccgaaaac cttcttcact cacgcggcgt tgctccatca gacttgcgtc cattgtggaa 1080
gattccctac tgctgcctcc cgtaggagtt tgggccgtgt ctcagtccca atgtggccga 1140
tcagtctctc aactcggcta tgcatcattg ccttggtaag ccgttacctt accaactagc 1200
taatgcaccg cggggccatc ccatagcgac agcttacgcc gccttttata agctgatcat 1260
gcgatctgct ttcttatccg gtattagcac ctgtttccaa gtggtatccc agactatggg 1320
gcaggttccc cacgtgttac tcacccatcc gccgctcgcg tccccagcgt cattaccgaa 1380
gtaaatctgc tggttctgct cgctcg 1406
Claims (9)
1.一株高产辛酸的瑞士乳杆菌(Lactobacillus helveticus),已于2018年1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,保藏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.15147。
2.含有权利要求1所述的瑞士乳杆菌的菌剂。
3.根据权利要求2所述的菌剂,其特征在于,含有权利要求1所述的瑞士乳杆菌(Lactobacillus helveticus)和食品用载体。
4.根据权利要求2或3所述的菌剂,其特征在于,所述菌剂的形式包括液体、固体、冻干制剂、粉末、凝胶或薄膜。
5.权利要求1所述的瑞士乳杆菌(Lactobacillus helveticus)在制备食品方面的应用。
6.根据权利要求5所述的应用,其特征在于,所述食品包括发酵食品。
7.根据权利要求5所述的应用,其特征在于,所述应用包括制备烘焙食品或乳制品。
8.一种提升乳制品风味的方法,其特征在于,向乳制品生产过程中接种含有权利要求1所述瑞士乳杆菌的微生物。
9.应用权利要求1所述瑞士乳杆菌(Lactobacillus helveticus)制备的食品或饮料。
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CN111000247A (zh) * | 2019-12-31 | 2020-04-14 | 扬州江大食生一生食品有限公司 | 一种复合益生菌粉的制备方法 |
CN115305225A (zh) * | 2022-09-16 | 2022-11-08 | 微康益生菌(苏州)股份有限公司 | 一种能够快速产酸的菌种组合、包含其的冻干菌粉及其应用 |
CN115305225B (zh) * | 2022-09-16 | 2024-07-02 | 微康益生菌(苏州)股份有限公司 | 一种能够产酸的菌种组合、包含其的冻干菌粉及其应用 |
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