CN108004176B - 一种鼠李糖乳杆菌lrh09、由其制备的发酵剂及发酵剂的应用 - Google Patents
一种鼠李糖乳杆菌lrh09、由其制备的发酵剂及发酵剂的应用 Download PDFInfo
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Abstract
本发明公开了一种鼠李糖乳杆菌LRH09、由其制备的发酵剂及发酵剂的应用,本发明发酵剂发酵温度宽泛。鼠李糖乳杆菌LRH09,已于2017年11月27日寄存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.14962。本发明鼠李糖乳杆菌LRH09可用于制备发酵剂,所得的发酵剂,产酸快、产酸量大、产酸温度宽。
Description
技术领域
本发明涉及一种鼠李糖乳杆菌LRH09、由其制备的发酵剂及发酵剂的应用,属于微生物领域。
背景技术
乳酸菌是一类可以代谢乳糖产生乳酸、降低发酵产品pH值的一类微生物。乳酸菌在乳品发酵中的主要作用是产酸和产生风味物质。类似发酵类产品的嗜温性,发酵剂要求具有良好的产酸、产香特性,以满足喜欢食用柔和风味酸奶的消费者。对于发酵食品类工作者、特别是利用乳酸菌作为发酵剂的生产厂家,产酸快、产酸量大是评估发酵剂性能的重要的指标,特别是用于酸奶生产的发酵剂尤其要求产酸快、产酸量大。
由于现阶段有很多食品工厂生产设备中的发酵罐还没有恒温系统,发酵过程的发酵温度控制主要是靠发酵罐自身的保温体系夹套(夹层)等来实现发酵过程中的温度控制,虽然此保温系统存在能耗小、节能环保、设备投资小、使用成本低等特点,但是实际生产过程中受环境温度变化的影响较为明显,由于季节性的温度变化可能会导致发酵物料4℃左右的温度变化,然而现有的发酵剂4℃左右的温度变化会导致产酸速度和产酸量的明显变化,从而导致生产过程中会出现季节性的较难控制。为此开发一款对发酵温度较为宽泛的发酵剂,就显得很有必要。
发明内容
本发明提供一种鼠李糖乳杆菌LRH09、由其制备的发酵剂及发酵剂的应用,本发明发酵剂发酵温度宽泛。
为解决上述技术问题,本发明所采用的技术方案如下:
一种鼠李糖乳杆菌LRH09,已于2017年11月27日寄存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.14962。
本发明所述的鼠李糖乳杆菌LRH09是鼠李糖乳杆(Lactobacillus rhamnosus)LRH09,该菌株的16S rDNA序列为:
AAGCATTGCTCCCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACACAGTTACTCTGCCGACCATTCTCTCACACAGAGTTTACGACCCGAAAGCTCTCACTCACGCGGCGTGCTCATCAGACTGCGTCCATGTGAGATCCCTACTGCTGCCTCCCGTAGAGTTTGGGCCGTTTCTCAGTCCCAAT。
一种发酵剂,由瑞士乳杆菌LH43和上述鼠李糖乳杆菌LRH09混合而成。本申请鼠李糖乳杆菌LRH09可直接作为发酵剂,但与瑞士乳杆菌LH43混合后产酸效果更好、产酸温度更宽。
瑞士乳杆菌LH43,已寄存于中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号为CGMCC No.9885,该菌株的16S rDNA序列为:
TCTACTGTCTTAGACGGCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGTCGCCTTCGCCACTGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGAATTCACTCTCTCTTCTGCACTCAGAAAAACAGTTTCGATGCAGTTCCTCGTTAGCCGAGGCTTTCACATCAGACTTATTCTTCGCTGCGCCTCGCTTACGCCAATAATCGACACGCTGCCACTACGTATTACCGCGCTGCTGCACGTAGTAGCTGACTTTCTGTGATACGTCATACGGCAGTACTACTCTATCTCTCCACACGAGCTACGATCGAAACTCTATCACGCTGCTCATCGACTGCTCATTGGAGATCTCGTGCTCGTAGATGCGTCAGTCATTGCGACGTTCATCGCATGCTCATGCTGTGAACCGTACTACA。
上述发酵剂由瑞士乳杆菌LH43和鼠李糖乳杆菌LRH09按照菌数比例3:4~4:3混合。
上述发酵剂的发酵温度为37℃~43℃。申请人经研究发现,在37℃~43℃的范围内,上述发酵剂的产酸能力受温度的影响很小,可忽略。
上述发酵剂可用于制备风味发酵乳,优选,其投菌量是总量0.05%。所制备的风味发酵乳为较为愉悦的天然的发酵风味。制备方法包括奶水制备、灭菌、冷却、接菌、发酵和灌装,具体参见现有发酵乳的制备。
上述发酵剂可用于制备发酵果汁,优选,其投菌量是总量0.05%。上述发酵剂发酵产生的发酵风味能与果汁的香气较为合适的融合而产生较为舒适的整体风味。制备方法包括果汁制备、灭菌、冷却、接菌、发酵和灌装,具体参见现有发酵果汁的制备。
上述发酵剂可用于制备发酵豆奶,优选,其投菌量是总量0.05%。所制备的豆奶发酵乳为较为愉悦的天然的发酵风味。制备方法包括豆浆制备、灭菌、冷却、接菌、发酵和灌装,具体参见现有发酵豆奶的制备。
本发明未提及的技术均参照现有技术。
本发明鼠李糖乳杆菌LRH09可用于制备发酵剂,所得的发酵剂,产酸快、产酸量大、产酸温度宽。
附图说明
图1为瑞士乳杆菌LH43产酸能力图;
图2为鼠李糖乳杆菌LRH09产酸能力图;
图3为瑞士乳杆菌LH43和鼠李糖乳杆菌LRH09混合物产酸能力图;
具体实施方式
为了更好地理解本发明,下面结合实施例进一步阐明本发明的内容,但本发明的内容不仅仅局限于下面的实施例。
实施例中,鼠李糖乳杆菌LRH09,已于2017年11月27日寄存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.14962;瑞士乳杆菌LH43,已寄存于中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号为CGMCC No.9885;
瑞士乳杆菌LH43的培育方式及冻干粉的制备参见申请号为2014107498412的专利;
鼠李糖乳杆菌LRH09的培育方式如下:
(1)制备MRS培养基
MRS培养基的具体成分如下:
制法:将上述成分加入到1000mL蒸馏水中,加热溶解,调节pH至6.2±0.2,分装后121℃高压灭菌15min~20min,得MRS培养基。
(2)从酸奶(也可以是其它乳制品)中分离得到鼠李糖乳杆菌LRH09:
第一步骤:取市场酸奶做为乳酸菌筛选来源样品,样品接种于10wt%脱脂奶粉培养液中,先进行37℃,18小时之集殖培养(Enrichment culture),并筛选出产生凝乳现象之菌株;
第二步骤:取集殖培养过后的样品进行序列稀释及平板划线培养,并于37℃厌氧条件下培养3天,以挑选出不同菌相之单一菌落;
第三步骤:将该具有单一菌落进行革兰氏染色及触酶反应测试,并筛选出革兰氏染色呈阳性且触酶反应呈阴性之数株菌株,即为初步筛选之乳酸菌菌株,并将该初步筛选出的乳酸菌菌株进行菌种之鉴定,该初步筛选出的数株乳酸菌菌株为鼠李糖乳杆菌,将该菌株命名为鼠李糖乳杆菌LRH09;
(3)鼠李糖乳杆菌LRH09加无菌生理盐水进行稀释;
(4)选择3个稀释度(10-6,10-7,10-8),每个浓度各以0.1mL分别置于2个MRS琼脂平板(每个平板0.1mL),使用L形棒进行表面涂布,37℃,厌氧培养48h;
(5)用接种环挑取平板上的菌落(在菌落分布均匀,菌落在30-100个的平板上挑取),接种到发酵培养基(发酵培养基的具体组成为:葡萄糖25g/L,大豆蛋白胨10g/L,酵母粉8g/L,K2HPO4 2g/L,CH3COONa 7g/L,柠檬酸钠3g/L,MgSO4 0.2g/L,FeCl2 0.1g/L,MnSO40.05g/L;)中高密度培养,在温度37℃的条件下发酵12h;然后离心分离得到菌泥,然后加入冷冻保护剂冷冻干燥制得鼠李糖乳杆菌LRH09冻干粉。
实施例1
1、瑞士乳杆菌LH43产酸能力测试:测试瑞士乳杆菌LH43冻干粉在10wt%脱脂奶粉培养液中分别在37℃、40℃、43℃的稳定环境中监测发酵基底的pH变化,pH变化越大说明其发酵产产酸能力越强。其变化如图1所示。
2、鼠李糖乳杆菌LRH09产酸能力测试:测试鼠李糖乳杆菌LRH09冻干粉在10wt%脱脂奶粉培养液中分别在37℃、40℃、43℃的稳定环境中监测发酵基底的pH变化,pH变化越大说明其发酵产产酸能力越强。其变化如图2所示。
3、瑞士乳杆菌LH43和鼠李糖乳杆菌LRH09混合物产酸能力测试:将瑞士乳杆菌LH43冻干粉和鼠李糖乳杆菌LRH09冻干粉按照菌数比例3:4混合,并在10wt%脱脂奶粉培养液中分别在37℃、40℃、43℃的稳定环境中监测发酵基底的pH变化,pH变化越大说明其发酵产产酸能力越强。其变化如图3所示。
由图1-3可明显看出:瑞士乳杆菌LH43和鼠李糖乳杆菌LRH09混合物的产酸能力明显好于瑞士乳杆菌LH43的产酸能力,也明显好于鼠李糖乳杆菌LRH09的产酸能力,且由图也可明显看出,瑞士乳杆菌LH43、鼠李糖乳杆菌LRH09单独产酸时,产酸能力受温度的影响较大,而瑞士乳杆菌LH43和鼠李糖乳杆菌LRH09的混合物则在不同温度37℃、40℃、43℃下的产酸能力基本一致。
综上,本发明通过瑞士乳杆菌LH43和鼠李糖乳杆菌LRH09进行复配,降低了因发酵温度的变化而导致的发酵剂产酸能力的变化,提高了产酸能力,可广泛应用于酸奶发酵、果汁发酵和豆奶发酵等。
实施例2
利用发酵剂制发酵乳的方法,包括如下步骤:
(1)奶水制备
将设备灭菌处理后按照配方要求称量足量的如下原料混合均匀:牛乳91.3%,白砂糖7%,变性淀粉0.4%,果胶0.1%和琼脂1.2%;
(2)灭菌
灭菌的温度为92℃,时间为5min,然后冷却至43℃;
(3)接菌;
在经步骤2)处理的奶液中接入占总重量0.05%的发酵剂(瑞士乳杆菌LH43冻干粉和鼠李糖乳杆菌LRH09冻干粉按照菌数比例3:4的混合物),混合冻干粉菌数≥1.0×1010cfu/g,
(4)发酵
于43℃进行发酵24h,搅拌形成发酵液;
(5)灌装,得到天然的发酵风味的发酵乳。
实施例3
利用发酵剂制发酵胡萝卜汁的方法,包括如下步骤:
(1)果汁制备
用胡萝卜榨汁成胡萝卜汁100%
(2)灭菌
灭菌的温度为92℃,时间为5min;冷却至43℃;
(3)接菌;
在经步骤2)处理的胡萝卜汁中接入占总重量0.05%的发酵剂(瑞士乳杆菌LH43冻干粉和鼠李糖乳杆菌LRH09冻干粉按照菌数比例3:4的混合物),混合冻干粉菌数≥1.0×1010cfu/g,
(4)发酵
于37℃进行发酵24h,搅拌形成发酵液;
(5)灌装,得胡萝卜风味发酵果汁。
实施例4
利用发酵剂制发酵豆奶的方法,包括如下步骤:
(1)豆浆制备
黄豆与水按照质量比例为1:3浸泡12h后,经磨浆机加水磨浆形成豆浆,其Brix(产品中的可溶性固形物的含量)为9%。
(2)灭菌
灭菌的温度为95℃,时间为15min;冷却至43℃;
(3)接菌;
在经步骤2)处理的豆奶中接入占总重量0.05%的发酵剂(瑞士乳杆菌LH43冻干粉和鼠李糖乳杆菌LRH09冻干粉按照菌数比例4:3的混合物),混合冻干粉菌数≥1.0×1010cfu/g,
(4)发酵
于37℃进行发酵24h,搅拌形成发酵液;
(5)灌装,得天然风味的发酵豆奶。
序列表
<110> 生合生物科技(南京)有限公司 生合生物科技(扬州)有限公司
<120> 一种鼠李糖乳杆菌LRH09、由其制备的发酵剂及发酵剂的应用
<130> 1
<141> 2017-12-28
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1154
<212> DNA
<213> Lactobacillus rhamnosus
<400> 1
aagcattgct cccttagacg gctcgctccc taaaagggtt acgccaccgg cttcgggtgt 60
tacaaactct catggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg 120
cgtgctgatc cgcgattact agcgattccg acttcgtgta ggcgagttgc agcctacagt 180
ccgaactgag aatggcttta agagattagc ttgacctcgc ggtctcgcaa ctcgttgtac 240
catccattgt agcacgtgtg tagcccaggt cataaggggc atgatgattt gacgtcatcc 300
ccaccttcct ccggtttgtc accggcagtc ttactagagt gcccaactaa atgctggcaa 360
ctagtcataa gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga 420
cgacaaccat gcaccacctg tcattttgcc cccgaagggg aaacctgatc tctcaggtga 480
tcaaaagatg tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc 540
caccgcttgt gcgggccccc gtcaattcct ttgagtttca accttgcggt cgtactcccc 600
aggcggaatg cttaatgcgt tagctgcggc actgaagggc ggaaaccctc caacacctag 660
cattcatcgt ttacggcatg gactaccagg gtatctaatc ctgttcgcta cccatgcttt 720
cgagcctcag cgtcagttac agaccagaca gccgccttcg ccactggtgt tcttccatat 780
atctacgcat ttcaccgcta cacatggagt tccactgtcc tcttctgcac tcaagtttcc 840
cagtttccga tgcacttcct cggttaagcc gagggctttc acatcagact taaaaaaccg 900
cctgcgctcg ctttacgccc aataaatccg gataacgctt gccacctacg tattaccgcg 960
gctgctggca cgtagttagc cgtggctttc tggttggata ccgtcacgcc gacacagtta 1020
ctctgccgac cattctctca cacagagttt acgacccgaa agctctcact cacgcggcgt 1080
gctcatcaga ctgcgtccat gtgagatccc tactgctgcc tcccgtagag tttgggccgt 1140
ttctcagtcc caat 1154
Claims (7)
1.一种鼠李糖乳杆菌(Lactobacillus rhamnosus)LRH09,其特征在于:已于2017年11月27日寄存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.14962。
2.一种发酵剂,其特征在于:由瑞士乳杆菌LH43制备的冻干粉和权利要求1所述的鼠李糖乳杆菌LRH09制备的冻干粉混合而成,所述瑞士乳杆菌LH43已寄存于中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号为CGMCC No. 9885。
3.如权利要求2所述的发酵剂,其特征在于:瑞士乳杆菌LH43制备的冻干粉和鼠李糖乳杆菌LRH09制备的冻干粉按照菌数比例3:4~4:3混合。
4.如权利要求2所述的发酵剂,其特征在于:其发酵温度为37℃~43℃。
5.权利要求2-4任意一项所述的发酵剂的应用,其特征在于:用于制备风味发酵乳,发酵剂用量为风味发酵乳质量的0.05±0.01%。
6.如权利要求5所述的应用,其特征在于:用于制备发酵果汁,发酵剂用量为发酵果汁质量的0.05±0.01%。
7.如权利要求5所述的应用,其特征在于:用于制备发酵豆奶,发酵剂用量为发酵豆奶质量的0.05±0.01%。
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