CN112195119B - 一种能降解生物胺且耐盐的植物乳杆菌及其应用 - Google Patents
一种能降解生物胺且耐盐的植物乳杆菌及其应用 Download PDFInfo
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- CN112195119B CN112195119B CN202011004901.XA CN202011004901A CN112195119B CN 112195119 B CN112195119 B CN 112195119B CN 202011004901 A CN202011004901 A CN 202011004901A CN 112195119 B CN112195119 B CN 112195119B
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Abstract
本发明公开了一种能降解生物胺且耐盐的植物乳杆菌和包含该植物乳杆菌的一种菌剂,及它们在降解生物胺中的应用,以及在制备发酵食品中中的应用;所述植物乳杆菌在中国典型培养物保藏中心进行保藏,保藏地址为湖北省武汉市,保藏编号为CCTCC NO:M 2019757。该植物乳杆菌SHY610能降解食品中的生物胺,降解率高,对尸胺、组胺、酪胺的降解率分别达到了62.42%、74.32%和89.97%;SHY610还具有良好的耐盐特性,在含盐量高达9.00%的环境中也能够较好的生长繁殖,也适合于含盐量较高的发酵食品生产。
Description
技术领域
本发明涉及微生物及食品发酵技术领域,具体涉及一种能降解生物胺且耐盐的植物乳杆菌及其应用。
背景技术
传统牦牛酸奶是青藏高原地区牧民用牦牛乳经传统自然发酵的方法制作而成,是青藏高原地区牧民日常食用的特有美食,具有悠久历史,经过长时间的自然选择,部分具有益生特性和优良品质的菌种被保留下来。牦牛乳中不仅含有丰富的维生素同时也包含人体所需的8种必需氨基酸,而且含有丰富的乳酸菌资源。
乳酸菌是一类对宿主有益且能在碳水化合物中发酵产生大量乳酸的细菌的统称。研究表明,益生乳酸菌具有改善和调节肠道菌群、抑制致病菌的生长、调节机体血糖平衡、降低血清胆固醇、预防肿瘤发生等功效。目前我国市场销售的益生菌发酵乳制品的发酵菌种大部分采用嗜热链球菌和保加利亚乳杆菌。上述两种菌种的商品化使用,使得市售酸奶中的乳酸菌无法呈现多样性。此外,我国乳制品行业大多采用国外益生菌发酵剂,成本高的同时也制约我国益生乳酸菌产业的发展。对于这些国外的菌种我国乳制品企业都没有自主知识产权,只有使用权,解决这一问题应从研发具有自主知识产权的益生乳酸菌菌种和发酵剂相关技术及产品出发。因此,合理充分利用我国青藏高原等地区的乳酸菌资源,筛选出具有优良特性的菌种,对提高我国益生菌品质和发酵食品的国际竞争力、开发自主知识产权的益生菌具有重要意义。
生物胺是一类具有生物活性、含氮低分子量的有机化合物,主要由微生物的氨基酸脱羧酶对氨基酸(即组氨酸、酪氨酸、鸟氨酸、赖氨酸、苯丙氨酸和色氨酸)的脱羧作用而形成的有机化合物(即组胺、酪胺、腐胺、尸胺、苯乙胺和色胺)。研究表明摄入适量浓度的生物胺能够促进生长、增强代谢活力、加强肠道免疫系统、控制血压和消除自由基等,但生物胺也是生成致癌物质和亚硝酸物质的前体,摄入的浓度过高则会对人体产生毒害作用,造成心血管疾病和神经系统的损伤等。尸胺虽然本身的毒性较低,但是它可以抑制组胺和酪胺代谢酶,使组胺和酪胺的毒性增强。
目前生产上控制生物胺一般采用超高压处理、使用食品添加剂和低温保藏等方法,这些方法均能够在一定程度上减少食品中生物胺含量,但不能够降解已经产生的生物胺,而且这些方法会对食品的风味或质量产生影响。利用发酵剂来控制发酵食品中的生物胺含量具有高效安全、成本低等特点。已有研究表明,传统发酵食品中乳酸菌具有降解生物胺的能力。
发明内容
本发明的目的是针对上述问题,提供一种能降解生物胺且耐盐的植物乳杆菌及其应用。
本发明为了实现其目的,采用的技术方案是:
一种能降解生物胺且耐盐的植物乳杆菌,所述植物乳杆菌在中国典型培养物保藏中心进行保藏,保藏编号为CCTCCNO:M 2019757。
一种菌剂,所述菌剂包含上述的植物乳杆菌。
上述的植物乳杆菌或菌剂在降解生物胺中的应用。
所述生物胺为尸胺、组胺和酪胺。
上述的植物乳杆菌或菌剂在制备发酵食品中中的应用。
所述发酵食品包括发酵乳、泡菜、豆豉、芽菜、豆瓣、大酱、鱼露、腌鱼、腊肉、火腿。
本发明的有益效果是:提供的植物乳杆菌SHY610能降解食品中的生物胺,降解率高,对尸胺、组胺、酪胺的降解率分别达到了62.42%、74.32%和89.97%;SHY610还具有良好的耐盐特性,在含盐量高达9.00%的环境中也能够较好的生长繁殖,也适合于含盐量较高的发酵食品生产。该乳杆菌是从发酵牦牛酸乳中筛选得到的,具有优良益生特性,因此该乳杆菌尤其适合于干酪和豆豉等食品的生产,对于功能性乳酸菌发酵剂的研发、建立乳酸菌菌种资源库以及一定程度解决食品安全问题具有重要的现实意义。
附图说明
图1为植物乳杆菌SHY610观察图,其中,(a)为菌落形态图,(b)为革兰氏染色结果。
图2为植物乳杆菌SHY610的API 50 CH反应结果。
图3为显色培养试验结果图。
图4为生物胺HPLC标准品谱图,图中,1为尸胺,2为组胺,3为酪胺。
图5为生物胺含量标准曲线图。
图6为植物乳杆菌SHY610在不同含盐量下耐盐性结果图。
具体实施方式
下面结合实施例对本发明作进一步说明,但并不因此而限制本发明。
下述实施例中的实验方法,如无特别说明,均为常规方法。
主要试剂来源或成分:
碳水化合物鉴定试剂盒API 50 CH:品牌梅里埃;
生物胺标准品购于Sigma公司;
MRS肉汤:酪蛋白酶消化物10.0g/L,牛肉膏粉10.0g/L,酵母膏粉4.0g/L,葡萄糖20.0g/L,硫酸镁0.2g/L,乙酸钠5.0g/L,柠檬酸三铵2.0g/L,磷酸氢二钾2.0g/L,硫酸锰0.05g/L,吐温801.08g/L,最终pH为5.7±0.2。
MRS液体培养基:酪蛋白酶消化物10.0g/L,牛肉膏粉10.0g/L,酵母膏粉4.0g/L,葡萄糖20.0g/L,硫酸镁0.2g/L,乙酸钠5.0g/L,柠檬酸三铵2.0g/L,磷酸氢二钾2.0g/L,硫酸锰0.05g/L,吐温801.08g/L,最终pH为5.7±0.2。
其余试剂如未表明,均为本领域常规试剂,可商购获得。
实施例1植物乳杆菌SHY610的鉴定与降解生物胺、耐盐实验
1实验材料
从四川省阿坝藏族羌族自治州红原县地区牧民家中采集的传统发酵牦牛酸乳分离筛选得到的乳酸菌。
2实验方法
2.1菌株鉴定试验
将保藏在安瓿管中的乳酸菌接种到MRS肉汤中,活化两代后进行平板划线和革兰氏染色镜检,若菌株为纯种则采用细菌基因组DNA提取试剂盒提取菌株的DNA。使用细菌通用引物27F(5’-AGA GTT TGA TCC TGG CTC AG-3’,SEQ ID No.1)和1492R(5’-TAC GGY TACCTT GTT ACG ACT T-3’,SEQ ID No.2)、采用25μL反应体系对其16S rDNA进行PCR扩增,反应结束后利用琼脂糖凝胶电泳进行检测。合格后委托华大基因科技有限公司对PCR扩增产物进行双向测序,测序结果通过NCBI中的BLAST程序进行同源性比对分析。将该菌株命名为SHY610。
2.2 API试剂盒鉴定
将菌株在37℃培养18h,于4 000r/min、10min的条件下离心收集菌体,用无菌生理盐水洗涤菌体后重悬为菌悬液。采用碳水化合物鉴定试剂条(API 50 CH)鉴定菌株的生化特性,具体操作步骤参考试剂条说明书进行操作。
2.3显色培养试验
取活化好的菌悬液(107cfu/mL)以2%的接种量接入到显色培养基中,37℃培养48h,记录实验结果,显紫色的为阳性,显黄色即不变色的为阴性。做3组平行,空白培养基作对照组。若结果为阴性则进行下一步实验。
2.4生物胺测定试验
2.4.1柱前衍生
2mL的生物胺标准混合溶液(尸胺、组胺、酪胺),加入1mL 2mol/L NaOH溶液、10μL苯甲酰氯,然后旋涡振荡30s,置于30℃水浴条件下避光反应40min,期间每隔10min振荡一次。反应完后用2mL饱和NaCl溶液中断反应。然后用3mL乙醚萃取,1200r/min离心10min,用移液枪移取乙醚层,再重复一次,将乙醚层移置于10mL的离心管中,重复一次上述操作,35℃氮气吹干,最后用1mL甲醇(色谱级)溶解,用0.22μm的滤膜过滤后置于为10mL的离心管中,保存于4℃冰箱中待测。
2.4.2标准曲线绘制
准确称取尸胺、组胺、酪胺标准品各50mg,用0.1mol/L HCl溶液定容至50mL,制成浓度为1000μg/mL混合标准溶液备用。分别取以上标准储备液,用0.1mol/L HCl溶液配制成浓度分别为1.0、2.0、5.0、10、20、40、80μg/mL的混合标准溶液。然后进行2.3.1柱前衍生处理,分析检测后绘制标准曲线。
2.4.3生物胺的含量测定
取活化好的菌悬液(107cfu/mL)以2%的接种量分别接入到含三种生物胺(酪胺、尸胺、组胺)各500μg/mL的MRS液体培养基中,37℃培养48h后,分别取1mL菌液加入4mL0.1mol/L HCl溶液,然后按照2.4.1柱前衍生的方法进行处理,最后进行高效液相色谱测定生物胺含量。做3组平行,空白液体培养基作对照组。
2.4.4色谱条件
色谱柱为AgilentXDB-C18(250minX4.6mm,5μm),流动相A为超纯水,流动相B为甲醇,采用梯度洗脱,洗脱程序见下表。流速为1.0mL/min,紫外检测波长为254nm,进样量20μL,梯度洗脱程序如表1所示。
表1 梯度洗脱程序表
2.5耐盐性试验
取活化好的显色培养试验阴性菌株菌悬液(107cfu/mL)以2%的接种量分别接入到含不同NaCl浓度的MRS培养基中,NaCl质量百分比浓度的梯度设置为:0.00%、5.00%、7.00%、9.00%、11.00%、13.00%,37℃培养48h后,测定600nm下的OD值,并绘制成曲线。
2.6统计分析
试验数据通过Excel软件进行数据处理和作图。
3结果与分析
3.1菌株鉴定结果
SHY610菌落形态如图1(a)所示,革兰氏染色结果如图1(b)所示。菌株的生理生化性质如下:革兰氏阳性菌,细胞形态为短杆形,菌落形态:乳白色、圆形、突起、表面光滑湿润。
16S rDNA同源性分析结果显示,SHY610的16S rDNA基因扩增产物的序列如SEQ IDNo.3所示,与Gene Bank数据库中已知植物乳杆菌(Lactobacillus plantarum,Accessionnumber:MT645511.1)的同源性达100%。
3.2菌株的生化特性鉴定结果
乳酸杆菌种水平的表型鉴定主要依据碳水化合物发酵试验。API 50 CH试剂盒是通过菌株对49种不同碳水化合物的利用情况来进行鉴定。
图2示出了编号为SHY610的菌株的API 50 CH反应结果。表2示出了菌株SHY610对49种碳水化合物发酵试验结果。由图2和表2可知,在供试的49种碳源中,菌株SHY610可以利用其中24种碳水化合物。经API lab plus系统最终鉴定,编号为SHY610的菌株为植物乳杆菌(Lactobacillus plantarum),其ID值为99.90%,T值为0.96,达到鉴定要求(ID值≥99.0%且T值≥0.5)。
表2 SHY610对49种碳水化合物发酵试验结果
注:“+”代表反应阳性;“-”代表反应阴性。
3.3显色试验结果
乳酸菌在相对低酸或营养匮乏的条件下为了维持自身繁殖发育,受胁迫机制调控具有氨基酸脱羧酶活性的菌株将氨基酸脱羧形成相应的生物胺(一种碱性物质)。溴甲酚紫在酸性条件下显示黄色,表明菌株不具有氨基酸脱羧酶活性即为不产生物胺的阴性菌株,在碱性条件下显示紫色,表明菌株具有氨基酸脱羧酶活性即为产生物胺的阳性菌株。
根据液体培养基的显色情况(如图3所示)初步判断植物乳杆菌SHY610为阴性菌株。
3.4生物胺测定结果
3.4.1标准曲线的绘制
取标准溶液采用前述方法衍生,7种不同浓度混合标准溶液重复测定3次。将三种生物胺以生物胺浓度为横坐标,峰面积为纵坐标进行线性回归,绘制标准曲线。
从图4中可以看出,各种生物胺标准品在高效液相色谱上分离明显、出峰速度快,20分钟以内三种生物胺全部出峰,分别为:尸胺在7.1分钟左右出峰,组胺在16.7分钟左右出峰,酪胺在18.9分作左右出峰。由图5可以看出三种生物胺的峰面积与其相应浓度分别呈现良好的线性关系,线性回归方程及相关系数R2见表2,相关系数R2均大于0.99,证明该方法测定三种生物胺具有良好的可靠性。
表2 线性回归方程表
3.4.2生物胺降解能力结果
前述显色试验是一种初步的筛选方法,而HPLC是定量检测生物胺的方法,从而保证了实验结果的准确性和严谨性,可以对菌株降解生物胺的能力做出准确的评价。
HPLC检测结果见表3,植物乳杆菌SHY610对三种生物胺均有不错的降解能力,是一株较理想的高效生物胺降解菌株。
表3 植物乳杆菌SHY610对生物胺的降解率
3.5耐盐性试验结果
我国大部分发酵食品具有高盐环境,因此若想将生物胺降解菌株运用于发酵食品中就需要菌株能够适应高盐高渗透压的环境。
对菌株SHY610进行耐盐性试验,测定其OD600nm,结果如图6所示,发现菌株随着盐浓度增加,生长逐渐受到抑制。菌株受到抑制是因为高浓度含盐量的环境对乳酸菌的生长繁殖具有盐胁迫作用,抑制了乳酸菌的生长繁殖,同时影响乳酸菌的存活率。同时高浓度含盐量的环境会增加溶液的渗透压,使乳酸菌细胞内水分外流,细胞胞质分离,从而造成细胞在结构和生理上损伤,导致细胞停止生长甚至死亡。
常见发酵食品的含盐量范围一般为2.50%-10.00%,SHY610可在此范围内良好的生长,因此可运用于发酵食品。
将菌株SHY610于2019年9月送中国典型培养物保藏中心(简称CCTCC)进行保藏,地址为湖北省武汉市武昌区八一路299号武汉大学校内,保藏日期为2019年10月11日,保藏编号为CCTCC NO:M 2019757,分类名为植物乳杆菌SHY610 LactobacillusplantarumSHY610。
实施例2植物乳杆菌SHY610菌剂用于花椒芽菜制备
制备植物乳杆菌SHY610菌剂:取实施例1中MRS肉汤中活化好的培养液,4 000r/min离心10min,弃去上清,收集到的菌体沉淀用无菌生理盐水洗涤得到活性菌泥,每10mL培养液的得到菌泥加入1mL冻干保护剂(10%脱脂乳+0.1%谷氨酸钠),置于超低温冰箱中速冻后取出,真空冷冻干燥得到植物乳杆菌SHY610菌剂。
将50重量份花椒芽菜剔除杂质和不可食的部分,清水洗净泥沙和污物,使用不锈钢隧道式蒸汽杀青设备进行蒸汽杀青,使青花椒芽叶(苗)杀透变软,花椒芽叶(苗)堆放厚度4-8cm,杀青时间3-5min,隧道内温度在90℃-100℃,杀青后快速摊凉到15℃-25℃。将杀青后花椒芽叶(苗)与混合盐(混合盐用量按杀青后花椒芽叶质量计算,食盐8%,氯化钙0.1%)拌合均匀,按2%的接种量接种植物乳杆菌SHY610菌剂,接种室温下在不锈钢大桶里压紧腌制4-7天。从不锈钢大桶里取出花椒芽叶(苗)菜,压榨适当脱水。将第一次翻池后的花椒芽叶(苗)菜与剩余盐(按杀青后花椒芽叶质量计算4-8%)拌合均匀,室温下在不锈钢大桶里压紧腌制7-14天。从不锈钢大桶或腌制池里再次取出花椒芽叶(苗)菜,压榨适当脱水。重新放入大不锈钢桶,压紧,桶口覆盖双层食品级塑料薄膜后,用细沙或食盐封口,即得到花椒芽菜成品。
序列表
<110> 西南大学
<120> 一种能降解生物胺且耐盐的植物乳杆菌及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tacggytacc ttgttacgac tt 22
<210> 3
<211> 1382
<212> DNA
<213> 植物乳杆菌SHY610(Lactobacillus plantarum SHY610)
<400> 3
tgattggtgc ttgcatcatg atttacattt gagtgagtgg cgaactggtg agtaacacgt 60
gggaaacctg cccagaagcg ggggataaca cctggaaaca gatgctaata ccgcataaca 120
acttggaccg catggtccga gtttgaaaga tggcttcggc tatcactttt ggatggtccc 180
gcggcgtatt agctagatgg tggggtaacg gctcaccatg gcaatgatac gtagccgacc 240
tgagagggta atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc 300
agtagggaat cttccacaat ggacgaaagt ctgatggagc aacgccgcgt gagtgaagaa 360
gggtttcggc tcgtaaaact ctgttgttaa agaagaacat atctgagagt aactgttcag 420
gtattgacgg tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 480
cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggtttttta 540
agtctgatgt gaaagccttc ggctcaaccg aagaagtgca tcggaaactg ggaaacttga 600
gtgcagaaga ggacagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga 660
acaccagtgg cgaaggcggc tgtctggtct gtaactgacg ctgaggctcg aaagtatggg 720
tagcaaacag gattagatac cctggtagtc cataccgtaa acgatgaatg ctaagtgttg 780
gagggtttcc gcccttcagt gctgcagcta acgcattaag cattccgcct ggggagtacg 840
gccgcaaggc tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 900
tttaattcga agctacgcga agaaccttac caggtcttga catactatgc aaatctaaga 960
gattagacgt tcccttcggg gacatggata caggtggtgc atggttgtcg tcagctcgtg 1020
tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttattatcag ttgccagcat 1080
taagttgggc actctggtga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1140
aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggatg gtacaacgag 1200
ttgcgaactc gcgagagtaa gctaatctct taaagccatt ctcagttcgg attgtaggct 1260
gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1320
tacgttcccg ggccttgtac acaccgcccg tcacaccatg agagtttgta acacccaaag 1380
tc 1382
Claims (6)
1.一种能降解生物胺且耐盐的植物乳杆菌(Lactobacillus plantarum)菌株SHY610,其特征在于:所述植物乳杆菌菌株在中国典型培养物保藏中心进行保藏,保藏编号为CCTCCNO:M 2019757。
2.一种菌剂,其特征在于:所述菌剂包含权利要求1所述的植物乳杆菌菌株SHY610。
3.权利要求1所述的植物乳杆菌菌株SHY610或权利要求2所述的菌剂在体外降解生物胺中的应用。
4.如权利要求3所述的应用,其特征在于:所述生物胺为尸胺、组胺和酪胺。
5.权利要求1所述的植物乳杆菌菌株SHY610或权利要求2所述的菌剂在制备发酵食品中的应用。
6.如权利要求5所述的应用,其特征在于:所述发酵食品为发酵乳、泡菜、豆豉、芽菜、豆瓣、大酱、鱼露、腌鱼、腊肉、火腿。
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