CN110713958A - 一株酪丁酸梭菌及其应用 - Google Patents
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Abstract
本发明公开了一株酪丁酸梭菌及其应用。该菌株保藏编号为GDMCC NO:60753,筛选自新疆地区奶牛的粪便,该菌株经过发酵培养,菌悬液和细胞破碎液均可以有效降解α‑乳白蛋白和β‑乳球蛋白,因而可用于去除牛乳中的过敏原,降低过敏反应的发病率,还可发酵生产低致敏乳源蛋白基料、制备低致敏性奶制品,降低婴儿乳品过敏的风险。
Description
技术领域
本发明属于食品生物技术领域,具体涉及一株酪丁酸梭菌及其应用。
背景技术
牛乳过敏(CMA)是一种最常出现的食物过敏反应,3岁以下的儿童发病约占25%,占所有确诊的食物过敏病例的9%。牛乳过敏主要是牛奶蛋白引起的过敏,牛乳过敏在临床上是对乳蛋白的异常免疫反应,这可能是某种或多种蛋白产生免疫作用,与IgE抗体结合并造成迅速的IgE参与的反应。婴儿牛奶过敏现象目前已经引起了人们广泛关注,经过研究发现牛乳中主要的致敏蛋白是ɑ-乳白蛋白和β-乳球蛋白。
为了降低牛乳蛋白的致敏性,目前已有很多方法,比如热处理、蛋白酶水解、乳酸发酵液降解等等。酪丁酸梭菌是一种人和动物肠道正常分布的革兰氏阳性芽孢杆菌,较非芽胞益生菌制剂具有耐热、耐酸和耐多种抗生素等生物学特性。酪丁酸梭菌能合成丁酸、乙酸等短链脂肪酸,以及多种氨基酸、B族维生素和维生素K,能分解胺类、吲哚类、硫化氢等有害物质,还能促进双歧杆菌、乳酸菌等益生菌的生长;而酪丁酸梭菌用于降解致敏蛋白的研究未见公开报道。
发明内容
本发明的目的提供一株酪丁酸梭菌及其应用。该菌株可以降解牛奶中的主要致敏蛋白,尤其是过敏原中的β-乳球蛋白。
第一方面,本发明提供一株酪丁酸梭菌,其保藏编号为GDMCC NO:60753。
第二方面,本发明提供上述酪丁酸梭菌的培养方法。具体为,将酪丁酸梭菌接种至培养基,在37℃条件下厌氧培养。
进一步的,所述培养基为RCM培养基,其成分为:蛋白胨1% (w/v),牛肉粉1%(w/v),酵母粉0.3% (w/v),葡萄糖0.5% (w/v) ,可溶性淀粉0.1% (w/v) ,氯化钠0.5% (w/v) ,醋酸钠0.3% (w/v) ,L-半胱氨酸盐酸盐0.05% (w/v) ,琼脂0.05% (w/v),pH 6.0。
进一步的,接种量为5%。
第三方面,本发明提供上述酪丁酸梭菌在降解牛奶中致敏蛋白中的应用。
所述致敏蛋白包括β-乳球蛋白、α-乳白蛋白。
本发明提供了一种具体应用方法,包括:将酪丁酸梭菌接入RCM培养基培养活化,取活化好的酪丁酸梭菌转接到新鲜的RCM培养基中培养,离心去除上清液,用磷酸缓冲液将菌体重悬,形成不再增殖的菌悬液;
将菌悬液与分离乳清蛋白混合反应。
或将酪丁酸梭菌接入RCM培养基培养活化,取活化好的酪丁酸梭菌转接到新鲜的RCM培养基中培养,离心去除上清液,将离心得到的菌体加入缓冲液后进行细胞破碎得到细胞破碎液;
将细胞破碎液与分离乳清蛋白混合反应。
进一步的,酪丁酸梭菌培养条件为:37℃厌氧条件下培养24 h。
进一步的,所述分离乳清蛋白成分包括β-乳球蛋白和α-乳白蛋白。
进一步的,菌悬液/细胞破碎液与分离乳清蛋白混合反应的条件为:20~50℃条件下反应24 h;优选37℃。
本发明的酪丁酸梭菌是从新疆地区褐牛的粪便中分离得到,通过选择性富集筛选可获得水解牛乳蛋白的菌株。该菌株经过发酵后能有效降解β-乳球蛋白、α-乳白蛋白,因而可用于去除牛乳中的过敏原,降低过敏反应的发病率,还可发酵生产低致敏乳源蛋白基料、制备低致敏性奶制品,降低婴儿乳品过敏的风险。
本发明所述的生物材料,其分类命名为酪丁酸梭菌(Clostridium tyrobutyricum)Z816,于2019年8月28日保藏于广东省微生物菌种保藏中心(GDMCC),保藏编号为GDMCC NO:60753,地址:广州市先烈中路100号大院59号楼5楼。
具体实施方式
梭菌增殖培养基(RCM培养基):蛋白胨1.0% (w/v),牛肉粉1.0%(w/v),酵母粉0.3%(w/v),葡萄糖0.5% (w/v) ,可溶性淀粉0.1% (w/v) ,氯化钠0.5% (w/v) ,醋酸钠0.3%(w/v) ,L-半胱氨酸盐酸盐0.05% (w/v) ,pH 6.0。
梭菌选择性培养基(TSN培养基):蛋白胨1.0% (w/v),酵母粉0.3% (w/v) ,亚硫酸钠0.1% (w/v),新生霉素0.003% (w/v),多粘菌素0.003% (w/v),pH 6.0。
实施例1 酪丁酸梭菌的筛选方法
取新疆褐牛的粪便样品10 g,混匀后加入90 mL PBS缓冲液中,置于100℃水浴10 min,杀死非芽孢菌,转接至RCM培养基中37℃厌氧培养24 h;在生长过程的对数期补料分批加入淀粉、木质纤维素等碳源,酪丁酸梭菌可分解利用多种碳源进行富集培养,提升自身活力的同时还可降低粪便中大肠杆菌的数量,培养24 h后将上述培养液转接入TSN培养基中,37℃厌氧培养24 h;酪丁酸梭菌具有一定的抗逆性,通过选择培养基可以进行二次筛选,将得到的样品分别稀释到10-2、10-4、10-6,取100 μL涂布到分离乳清蛋白平板上,37℃培养48 h;挑选有水解圈产生的菌落,再次转接到新的分离乳清蛋白平板上,37℃静置培养24 h。观察水解圈大小及菌落形态,筛选水解圈最大的单菌落,转接于RCM培养基中活化12 h后,再转接于含30%甘油的RCM培养基中藏于-80℃培养。
该菌株的16S rRNA序列如SEQ ID NO:1,菌株鉴定结果表明是酪丁酸梭菌。
实施例2 酪丁酸梭菌菌悬液对α-乳白蛋白、β-乳球蛋白的降解效果
标准曲线的绘制:准确称量β-乳球蛋白标准品、α-乳白蛋白标准品 8 mg,加入 2 mLPBS进行溶解,配置成 4 mg/mL 的标准原液,吸取适量原液,加入PBS进行稀释,稀释成4、2、1、0.5、0.25、0.125、0.0625、0.03125 mg/mL浓度梯度的标准溶液进行测定。色谱柱: Bio-C8 (250mm × 4.6mm,5μ m);流动相:0.1%三氟乙酸(TFA)水(A),0.1% 三氟乙酸(TFA)乙腈(B),梯度洗脱条件: 0~17.5 min,B 70%~50%; 17.5~20 min,B 50%~70%。柱温:35℃;流速:1.0 mL/min;进样量:10 μ L;检测波长:280 nm。根据标准品浓度和峰面积绘制标准曲线。
将保藏于-80℃甘油管中的酪丁酸梭菌(Clostridium tyrobutyricum)Z816以5%的接种量接入RCM培养基,在37℃厌氧条件下培养24 h进行活化,以2%的接种量分别取活化好的酪丁酸梭菌转接到新鲜的RCM培养基中,在37℃厌氧条件下培养24 h, 8000 rpm条件下离心10 min,去除含有培养基的上清液,用磷酸缓冲液将菌体重悬,形成不再增殖的菌悬液。
将菌悬液与3mg/mL的分离乳清蛋白样品(含有45%β-乳球蛋白和15%α-乳白蛋白)1:1进行混合,在37℃条件下有氧反应24 h,8000 rpm条件下离心10 min,取上清液。以不加菌悬液的样品为对照,利用高效液相色谱法在所述色谱条件下分别检测样品中β-乳球蛋白和α-乳白蛋白的浓度。每个样品重复测定5次。
表1. 酪丁酸梭菌菌悬液对β-乳球蛋白、α-乳白蛋白的降解效果
样品(不添加菌体) | 样品(添加菌体) | 去除率 | |
β-乳球蛋白浓度(mg/mL) | 1.35 | 0.20 | 85.2% |
α-乳白蛋白浓度(mg/mL) | 0.45 | 0.22 | 51.1% |
实施例3 酪丁酸梭菌细胞破碎液对α-乳白蛋白、β-乳球蛋白的降解效果
标准曲线的绘制:准确称量β-乳球蛋白标准品、α-乳白蛋白标准品 8 mg,加入2 mLPBS进行溶解,配置成4 mg/mL 的标准原液,吸取适量原液,加入PBS进行稀释,稀释成4、2、1、0.5、0.25、0.125、0.0625、0.03125 mg/mL浓度梯度的标准溶液进行测定。色谱柱:Bio-C8(250 mm × 4.6 mm,5 μ m);流动相: 0.1%三氟乙酸(TFA)水(A),0.1% 三氟乙酸(TFA)乙腈(B),梯度洗脱条件: 0~17.5 min,B 70%~50%; 17.5~20 min,B 50%~70%。柱温:35℃;流速:1.0mL/min;进样量:10 μ L;检测波长:280 nm。根据标准品浓度和峰面积绘制标准曲线。
将保藏于-80℃甘油管中的酪丁酸梭菌(Clostridium tyrobutyricum) Z816以2%的接种量接入RCM培养基,在37℃厌氧条件下培养24 h进行活化,以2%的接种量分别取活化好的酪丁酸梭菌转接到新鲜的RCM培养基中,在37℃厌氧条件下培养24 h,8000 rpm条件下离心10 min,去除含有培养基的上清液,将离心得到的菌体加入PBS后进行细胞破碎20分钟得到细胞破碎液。
将细胞破碎液与3mg/mL的分离乳清蛋白样品(主要含有45%β-乳球蛋白和15%α-乳白蛋白)1:1进行混合,在37℃条件下有氧反应24 h,8000 rpm条件下离心10 min,取上清液。以不加细胞破碎液的样品为对照,利用高效液相色谱法在所述色谱条件下分别检测样品中β-乳球蛋白和α-乳白蛋白的浓度。每个样品重复测定5次。
表2. 酪丁酸梭菌细胞破碎液对β-乳球蛋白、α-乳白蛋白的降解效果
样品(不添加菌体) | 样品(添加菌体) | 去除率 | |
β-乳球蛋白浓度(mg/mL) | 1.35 | 0.10 | 92.6% |
α-乳白蛋白浓度(mg/mL) | 0.45 | 0.04 | 91.1% |
实施例4 不同温度下酪丁酸梭菌菌悬液对α-乳白蛋白、β-乳球蛋白的降解效果
采用实施例2的方法制备菌悬液。将菌悬液与3mg/mL的分离乳清蛋白样品(含有45%β-乳球蛋白和15%α-乳白蛋白)1:1进行混合,分别在20℃、30℃、37℃、40℃、50℃条件下有氧反应24 h,8000 rpm条件下离心10 min,取上清液。以不加菌悬液的样品为对照,利用高效液相色谱法在所述色谱条件下分别检测样品中β-乳球蛋白和α-乳白蛋白的浓度以计算致敏蛋白的去除率,每个样品重复测定5次。结果如下表3所示。
表3 不同温度下酪丁酸梭菌菌悬液对α-乳白蛋白、β-乳球蛋白的降解效果
20℃ | 30℃ | 37℃ | 40℃ | 50℃ | |
β-乳球蛋白去除率(%) | 78.6 | 83.1 | 85.2 | 82.1 | 77.6 |
α-乳白蛋白去除率(%) | 48.2 | 50.4 | 51.1 | 49.8 | 47.6 |
可以看出,菌悬液在20~50℃下均可稳定去除致敏蛋白。
序列表
<110> 南京工业大学
<120> 一株酪丁酸梭菌及其应用
<130> xb19112601
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1371
<212> DNA
<213> 酪丁酸梭菌Z816(Clostridium tyrobutyricum)
<400> 1
tgcagtcgag cgatgaaccc cttcgggggt ggattagcgg cggacgggtg agtaacacgt 60
gggtaacctg cctcaaagtg ggggatagcc ttccgaaagg aagattaata ccgcataaag 120
ccaagtttca catggaattt ggatgaaagg agtaattcgc tttgagatgg acccgcggcg 180
cattagttag ttggtggggt aatggcctac caagacagcg atgcgtagcc gacctgagag 240
ggtgatcggc cacattggaa ctgagatacg gtccagactc ctacgggagg cagcagtggg 300
gaatattgca caatgggcga aagcctgatg cagcaacgcc gcgtgagtga tgaaggtctt 360
cggattgtaa agctctgtct tttgggacga taatgacggt accaaaggag gaagccacgg 420
ctaactacgt gccagcagcc gcggtaatac gtaggtggcg agcgttgtcc ggatttactg 480
ggcgtaaagg gtgcgtaggc ggatgtttaa gtgagatgtg aaatacccgg gcttaacttg 540
ggtgctgcat ttcaaactgg atatctagag tgcaggagag gagaatggaa ttcctagtgt 600
agcggtgaaa tgcgtagaga ttaggaagaa caccagtggc gaaggcgatt ctctggactg 660
taactgacgc tgaggcacga aagcgtgggt agcaaacagg attagatacc ctggtagtcc 720
acgccgtaaa cgatgagtac taggtgtagg aggtatcgac cccttctgtg ccgcagtaaa 780
cacattaagt actccgcctg ggaagtacga tcgcaagatt aaaactcaaa ggaattgacg 840
ggggcccgca caagcagcgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 900
tggacttgac atcccctgaa taacctagag ataggcgaag cccttcgggg cagggagaca 960
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt aggttaagtc ctgcaacgag 1020
cgcaaccctt attgttagtt gctaacattc agttgagcac tctaacgaga ctgccgcggt 1080
taacgcggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgtc cagggcaaca 1140
cacgtgctac aatgggcaga acaaagagaa gcaataccgc gaggtggagc caaactcaaa 1200
aactgctctc agttcggatt gcaggctgaa actcgcctgc atgaagctgg agttgctagt 1260
aatcgcgaat cagcatgtcg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1320
caccatgaga gctggcaaca cccgaagtcc gtagtctaac gtaagaggac g 1371
Claims (10)
1.一株酪丁酸梭菌,其特征在于,保藏编号为:GDMCC NO:60753。
2.权利要求1所述酪丁酸梭菌的培养方法,其特征在于,将酪丁酸梭菌接种至培养基,在37℃条件下厌氧培养。
3.权利要求2所述酪丁酸梭菌的培养方法,其特征在于,所述培养基为RCM培养基。
4.权利要求1所述酪丁酸梭菌在降解牛奶中致敏蛋白中的应用。
5.根据权利要求4所述的应用,其特征在于,所述致敏蛋白包括β-乳球蛋白、α-乳白蛋白。
6.根据权利要求5所述的应用,其特征在于,包括:
将酪丁酸梭菌接入RCM培养基培养活化,取活化好的酪丁酸梭菌转接到新鲜的RCM培养基中培养,离心去除上清液,用磷酸缓冲液将菌体重悬,形成不再增殖的菌悬液;
将菌悬液与分离乳清蛋白混合反应。
7.根据权利要求5所述的应用,其特征在于,包括:
将酪丁酸梭菌接入RCM培养基培养活化,取活化好的酪丁酸梭菌转接到新鲜的RCM培养基中培养,离心去除上清液,将离心得到的菌体加入缓冲液后进行细胞破碎得到细胞破碎液;
将细胞破碎液与分离乳清蛋白混合反应。
8.根据权利要求6或7所述的应用,其特征在于,酪丁酸梭菌培养条件为:37℃厌氧条件下培养24 h。
9.根据权利要求6或7所述的应用,其特征在于,所述分离乳清蛋白成分包括β-乳球蛋白和α-乳白蛋白。
10.根据权利要求6或7所述的应用,其特征在于,菌悬液/细胞破碎液与分离乳清蛋白混合反应的条件为:20~50℃条件下反应24 h;优选37℃。
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