CN114437969A - 一种嗜酸乳杆菌md-286及其应用 - Google Patents
一种嗜酸乳杆菌md-286及其应用 Download PDFInfo
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Abstract
本发明公开一种新的嗜酸乳杆菌Lactobacillus acidophilus MD‑286,该菌株由产消化酶的方法两次筛选得到,具有优良的淀粉酶、蛋白酶、纤维素酶的生产能力,可制备成菌粉,用于具有益生功能的制品;同时,该菌株还具备促进骨细胞(C3H10T1/2)生长的功能,可进一步应用在促消化以外的保健功效上,拓展菌株的来源对社会经济生态效益产生积极作用。
Description
技术领域
本发明属于微生物工程领域,具体涉及一种嗜酸乳杆菌MD-286及其应用。
背景技术
嗜酸乳杆菌(Lactobacillus acidophilus)属于乳杆菌属(Lactobacillus),革兰氏阳性,不产芽抱,无鞭毛,不运动,同型发酵乳糖,不液化明胶,接触酶阴性,最适生长温度为35℃~38℃,最适pH值为5.5-6.4,G+C含量为36.0%-37.4%,是一类厌氧或兼性厌氧的微生物。嗜酸乳杆菌是栖息在人体肠道内的有益菌之一。
随着人们生活水平的日益提高,生活方式发生改变,膳食中高脂肪、高蛋白过量摄入,其引发的心脑血管疾病、肥胖等问题日益严重,成为威胁人们健康的主要因素之一。益生菌是用于提高人、动物和植物健康水平的活的微生物及其代谢产物,是改善宿主肠道微生物菌群平衡的核心功能因子。研究表明,益生菌具有调节肠道菌群平衡、预防和缓解多种慢性疾病的功能。
因此,拓展菌株的来源,选择具有一定益生功能的乳酸菌菌株并开发益生菌食品,将带来巨大的经济效益、社会效益和生态效应。
发明内容
为达到上述目的,本发明的技术方案提供一种新的嗜酸乳杆菌,所述嗜酸乳杆菌菌株编号为MD-286,分类命名为嗜酸乳杆菌Lactobacillus acidophilus;保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC;保藏地址:北京市朝阳区北辰西路1号院3号;保藏日期:2021年11月15日;保藏编号:CGMCC NO.23901。该菌株经全基因组测序,Blast搜索比对后未发现与其基因组完全一样的菌株。
上述嗜酸乳杆菌Lactobacillus acidophilus MD-286具有产蛋白酶、淀粉酶和纤维素酶的功能。
本发明还提供一种具有产蛋白酶、淀粉酶和纤维素酶功能的嗜酸乳杆菌Lactobacillus acidophilus MD-286菌粉,所述嗜酸乳杆菌菌粉是由权利1或2任一项所述嗜酸乳杆菌Lactobacillus acidophilus MD-286的发酵产物经冷冻干燥(真空度0.08MPa,冷肼温度-60℃)制备而成;所述菌粉具有超高细胞浓度,菌落形成单位大于1×1011CFU/g。
具体的,嗜酸乳杆菌Lactobacillus acidophilus MD-286菌粉的制备方法包括如下步骤:
1)将所述嗜酸乳杆菌Lactobacillus acidophilus MD-286菌粉接种在MRS培养液中在36~38℃下发酵12~36h,得到嗜酸乳杆菌Lactobacillus acidophilus MD-286发酵液;
2)将所述步骤1)的嗜酸乳杆菌Lactobacillus acidophilus MD-286发酵液离心,收集沉淀物,得到嗜酸乳杆菌Lactobacillus acidophilus MD-286菌泥;
3)将所述步骤2)的嗜酸乳杆菌Lactobacillus acidophilus MD-286菌泥进行真空冷冻干燥,得到嗜酸乳杆菌Lactobacillus acidophilus MD-286菌粉。
本发明还提供一种上述任一项所述嗜酸乳杆菌在促进骨细胞(C3H10T1/2)生长上的应用。
与现有技术相比,本发明的有益效果包括:1)提供一种新的嗜酸乳杆菌Lactobacillus acidophilus MD-286,由产消化酶的方法两次筛选得到,具有优良的淀粉酶、蛋白酶、纤维素酶的生产能力,可制备成菌粉,用于具有益生功能的制品;2)另外,该菌株还具备促进骨细胞(C3H10T1/2)生长的功能,可进一步应用在促消化以外的保健功效上。
附图说明
图1为实施例2中二次筛选4种菌株的产消化酶的检测结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1嗜酸乳杆菌MD-286的分离与鉴定
将5岁左右健康小孩粪便1g加入9ml无菌生理盐水中,混匀取1ml混悬液再加入到9ml无菌生理盐水中,经过一系列十倍稀释,分别取浓度为10-6,10-7,10-8的菌液各0.1ml涂布MRS平板,每个稀释度作3个重复,37℃厌氧培养48h,挑去单菌落划线厌氧培养。
最终在乳酸细菌培养基(MRS)中分离得到的单菌落,利用细菌基因组DNA提取试剂盒提取菌株DNA,通过PCR技术扩增16S rDNA,进行测序分析,鉴定菌株的种属。
对筛选出的菌株革兰氏染色后于显微镜下观察其形态,染色后菌体呈蓝色则为革兰氏阳性菌株,菌体呈红色则为革兰氏阴性菌株。
实验结果:从5岁左右健康小孩粪便筛选出多株微生物菌株,一共有64种菌株,经16S rDNA测序分析和形态观察,其中菌株5B5Y5被鉴定为嗜酸乳杆菌,其16S rDNA序列(SEQID NO:1所示)与已报道的嗜酸乳杆菌(Lactobacillus acidophilus FSI4)的序列相似性为99%。该菌株已在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)进行了专利保藏,保藏编号为CGMCC NO.23901。
实施例2产四种基本消化酶检测
第一次筛选:将MRS初步筛选得到的64种菌株进行产消化酶的检测,检测方法及培养基如下:
(1)蛋白水解培养基:脱脂奶粉10g,蛋白胨10g,琼脂粉15g,1000ml无菌水,PH7.0-7.2。
(2)脂肪水解培养基:13mM Tris-HCL、75mM NaCl和1.3mM CaCl2(pH8.0)、琼脂15g、10mg罗丹明B,聚乙烯橄榄油乳化液31.25ml,pH7.0
(3)淀粉水解培养基:酵母浸膏5g,可溶性淀粉15g,琼脂粉15g,1000ml无菌水,PH7.0-7.2。
(4)纤维素水解培养基:酵母膏5g,羧甲基纤维素5g,琼脂粉15g,1000ml无菌水,PH7.0-7.2。
将各菌株的菌液分别用接种棒点种于蛋白水解培养基、脂肪水解培养基、淀粉水解培养基和纤维素水解培养基上,每个菌液做4个重复,置于37℃培养24h。蛋白酶检测需要用10%HCl溶液淹没平板进行透明圈的判断;淀粉酶检测需要滴加少量的卢戈尔氏碘液于平板上,稍等片刻,观察其菌落周围形成的透明圈。检测结果如下:
第二次筛选:从这六十多菌株初步筛选出25、51、54、57号菌株,进行进一步的筛选确认。
25、51、54、57号菌株均在MRS培养基中,pH 6.2-6.6,在37℃恒温培养箱静置过夜培养。
优先选择蛋白酶水解直径大的、淀粉酶两个+号的,产纤维素的,同时符合这些条件的情况下,初步筛选出了51号菌株5B5Y5,即命名的Lactobacillus acidophilus MD-286菌株。二次筛选的结果如下:
图1可以看出,51号菌株除了具有淀粉酶、纤维素酶和脂肪酶活性外,还具有良好的消化蛋白的蛋白酶活性。
实施例3菌株促进大鼠背根神经节神经元细胞(DRG)生长的检测
方法:培养大鼠背根神经节神经元细胞(DRG),使用待测灭活菌株上清液刺激,用MTT法检测细胞生长。
结果如下:
实施例4菌株促进大鼠垂体瘤GH3细胞产生生长激素的检测
方法:培养大鼠垂体瘤GH3细胞,使用待测灭活菌株上清液刺,用大鼠GH的ELISA试剂盒检测生长激素。
结果如下:
NO. | 编号 | 名称 | 大鼠垂体瘤GH3产生长激素(ng/L) |
CK | 细胞 | 50.124 | |
空白 | MRS | 12.558 | |
25 | cb41Y5 | 长双歧杆菌 | 83.972 |
51 | 5B5Y5 | 嗜酸乳杆菌 | 20.963 |
54 | cb13m2 | 唾液乳杆菌 | 47.256 |
57 | cb18m1 | 乳酸乳球菌 | 82.077 |
实施例5菌株促进胚胎干细胞CE3生长的检测
方法:培养胚胎干细胞CE3,使用待测灭活菌株上清液刺激,用MTT法检测细胞生长。
结果如下:
NO. | 编号 | 名称 | 胚胎干细胞CE3生长率(%) |
CK | 细胞 | 100.00% | |
空白 | MRS | 95.73% | |
25 | cb41Y5 | 长双歧杆菌 | 113.53% |
51 | 5B5Y5 | 嗜酸乳杆菌 | 81.67% |
54 | cb13m2 | 唾液乳杆菌 | 128.77% |
57 | cb18m1 | 乳酸乳球菌 | 126.54% |
实施例6菌株促进骨细胞(C3H10T1/2)生长的检测
方法:培养骨细胞(C3H10T1/2),使用待测灭活菌株上清液刺激,用MTT法检测细胞生长。
结果如下:
NO. | 编号 | 名称 | 骨细胞C3H10T1/2生长率(%) |
CK | 细胞 | 100.00% | |
空白 | MRS | 111.43% | |
25 | cb41Y5 | 长双歧杆菌 | 133.92% |
51 | 5B5Y5 | 嗜酸乳杆菌 | 140.14% |
54 | cb13m2 | 唾液乳杆菌 | 131.35% |
57 | cb18m1 | 乳酸乳球菌 | 81.72% |
实施例7嗜酸乳杆菌Lactobacillus acidophilus MD-286菌粉的制备
1、菌种活化:将甘油管中保存的嗜酸乳杆菌Lactobacillus acidophilus MD-286解冻后,无菌条件下,在MRS平板上进行划线分离,37℃厌氧培养48h,从MRS平板上挑取单菌落进行斜面划线,37℃厌氧培养16h,保藏备用。
2、一级培养:分别取生长旺盛的斜面,用灭菌后的接种环刮取1环菌于100ml的一级培养基中,37℃厌氧培养10h-20h,获得嗜酸乳杆菌的一级培养液。
3、二级培养:将嗜酸乳杆菌的一级培养液接种于5L发酵罐中的二级培养基中,接种量为1%-5%,装液量为2L-4L,发酵温度30℃-38℃,转速50rpm-120rpm,培养8h-20h得到二级种子液。
4、嗜酸乳杆菌菌粉制备:将二级种子液通过离心(5000g,10min)获得嗜酸乳杆菌活性菌泥,与脱脂乳(20%,w/v)按照1:1.5混合30min,获得高浓度嗜酸乳杆菌菌悬液,经冷冻干燥制成嗜酸乳杆菌菌粉。
结果:经平板计数,嗜酸乳杆菌菌粉的活菌量(菌落形成单位)大于1×1011CFU/g。
以上所述本发明的具体实施方式,并不构成对本发明保护范围的限定。任何根据本发明的技术构思所做出的各种其他相应的改变与变形,均应包含在本发明权利要求的保护范围内。
SEQUENCE LISTING
<110> 美益添生物医药(武汉)有限公司
<120> 一种嗜酸乳杆菌MD-286及其应用
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1463
<212> DNA
<213> 16S rDNA
<400> 1
cggggatctg ctatactgca gtcgagcgag ctgaaccaac agattcactt cggtgatgac 60
gttgggaacg cgagcggcgg atgggtgagt aacacgtggg gaacctgccc catagtctgg 120
gataccactt ggaaacaggt gctaataccg gataagaaag cagatcgcat gatcagctta 180
taaaaggcgg cgtaagctgt cgctatggga tggccccgcg gtgcattagc tagttggtag 240
ggtaacggcc taccaaggca atgatgcata gccgagttga gagactgatc ggccacattg 300
ggactgagac acggcccaaa ctcatacggg aggcagcagt agggaatctt ccacaatgga 360
cgaaagtctg atggagcaac gccgcgtgag tgaagaaggt tttcggatcg taaagctctg 420
ttgttggtga agaaggatag aggtagtaac tggcctttat ttgacggtaa tcaaccagaa 480
agtcacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg 540
atttattggg cgtaaagcga gcgcaggcgg aagaataagt ctgatgtgaa agccctcggc 600
ttaaccgagg aactgcatcg gaaactgttt ttcttgagtg cagaagagga gagtggaact 660
ccatgtgtag cggtggaatg cgtagatata tggaagaaca ccagtggcga aggcggctct 720
ctggtctgca actgacgctg aggctcgaaa gcatgggtag cgaacaggat tagataccct 780
ggtagtccat gccgtaaacg atgagtgcta agtgttggga ggtttccgcc tctcagtgct 840
gcagctaacg cattaagcac tccgcctggg gagtacgacc gcaaggttga aactcaaagg 900
aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga 960
accttaccag gtcttgacat ctagtgcaat ccgtagagat acggagttcc cttcggggac 1020
actaagacag gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080
cgcaacgagc gcaacccttg tcattagttg ccagcattaa gttgggcact ctaatgagac 1140
tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcatcatgcc ccttatgacc 1200
tgggctacac acgtgctaca atggacagta caacgaggag caagcctgcg aaggcaagcg 1260
aatctcttaa agctgttctc agttcggact gcagtctgca actcgactgc acgaagctgg 1320
aatcgctagt aatcgcggat cagcacgccg cggtgaatac gttcccgggc cttgtacaca 1380
ccgcccgtca caccatggga gtctgcaatg cccaaagccg gtggcctaac cttcgggaag 1440
gagccgtcta aggcaggcag ata 1463
Claims (5)
1.一种嗜酸乳杆菌,其特征在于:所述嗜酸乳杆菌菌株编号为MD-286,分类命名为嗜酸乳杆菌Lactobacillus acidophilus;保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC;保藏地址:北京市朝阳区北辰西路1号院3号;保藏日期:2021年11月15日保藏编号:CGMCC NO.23901。
2.根据权利要求1所述嗜酸乳杆菌Lactobacillus acidophilus MD-286具有产蛋白酶、淀粉酶和纤维素酶的功能。
3.一种具有产蛋白酶、淀粉酶和纤维素酶功能的嗜酸乳杆菌Lactobacillusacidophilus MD-286菌粉,其特征在于:所述嗜酸乳杆菌菌粉是由权利要求1或2任一项所述嗜酸乳杆菌Lactobacillus acidophilus MD-286的发酵产物经冷冻干燥(真空度0.08MPa,冷肼温度-60℃)制备而成;所述菌粉具有超高细胞浓度,菌落形成单位大于1×1011CFU/g。
4.根据权利要求3所述嗜酸乳杆菌Lactobacillus acidophilus MD-286菌粉,其特征在于,嗜酸乳杆菌Lactobacillus acidophilus MD-286菌粉的制备方法包括如下步骤:
1)将所述嗜酸乳杆菌Lactobacillus acidophilus MD-286菌粉接种在MRS培养液中在36~38℃下发酵12~36h,得到嗜酸乳杆菌Lactobacillus acidophilus MD-286发酵液;
2)将所述步骤1)的嗜酸乳杆菌Lactobacillus acidophilus MD-286发酵液离心,收集沉淀物,得到嗜酸乳杆菌Lactobacillus acidophilus MD-286菌泥;
3)将所述步骤2)的嗜酸乳杆菌Lactobacillus acidophilus MD-286菌泥进行真空冷冻干燥,得到嗜酸乳杆菌Lactobacillus acidophilus MD-286菌粉。
5.一种权利要求1或2任一项所述嗜酸乳杆菌在促进骨细胞(C3H10T1/2)生长上的应用。
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