CN114657095A - 一株发酵乳杆菌ncu001464 - Google Patents
一株发酵乳杆菌ncu001464 Download PDFInfo
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- CN114657095A CN114657095A CN202210287684.2A CN202210287684A CN114657095A CN 114657095 A CN114657095 A CN 114657095A CN 202210287684 A CN202210287684 A CN 202210287684A CN 114657095 A CN114657095 A CN 114657095A
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- Prior art keywords
- ncu001464
- lactobacillus fermentum
- strain
- lactobacillus
- hyperuricemia
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Links
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Images
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一株发酵乳杆菌NCU001464，已于2021年11月30日保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏编号为CGMCC No.24010。本发明发酵乳杆菌NCU001464具有优良的耐酸性能及耐胆盐性能，高β‑葡萄糖苷酶活性以及对尿酸前体物质肌苷和鸟苷有明显的降解作用，可以有效降低由饮食中嘌呤的摄入所导致的血尿酸水平偏高的问题。
Description
技术领域
本发明属于微生物技术领域，具体涉及一株发酵乳杆菌NCU001464。
背景技术
在过去的几十年里，高尿酸血症的风险在全球范围内都有所增加，即将成为继三高疾病之后的第四大常见慢性病，不断威胁着医疗保健系统，现在已经成为现代社会的重要医疗负担。高尿酸血症是肾脏疾病的重要危险因素，是痛风的诱因。目前高尿酸血症的治疗以药物治疗为主，主要分为尿酸合成抑制剂和尿酸排泄促进剂。其中，黄嘌呤氧化酶(XO)已被用作治疗高尿酸血症的药物靶点，但黄嘌呤药物抑制剂可诱发严重的副作用，如肾功能衰竭、肝功能障碍、过敏反应和肝毒性问题。乳酸菌(LAB)作为益生菌，可作为辅助食品，缓解高尿酸血症诱发的慢性肾病的发展。LAB通过抑制肠道内外嘌呤的吸收，有效修饰和恢复宿主肠道微生物群，从而降低血清UA浓度，缓解高尿酸血症。
膳食类黄酮因其有益的特性，包括清除自由基的能力和抗炎活性，越来越受到关注。近来研究发现，包括高良姜素、黑皮诺素、槲皮素、桑色素、杨梅素、山奈酚和葛根素在内的黄酮类化合物被认为是有效的黄嘌呤氧化酶抑制剂。然而，黄酮类化合物大部分是以与糖苷结合的黄酮糖苷的形式存在，黄酮结合型糖苷药物活性远远低于游离的苷元，需要通过去糖基定向修饰黄酮类化合物的分子结构，从而产生高生物活性的苷元。生物转化去糖基是最经济绿色的途径，目前利用最多的β-葡萄糖苷酶。因此，将含β-葡萄糖苷酶的益生菌应用于水果发酵，有利于开发缓解高尿酸血症的功能性食品。
发明内容
本发明的目的是提供一株发酵乳杆菌(Limosilactobacillus fermentum)NCU001464。
一株发酵乳杆菌NCU001464，该菌株已于2021年11月30日保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1 号院3号，中国科学院微生物研究所，保藏编号为CGMCC No.24010。
进一步地，所述发酵乳杆菌NCU001464的16S rRND序列如SEQ ID NO:1 所示。
进一步地，所述发酵乳杆菌NCU001464分离自发酵桑葚。
所述发酵乳杆菌菌株的获得方式为：将桑葚(250克)、葡萄糖(7.5克) 和蒸馏水(500毫升)加入发酵罐中。混合物在室温下培育7天。在此过程中，取出等分试样，用平皿法分离乳酸菌菌株，乳酸菌在MRS琼脂培养基(含0.04％溴甲酚紫)中生长24h，挑取单个菌落反复划线培养。然后将纯种乳酸菌加入到50毫升MRS肉汤中，然后在37℃培养过夜。随后，将2毫升培养液注入 100毫升MRS肉汤中，在37℃下生长18h。通过离心(6000g，5min，4℃) 收获乳酸菌细胞。
菌落形态：发酵乳杆菌NCU001464在MRS(含0.04％溴甲酚紫)琼脂培养基上的形态为黄色圆形凸起状、表面粗糙、边缘不整齐，产酸能力强，菌落直径2-3mm；革兰氏染色阳性菌，厌氧或兼性厌氧，短杆状，具体形态见附图1。
自凝集能力的测定：按体积分数2％的量接种于液体培养基再次活化，将培养24h后的乳杆菌于4℃、6000g下离心5min收集菌体，用无菌的PBS洗涤两次，随即用无菌PBS重悬菌体并调整A600＝0.60±0.05(A0)，漩涡振荡10s 后于37℃孵育24h。在2、4、18、24h取上层菌悬液，于600nm下测定吸光度(At)，每次测定三个平行，重复三次。按照下式计算细菌的自凝集百分比：细菌自凝集百分比＝(1-At/A0)×100％，其中，A0表示菌体初始吸光值，At代表t 时刻菌体的吸光值，具体见附图2。
发酵乳杆菌NCU001464对模拟肠上皮细胞的黏附性：将培养好的Caco-2 细胞用0.25％胰酶-EDTA消化液消化，再用DMEM完全培养液调整细胞浓度为2.5×105个/mL，装于6孔组织培养板中，每孔2mL，于CO2培养箱(5％CO2， 95％空气)中37℃孵育至细胞长至单层。弃去组织培养板中各孔的DMEM培养液，以无菌PBS缓冲液清洗培养板2次，其中1个孔用0.5mL胰酶-EDTA消化液进行消化后，加入0.5mL PBS，用巴氏吸管吹打，将细胞完全消化下来并使之混合均匀，血球计数板计算细胞浓度。其他5孔再加入1mL DMEM不完全培养液和1mL发酵乳杆菌菌悬液(108CFU/mL)，用移液枪吹打混匀，于37℃孵育2h。孵育后弃去组织培养板中各孔的混合液，用无菌PBS缓冲液洗涤5 次，以除去未黏附的菌体。加入0.5mL胰酶-EDTA消化液进行消化后加入0.5 mL无菌PBS缓冲液，进行梯度稀释，涂布于MRS固体平板上计算黏附的细菌数。黏附能力用下式计算：黏附率(％)＝Nt/N0×100％，其中，Nt是指黏附在Caco-2细胞上的细菌数；N0是指加入的乳杆菌总细菌数。
分子生物学鉴定：将得到的纯种培养物进行细菌基因组DNA的提取，提取方法如下:[1]将获得的纯培养物接种培养24h后取菌液1mL，10000rpm离心 10min，弃上清液；[2]加500μL TE缓冲液，微振；[3]加入70μL 50mg/mL 溶菌酶，40℃水浴每10min微振一次，4-6次即可；[4]加入50μL 5mg/mL蛋白酶，60℃水浴每10min微振一次，4-6次即可；[5]加入300μL SDS裂解液， 65℃水浴每10min微振一次，4-6次即可；[6]加入300μL 2g/50mL KCl溶液，微振，常温静置10min，微振，4℃静置30min；[7]10000rpm 4℃离心10min，取上清(推荐量850μL)转移至2mL离心管中，加入等体积异丙醇(-20℃预冷) 微振，4℃静置30min；10000rpm 4℃离心10min，去上清液；[8]加入500μL 冰乙醇(-20℃预冷)；10000rpm 4℃离心10min，去上清，晾干沉淀。
16S rRNA鉴定：将获得的疑似乳酸菌的基因组DNA，以细菌通用引物27F 进行PCR扩增，PCR反应体系和反应程序见表1和表2。将PCR产物送至上海生工公司进行测序，将所得结果在Gene Bank数据库中使用BLAST工具进行同源性比较，以此确定所分离菌株的种属。
表1 PCR反应体系
表2 PCR反应程序
鉴定结果：所得序列片段长度为1477，在NCBI中将所得16S rRNA序列经BLAST序列对比，得到结果与Limosilactobacillus fermentum同源性大于 99.80％。
进一步地，所述发酵乳杆菌NCU001464应用于功能性食品如发酵果蔬饮料、发酵果蔬原浆、发酵果蔬泥的生产加工。
进一步地，所述功能性食品用于缓解高尿酸血症。
进一步地，所述高尿酸血症是由饮食中嘌呤的摄入所导致。
本发明的有益效果是：
1、本发明发酵乳杆菌NCU001464具有优良的耐酸、耐胆盐能力；
2、本发明发酵乳杆菌NCU001464具有高β-葡萄糖苷酶活性；
3、本发明发酵乳杆菌NCU001464对尿酸前体物质肌苷和鸟苷有明显的降解效果，可以有效降低由饮食中嘌呤的摄入所导致的血尿酸水平偏高的问题。
附图说明
图1为菌落形态及镜检结果。
图2为自凝集备份占比。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合实施例对本发明的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
菌株优良的β-葡萄糖苷酶活性
将过夜培养18h的乳酸菌通过离心(6000g，5min，4℃)收获乳酸菌细胞，并用生理盐水洗涤两次。然后，用缓冲液稀释细胞沉淀物并用于进一步实验。首先，向上述细胞沉淀物中加入柠檬酸-磷酸氢钠缓冲液(100mM，pH 4.5)以获得细胞悬浮液。其次，2mL柠檬酸-磷酸氢钠缓冲液(100mM，pH 4.5)反应体系由5mg/mL的湿细胞组成，6mM的pNPG在37℃和160rpm下孵育30min。然后，向体系中加入2mL碳酸钠缓冲液(1moL/L)以终止反应。然后，将混合物离心(8000g，5min，4℃)以获得上清液，并用双光束紫外分光光度计(Puxi TU-1901)在400nm处测定上清液中对硝基苯酚的吸光度。
1U细胞β-葡萄糖苷酶活性定义为在37℃和160rpm下每分钟水解pNPG 产生1μmol对硝基苯酚的细胞数量。
表1 NCU001464的β-葡萄糖苷酶活性
发酵乳杆菌NCU001464全细胞催化反应得出β-葡萄糖苷酶活性为0.2077 U/mg湿细胞，参考Lorn等人通过全细胞催化反应分离出β-葡萄糖苷酶活性最高的植物乳杆菌C022-2B，为27nmol/min/mg干细胞。
实施例2
菌株优良的转化核苷类化合物能力
通过加入磷酸盐缓冲液(100mM，pH 7.0)制备收获的细胞上清液。将20 mg/mL湿细胞和20mM肌苷/6mM鸟苷加入到2mL磷酸盐缓冲液(100mM， pH 7.0)中。反应在37℃和160rpm下进行30min。然后，将混合物煮沸5min，在10000g和4℃下离心5min，得到上清液。核苷类化合物的浓度用高效液相色谱法测定。基于以下公式计算初始反应速率：V(mM/h)＝(C0-Ct)/t，其中C0和Ct分别是肌苷/鸟苷在0小时和t小时的水平；t是反应时间；v是初始反应速率。
高效液相色谱分析方法：反相色谱柱Hypersil ODS2 C18(4.6mm×250mm；流动相V(0.01mol/L KH2PO4)∶V(色谱甲醇)＝90:10；流速0.5mL/min，柱温35℃，测定波长254nm，洗脱时间10min。
表2
| 菌株编号 | 肌苷转化速率(mM/h) | 鸟苷转化速率(mM/h) |
| NCU001464 | 4.66±0.26 | 1.83±0.06 |
发酵乳杆菌NCU001464对肌苷与鸟苷的降解初速率分别为4.66mM/h与 1.83mM/h。
以上所描述的实施例仅为本发明优选实施例，并不用于限制本发明。对于本领域的技术人员来说，本发明可以有各种变化和更改，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 南昌大学
<120> 一株发酵乳杆菌NCU001464
<130> 2021.11.30
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1477
<212> DNA
<213> 发酵粘液乳杆菌（Limosilactobacillus fermentum）
<400> 1
aaggcaggcg ttgctataca tgcagtcgaa cgcgttggcc caattgattg atggtgcttg 60
cacctgattg attttggtcg ccaacgagtg gcggacgggt gagtaacacg taggtaacct 120
gcccagaagc gggggacaac atttggaaac agatgctaat accgcataac aacgttgttc 180
gcatgaacaa cgcttaaaag atggcttctc gctatcactt ctggatggac ctgcggtgca 240
ttagcttgtt ggtggggtaa tggcctacca aggcgatgat gcatagccga gttgagagac 300
tgatcggcca caatgggact gagacacggc ccatactcct acgggaggca gcagtaggga 360
atcttccaca atgggcgcaa gcctgatgga gcaacaccgc gtgagtgaag aagggtttcg 420
gctcgtaaag ctctgttgtt aaagaagaac acgtatgaga gtaactgttc atacgttgac 480
ggtatttaac cagaaagtca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540
gcaagcgtta tccggattta ttgggcgtaa agagagtgca ggcggttttc taagtctgat 600
gtgaaagcyt tcggcttaac cggagaagtg catcggaaac tggataactt gagtgcagaa 660
gagggtagtg gaactccatg tgtagcggtg gaatgcgtag atatatggaa gaacaccagt 720
ggcgaaggcg gctacctggt ctgcaactga cgctgagact cgaaagcatg ggtagcgaac 780
aggattagat accctggtag tccatgccgt aaacgatgag tgctaggtgt tggagggttt 840
ccgcccttca gtgccggagc taacgcatta agcactccgc ctggggagta cgaccgcaag 900
gttgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960
gaagctacgc gaagaacctt accaggtctt gacatcttgc gccaacccta gagatagggc 1020
gtttccttcg ggaacgcaat gacaggtggt gcatggtcgt cgtcagctcg tgtcgtgaga 1080
tgttgggtta agtcccgcaa cgagcgcaac ccttgttact agttgccagc attaagttgg 1140
gcactctagt gagactgccg gtgacaaacc ggaggaaggt ggggacgacg tcagatcatc 1200
atgcccctta tgacctgggc tacacacgtg ctacaatgga cggtacaacg agtcgcgaac 1260
tcgcgagggc aagcaaatct cttaaaaccg ttctcagttc ggactgcagg ctgcaactcg 1320
cctgcacgaa gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380
cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa agtcggtggg 1440
gtaacctttt aggagccagc cgcctaagtg acagatg 1477
Claims (6)
1.一株发酵乳杆菌NCU001464，其特征在于，该菌株已于2021年11月30日保藏在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.24010。
2.如权利要求1所述一株发酵乳杆菌NCU001464，其特征在于，所述发酵乳杆菌NCU001464的16S rRND序列如SEQ ID NO:1所示。
3.如权利要求1所述一株发酵乳杆菌NCU001464，其特征在于，所述发酵乳杆菌NCU001464分离自发酵桑葚。
4.如权利要求1所述一株发酵乳杆菌NCU001464应用于功能性食品如发酵果蔬饮料、发酵果蔬原浆、发酵果蔬泥的生产加工。
5.如权利要求4所述一株发酵乳杆菌NCU001464的应用，其特征在于，所述功能性食品用于缓解高尿酸血症。
6.如权利要求5所述一株发酵乳杆菌NCU001464的应用，其特征在于，所述高尿酸血症是由饮食中嘌呤的摄入所导致。
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