CN112795500B - 一株可缓解高尿酸血症的植物乳杆菌及其用途 - Google Patents
一株可缓解高尿酸血症的植物乳杆菌及其用途 Download PDFInfo
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Abstract
一株可缓解高尿酸血症的植物乳杆菌及其用途，该植物乳杆菌为植物乳杆菌grx03，从广西巴马长寿村人群肠道中分离所得，本发明菌株发酵传统药食同源中草药菊苣表现出良好的降尿酸能力，可以与其他益生乳酸菌复配开发降尿酸发酵乳产品或乳酸菌饮料。
Description
技术领域
本发明涉及生物技术领域，特别涉及一株可缓解高尿酸血症的植物乳杆菌及其用途。
背景技术
高尿酸血症与高血压、高血糖、高血脂共称为“四高”代谢性疾病。长期高尿酸血症会诱发痛风等多种并发症，严重影响人们身体健康和生活质量。别嘌呤醇等西药治疗高尿酸血症虽然疗效好，但副作用较大，如肾脏毒性和肠胃刺激等，在临床上的应用受到一定限制；因此，探索更加安全有效的降尿酸方案十分必要。
人体内约67％的尿酸由内源性产生，剩下33％的尿酸由饮食中的嘌呤代谢产生。由于人体缺乏有生物活性的尿酸氧化酶，所以尿酸为嘌呤在人体内代谢的终产物。不论由内源产生还是由饮食中嘌呤代谢产生的尿酸，在尿酸产生过程中，黄嘌呤氧化酶(xanthineoxidase，XO)都是催化尿酸生成的关键酶。因此，通过促进尿酸合成前体核苷的降解、通过抑制XO活性来减少尿酸生成，是防治高尿酸血症的主要方法。
乳酸菌是公认的食品安全微生物，作为人体肠道内正常菌群，具有降尿酸的能力，在治疗高尿酸血症上具有药物治疗无可比拟的优点，如无药物不良反应、无严格限制饮食、患者依从度高等。筛选降尿酸能力强的乳酸菌，作为辅助治疗高尿酸血症的手段，具有巨大的应用前景。
发明内容
本发明的目的是针对上述现有技术的不足，提供一株可缓解高尿酸血症的植物乳杆菌及其用途。
本发明的第一个目的在于提供一种降尿酸能力强的乳酸菌，即一株可缓解高尿酸血症的植物乳酸菌，该植物乳杆菌为植物乳杆菌grx03，已于本发明申请日前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)，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。
该菌株从广西巴马长寿村人群肠道中分离所得，经实验验证，本发明菌株具有优异的降尿酸能力，在MRS培养基中培养后对鸟苷和肌苷降解率均为100％，鸟苷降解速率大于1.35mmol/(L·h)。而且本发明菌株在MRS、牛乳中发酵24h后发酵液pH值分别为3.62和3.86，有较高的产酸能力。该菌株发酵MRS24h后的发酵上清液对黄嘌呤氧化酶抑制率大于94.0％，发酵菊苣浸提液24h后的发酵上清液对黄嘌呤氧化酶抑制率大于90.0％，发酵脱脂乳24h后的发酵上清液对黄嘌呤氧化酶抑制率大于55.0％。
本发明的第二个目的是提供降尿酸能力和产酸能力强的植物乳杆菌grx03在发酵乳、乳酸菌饮料及相关功能药品生产中的应用。
本发明菌株发酵传统药食同源中草药菊苣表现出良好的降尿酸能力，可以与其他益生乳酸菌复配开发降尿酸发酵乳产品或乳酸菌饮料。
具体实施方法
一、植物乳杆菌grx03的制备方法
本发明采用传统涂布平板分离法从广西巴马长寿村采集的样品中分离乳酸菌，通过测定MRS发酵液pH，确定产酸高的乳酸菌株；通过测定乳酸菌在鸟苷-肌苷磷酸盐缓冲液中的核苷降解率、核苷降解速率，测定乳酸菌发酵MRS、菊苣浸提液和脱脂乳后的黄嘌呤氧化酶抑制率，筛选出降尿酸能力较强的乳酸菌；最终确定了产酸高、降尿酸效果好的乳酸菌菌株。
1、乳酸菌的分离
本发明具有降尿酸功能的植物乳杆菌，是发明人从广西巴马长寿村长寿人群的肠道中筛选得到，经鉴定属于植物乳杆菌(Lactobacillus plantarum)，命名为grx03，已于本发明申请日前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)。该菌株最适生长温度为37℃，兼性厌氧，生长于MRS培养基中。
2、乳酸菌的产酸能力
将待试菌株在MRS固体平板(蛋白胨10.0g，三水醋酸钠3.0g，牛肉膏8.0g，柠檬酸三铵2.0g，酵母浸膏4.0g，七水硫酸镁0.2g，葡萄糖10.0g，一水合硫酸锰0.04g，吐温-801.0mL，磷酸氢二钾2.0g，硫乙醇酸钠2.0g，20g琼脂，蒸馏水1000mL。调pH至6.4，121℃灭菌15min，冷却倒板)上划线分离单菌落，挑单菌落接种至5mL液体MRS培养基，37℃静置培养24h，作为活化一代菌株；再将一代菌株以4％接种量转接至5mL液体MRS培养基中，37℃静置培养24h，作为活化二代菌株，取活化二代菌株用于后续试验。
取活化后乳酸菌株，按质量百分比为3％的比例接种到MRS液体培养基中，于37℃下培养24h，测定发酵液pH。结果表明，grx03在MRS基质中经过24h的发酵后发酵液pH值为3.62，表明该菌株具有良好的产酸能力。
3、乳酸菌鉴定
基于16s rDNA的菌株鉴定：以植物乳杆菌grx03基因组DNA作为PCR扩增模板，采用25μL反应体系进行PCR扩增。模板DNA 0.5uL，10mmol上下游引物(27F/1492R)各0.5μL，2×Taq酶12.5μL，以ddH2O补至25μL。PCR循环参数为：95℃预变性5min；95℃变性3min，57℃退火30s，72℃延伸2min，循环30次；72℃延伸10min。吸取PCR产物5μL，加样至1.0％(质量浓度)琼脂糖凝胶电泳，电压为80V。电泳结束后用Gold View染色20-30min，拍照。大小约1500bp的条带即为目标条带。PCR扩增产物送至上海生工生物工程技术服务有限公司进行测序，将测序得到的序列与NCBI的GenBank数据库进行同源比对分析，结果表明本发明菌株grx03为植物乳杆菌，16s rDNA序列如SEQ ID NO.1所示。
SEQ ID NO.1：
TATACATGCAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGAA
4、乳酸菌降解核苷能力评价
鸟苷-肌苷磷酸盐缓冲液：准确称取鸟苷0.02g、肌苷0.02g，用20mmol/LKH2PO4溶液(pH 7.0)定容至100mL并调节pH至7.0，最终缓冲液中鸟苷浓度为0.2g/L(0.706mmol/L)，肌苷浓度为0.2g/L(0.746mmol/L)。
采用磷酸盐缓冲液体系初步筛选具有降解鸟苷和肌苷能力的乳酸菌。取适量活化二代菌培养液，室温4500r/min离心3min，弃上清，收集菌体，菌体用无菌生理盐水(质量浓度为0.9％的NaCl溶液)洗涤2次，向清洗后的菌体中加入1mL鸟苷-肌苷磷酸盐缓冲液，混合均匀，控制缓冲液中菌浓OD600为1.5，37℃静置孵育4h。孵育后，取菌液4℃，4500r/min离心3min，取上清810μL，加入90μL反应终止剂0.1mol/L高氯酸溶液，混合均匀后4500r/min离心3min，取上清液使用0.22μL水相滤膜过滤后用于HPLC检测，分析反应液中鸟苷和肌苷的减少量。
植物乳杆菌grx03对鸟苷和肌苷具有很强的降解能力，可将鸟苷和肌苷100％降解。
5、乳酸菌降解核苷速率评价
将乳酸菌在MRS培养基中活化后，取活化二代菌培养液，离心收集菌体，用无菌生理盐水洗涤2次，重悬于鸟苷缓冲液中，每隔30min取样检测分析鸟苷的剩余量。植物乳杆菌grx03可在30min内将鸟苷完全降解，核苷降解速率大于1.40mmol/(L·h)。
6、MRS发酵液黄嘌呤氧化酶抑制率评价
黄嘌呤氧化酶体外抑制率测定：在1mL酶促反应体系中依次加入560μl pH 7.6磷酸盐缓冲液、40μl发酵上清液和200μl 0.2U/ml黄嘌呤氧化酶溶液，充分混匀，37℃温浴10min，以便让溶液达到均衡状态。然后加入200μl 37℃预温的0.2g/L的黄嘌呤溶液启动反应。采用紫外分光光度计的动力学软件记录一定时间内295nm波长下吸光值随时间的变化，每10s测定一次，以反应时间为横坐标，吸光度值为纵坐标作图，求出曲线斜率AS；以相同体积的空白MRS培养基代替样品作为空白对照，计算其酶促反应速率AC。黄嘌呤氧化酶的相对抑制率计算方法如下所示：黄嘌呤氧化酶的相对抑制率＝(1-AS/AC)×100％。
将乳酸菌活化一代后，以4％(质量百分比)的接种量接种至10mL MRS培养基中，37℃静置发酵24h；4℃，12000r/min离心5min，取上清液按上述方法测定XO抑制率。植物乳杆菌grx03 MRS发酵液对黄嘌呤氧化酶的体外抑制率为94.5％。
7、菊苣发酵液黄嘌呤氧化酶抑制率评价
取干菊苣根使用粉碎机粉碎后称取20g，加入去离子水600g，85℃超声浸提30min，经80目筛过滤2次后分装10mL到无菌试管中，95℃水浴杀菌5min，即得无菌菊苣浆液，冷却备用。
将乳酸菌活化二代后，以4％(质量百分比)的接种量接种至脱脂乳培养基中，37℃发酵24h，震荡破乳、混匀，然后以4％的接种量将发酵脱脂乳接种至无菌菊苣浆液中，37℃发酵48h，4℃、12000r/min离心5min，取上清液按上述方法测定XO抑制率。植物乳杆菌grx03菊苣发酵液对黄嘌呤氧化酶的体外抑制率为90.3％。
8、脱脂乳发酵液黄嘌呤氧化酶抑制率分析
全脂乳培养基：称取7％(质量百分比)的蔗糖溶于热水中，将全脂乳粉按12％(质量百分比)的比例溶于40-50℃的糖水中，95℃热处理5min，冷却备用。取活化二代菌株接种于上述全脂乳培养基中发酵24h，植物乳杆菌grx03的脱脂乳发酵液对黄嘌呤氧化酶抑制率达55.6％。
二、植物乳杆菌grx03的应用实施例
1、含该乳酸菌发酵乳的制作方法：
将鲜牛乳加热到50℃以上，加入6％(质量百分比)的白砂糖搅拌至完全溶解，预热到60～65℃，20Mpa压力均质，95℃热处理5-8分钟，冷却至42～45℃，接种该乳酸菌，接种量为2％～3％。40～42℃发酵至pH值为4.2～4.5，冷却，冷藏即制成含该乳酸菌的酸乳。经测定，发酵乳中活菌数高于109CFU/mL，且发酵乳离心上清液对黄嘌呤氧化酶的抑制率大于55％。
2、含该乳酸菌饮料的制作方法：
将应用示例1中的发酵乳，用无菌并冷却的薄荷、菊苣、荷叶、葛根、槐米或者桑叶等具有降尿酸作用的药食同源中草药单一浸提液或者其混合物稀释2-4倍，制成可直接饮用的乳酸菌饮料。该乳酸菌饮料口感酸甜，风味良好，且对黄嘌呤氧化酶的抑制率大于45％。
以上利用本发明菌制得的产品，具有体外抑制黄嘌呤氧化酶的特点。
序列表
<110> 扬州大学
<120> 一株可缓解高尿酸血症的植物乳酸菌及其用途
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1431
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tatacatgca gtcgaacgaa ctctggtatt gattggtgct tgcatcatga tttacatttg 60
agtgagtggc gaactggtga gtaacacgtg ggaaacctgc ccagaagcgg gggataacac 120
ctggaaacag atgctaatac cgcataacaa cttggaccgc atggtccgag cttgaaagat 180
ggcttcggct atcacttttg gatggtcccg cggcgtatta gctagatggt ggggtaacgg 240
ctcaccatgg caatgatacg tagccgacct gagagggtaa tcggccacat tgggactgag 300
acacggccca aactcctacg ggaggcagca gtagggaatc ttccacaatg gacgaaagtc 360
tgatggagca acgccgcgtg agtgaagaag ggtttcggct cgtaaaactc tgttgttaaa 420
gaagaacata tctgagagta actgttcagg tattgacggt atttaaccag aaagccacgg 480
ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg 540
ggcgtaaagc gagcgcaggc ggttttttaa gtctgatgtg aaagccttcg gctcaaccga 600
agaagtgcat cggaaactgg gaaacttgag tgcagaagag gacagtggaa ctccatgtgt 660
agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct gtctggtctg 720
taactgacgc tgaggctcga aagtatgggt agcaaacagg attagatacc ctggtagtcc 780
ataccgtaaa cgatgaatgc taagtgttgg agggtttccg cccttcagtg ctgcagctaa 840
cgcattaagc attccgcctg gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg 900
ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc 960
aggtcttgac atactatgca aatctaagag attagacgtt cccttcgggg acatggatac 1020
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080
gcgcaaccct tattatcagt tgccagcatt aagttgggca ctctggtgag actgccggtg 1140
acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1200
acacgtgcta caatggatgg tacaacgagt tgcgaactcg cgagagtaag ctaatctctt 1260
aaagccattc tcagttcgga ttgtaggctg caactcgcct acatgaagtc ggaatcgcta 1320
gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380
cacaccatga gagtttgtaa cacccaaagt cggtggggta accttttaga a 1431
Claims (2)
1.一株可缓解高尿酸血症的植物乳杆菌（Lactobacillus plantarum），其特征是，该植物乳杆菌为植物乳杆菌grx03，2020年11月30日保藏于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CGMCC No. 21268。
2.权利要求1所述的一株可缓解高尿酸血症的植物乳杆菌的用途，其特征是，所述植物乳杆菌grx03在发酵乳、乳酸菌饮料及相关功能药品生产中的应用。
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