CN116925946A - 具有高粘附性能和降尿酸功能发酵乳杆菌fm-lf-sr6及其用途 - Google Patents
具有高粘附性能和降尿酸功能发酵乳杆菌fm-lf-sr6及其用途 Download PDFInfo
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- CN116925946A CN116925946A CN202210353849.1A CN202210353849A CN116925946A CN 116925946 A CN116925946 A CN 116925946A CN 202210353849 A CN202210353849 A CN 202210353849A CN 116925946 A CN116925946 A CN 116925946A
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- lactobacillus fermentum
- strain
- uric acid
- lactobacillus
- fermentum
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- A—HUMAN NECESSITIES
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Abstract
本发明涉及一株具有高粘附性能和降尿酸功能发酵乳杆菌新菌株及其应用,属于生物工程技术领域。从传统发酵的贵州酸肉中分离、筛选到1株具有良好降尿酸功能的发酵乳杆菌新菌株,命名为FM‑LF‑SR6,经鉴定为发酵乳杆菌(Lactobacillus fermentum)FM‑LF‑SR6,该菌具有优良肠壁细胞粘附能力,对Caco‑2细胞的黏附数为60/cells以上;该菌株具有很强降解肌苷、鸟苷能力,降解能力都在98%以上;黄嘌呤氧化酶活抑制效果也达到50%以上,同时具有优良的发酵性能和较好的耐酸性能。该菌株于2022年1月17在中国微生物菌种保藏管理委员会普通微生物中心进行保藏,保藏号是CGMCC NO.24329,本发明的发酵乳杆菌(Lactobacillus fermentum)FM‑LF‑SR6,对提高其作为发酵菌种的发酵制品的降尿酸性能有很大的作用,具有广泛的应用前景。
Description
一、技术领域
本发明涉及一株具有高粘附性能好降尿酸功能发酵乳杆菌(Lactobacillusfermentum)FM-LF-SR6及其用途属于生物工程技术领域。
二、背景技术
随着经济水平的提高,高嘌呤食物的饮食导致高尿酸病症的发病率处于升高状态。高尿酸血症已经成为一种现代人常见的“富贵病”,继高血压、高血脂和高血脂之后的第四高症。高尿酸血症由嘌呤代谢紊乱引发,与高血压、动脉粥样硬化、高血脂等疾病的发生关系密切。目前对于此类疾病的治疗主要从促进尿酸排泄和控制尿酸生成两方面进行,但已知的目前在用治疗药物如别嘌呤醇和苯溴马隆的长期使用后对人体产生副作用,包括过敏、肠胃不适、皮肤发红、发烧,甚至上皮坏死,特别是肾功能不足的病患副作用更严重。别嘌呤醇副作用主要是过敏,有肾结石的患者不建议选用苯溴马隆,非布司他是一个相对安全的降尿酸药物,但是有部分患者在应用的过程中,也可以出现肝功升高,甚至部分病人血糖升高等。
XOD是参与嘌呤代谢的关键酶,通过抑制XOD的活性,可有效抑制尿酸的生成。别嘌呤醇和苯溴马隆是临床广泛使用的抗高尿酸血症药物,是一种有效的XOD抑制剂,但因其副作用较大,因此,近年来,越来越多研究者转向从天然产物中寻找 XOD抑制活性因子。
有研究表明,乳酸菌是一类具有长久安全食用历史的公认安全的食品级微生物,具有特定的生理功能和适宜生物加工的属性,是食品工业上常用的菌种,并且对人体具有多方面的保健功能,如提高免疫力、缓解乳糖不耐症、调节肠道菌群、增强抗过敏、降低胆固醇等。因此,乳酸菌已经成为食品与药物等相关产品的主要配料,用于生产功能性发酵食品、保健品或是抗衰老药品。有研究表明,乳酸菌可通过在肠道定植,减少小肠上皮细胞对尿酸的前体物质即核苷的吸收进而降低尿酸生成量,缓解高尿酸血症。LI等人发现乳杆菌DM9128-A能明显降低小鼠血清中尿酸水平及黄嘌呤氧化酶的活性。金方等人发现干酪乳杆菌ZM15能够高效降解核苷类物质,同时可能是通过与肠道上皮细胞竞争吸收相应核苷酸与核苷使高尿酸血症大鼠的尿酸水平显著降低。因此,开发具有自主知识产权的将尿素功能的乳酸菌种将会是今后科技攻关与产品创新的重点和难点,也是一个热点,已经成为一个很有实际意义的研究课题。
三、发明内容
技术问题 具有降尿酸能力的乳酸菌有干酪乳杆菌、嗜酸乳杆菌、乳双歧杆菌、乳酸片求菌、植物乳杆菌等,但真正成功利用于发酵食品、保健食品和药品为数不多的几株菌,日本的PA-3对嘌呤具有较强吸收能力,能够有效降低大鼠肠道对嘌呤的吸收,并能够显著降低大鼠和人的尿酸水平,日本明治株式会社的PA-3产品已经成功投入商业化开发使用。其他绝大部分停留于实验室阶段。针对这一现状,本发明提供一株具有优良高粘附性能和降解肌苷和鸟苷能力,同时具有优良的黄嘌呤氧化酶抑制活性的发酵乳杆菌FM-LF-SR6及其应用,用于降尿酸功能发酵食品、保健食品和药品的开发。
技术方案 本发明涉及一株具有优良高粘附性能和降解尿酸功能的发酵乳杆菌FM- LF-SR6及其用途,其内容和实施方案如下:
本发明以MRS培养基为筛选培养基,根据革兰氏染色和过氧化氢接触酶实验结果,从贵州酸肉中初步分离出乳酸菌,本研究运用HPLC方法筛选出一株降解肌苷和鸟苷能力最高的发酵乳杆菌SR6,并且检测菌株不同生长时段不同成分对XOD的抑制作用。该菌株具有良好的耐酸、耐胆盐、胃肠道转运及细胞黏附能力,可作为预防和辅助治疗高血尿酸症的微生态制剂的菌种资源,应用价值广阔。该菌株命名为FM-LF- SR6,通过测定16s rDNA序列进行系统发育分析和生理生化试验鉴定该菌株,鉴定结果是该菌株为发酵乳杆菌(Lactobacillus fermentum),该菌株于2022年1月17在中国微生物菌种保藏管理委员会普通微生物中心进行保藏,保藏号是CGMCC NO.24329,地址是北京市朝阳区北辰西路1号院3号。
有益效果
本发明提供一株新的发酵乳杆菌菌株FM-LF-SR6,该菌株具有优良的肌苷、鸟苷的降解能力,降解率在98%以上;其稳定期胞内提取物具有较强的黄嘌呤氧化酶(xanthineoxidase,XOD)的抑制活性,抑制率在50%以上;而且该菌株生长速度快,产酸能力强,耐酸和耐胆盐能力强,发酵性能优良,粘附能力强;在30-37℃条件下,12-14h进入稳定期,;可为降尿酸功能食品和保健食品的开发提供可靠的菌株资源。
本发明的主要优点和积极效果如下:
1.发酵乳杆菌菌株FM-LF-SR6,分离自贵州的传统贵州酸肉中,来源比较安全。
2.发酵乳杆菌菌株FM-LF-SR6,该菌株具有优良的肌苷、鸟苷的降解能力,降解率在98%以上;其稳定期胞内提取物具有较强的黄嘌呤氧化酶(xanthine oxidase,XOD)的抑制活性,抑制率在50%以上;而且该菌株生长速度快,在30-37℃条件下,12-14h即可进入稳定期,产酸能力强,耐酸和耐胆盐性能强,发酵性能优良,粘附能力强;可为降尿酸功能食品和保健食品的开发提供可靠的菌株资源。
四、附图说明
图1 FM-LF-SR6菌体电镜图片
图2 FM-LF-SR6细胞粘附能力
五、具体实施方式
下面详细叙述本发明的实施例,需要说明的是,本实施例是叙述性的,不是限定性的,不能以此限定本发明的保护范围。
1、菌株的筛选
利用梯度稀释的方法从风味贵州酸肉中筛选乳酸菌,结合菌落形态,革兰氏染色和接触酶试验,初筛获得36株乳酸菌。挑单菌落接种MRS液体培养基,35℃静止培养18h,4℃条件下5000rpm/min离心5min,收集菌体,37℃培养24h后,4℃ 4000r/min离心10min后取沉淀。用无菌生理盐水将菌体浓度调整为2.01×108 CFU/mL。取2mL菌体重悬液离心,将菌体重悬于750μL 500mg/L肌苷鸟苷混合标液,37℃ 120r/min震荡反应1h结束后,收集720μL上清液,加入80μL反应终止剂 0.1mol/L HClO4(上清液∶终止液=9∶1)混匀,0.22μm微孔滤膜过滤,取10μL用于 HPLC测定反应结束后剩余的中肌苷鸟苷含量。按如下公式计算肌苷鸟苷降解率及反应速率:α%(降解率)=(0.9C母液-C残留)/0.9C母液;v(降解速率)=(0.9C母液-C残留)/60Mr(肌苷/鸟苷);其中C为浓度(mg/L),v为降解速率(mmoL·L-1·min-1)。研究结果显示,不同的菌株对肌苷、鸟苷的降解率不同,降解速率也不同, 20株菌的鸟苷降解率范围是0~98.85%,肌苷的降解率在2.67~99.26%,可见不同的菌株之间功能差异很大。表中选取了降解都在85%上的3个菌株
表1 降解肌苷鸟苷85%以上的乳酸菌株
2、菌株的耐酸、耐胆盐能力测定
对上述筛选出的SR6、SR10和SR16菌株的耐酸和耐胆盐的能力进行测定,以3%的接种量分别接种pH为2.5的MRS培养基中和牛胆汁盐含量为0.5%(w/v)的MRS培养基中,4h后采用平板培养法测定菌体密度。结果显示SR6具有很强的耐酸和耐胆盐能力,在pH 2.5环境下,其4h后存活率为80%以上;在牛胆汁盐含量为0.5%(w/v)的 MRS中,其4h后存活率为75%以上。
根据前面的的研究结果筛选出SR6作为目标菌株,同时根据研究室菌株命名规则,把该菌株命名为FM-LF-SR6。
3、FM-LF-SR6菌株的耐肠胃液能力测定
在饱腹状态下,模拟胃环境消化2h后,FM-LF-SR6活菌数无减少;而后在肠道环境消化4h结束后,其活菌数仍能保持在7.46×106CFU/mL以上,符合微生态制剂活菌数的要求。
4、菌株的鉴定
提取FM-LF-SR6的基因组,利用16S r DNA通用引物,以基因组为模版进行PCR 扩增,扩增出的产物经纯化后送上海生物工程有限公司进行测序。用Blast搜索程序从GenBank数据库中调出相似性较高的相关菌株的16S rRNA基因序列,经过序列多重比对和系统发育进化分析,发现菌株FM-LF-SR6发酵乳杆菌(Lactobacillus fermentum)序列相似性高达99%,并且系统发育分析结果显示该菌与发酵乳杆菌(Lactobacillus fermentumOM320672.1)自然聚为一支,说明该菌株是发酵乳杆菌的成员。FM-LF-SR6 的菌体形态的电镜照片见图1。该菌在MRS培养基平板上培养36~48h的菌落圆形凸起,边缘整齐,颜色为乳白色;电镜图片显示菌体短杆状,多单个,无芽孢,大小 0.7~0.9μm×0.91~2.35μm;其生理学特性是革兰氏染色为阳性、氧化酶和过氧化氢酶试验为阴性、15℃条件下生长很弱。厌氧条件下能生长、能水解七叶灵、菌株能代谢果糖、半乳糖、葡萄糖、葡糖酸盐、乳糖、麦芽糖、甘露糖、棉子糖、核糖、蔗糖。不能利用七叶灵、甘露醇、松三糖、鼠李糖、水杨苷。结合形态及主要生物学特性把菌株FM-LF-SR6鉴定为发酵乳杆菌(Lactobacillus fermentum)。
5、黏附Caco-2细胞能力
体外Caco-2细胞黏附模型可评价乳酸菌的黏附能力。经革兰氏染色后,在显微镜下可观察到Caco-2细胞表面有明显附着的乳酸菌菌体。菌株FM-LF-SR6在黏附后的活菌数仍能达到106CFU/mL以上,黏附数为60个/cells以上。
6、菌株不同生长时期的不同成分对XOD酶活抑制能力
采用尿酸生成法,检测菌株不同生长时期的胞内提取物(cell-free extracts,CFE)、胞外代谢物(cell-free excretory supernatants,CFS)及菌体碎片沉淀(celldebris precipitation,CDP)对XOD酶活性抑制能力进行研究,结果表明FM-LF-SR6稳定时期的活性提取物对XOD的抑制率最高,抑制率为50%以上。
7、发酵乳杆菌(Lactobacillus fermentum)FM-LF-SR6对高尿酸血症小鼠血尿酸水平的影响:
高尿酸血症小鼠模型采用给小鼠腹腔注射氧嗪酸钾(30mg/100g)并辅以高嘌呤饮食(0.18g/mL)建立,实验动物采用雄性昆明小鼠32只,在室温下饲养,自由饮食采食,经1周适应性饲养后,随机分成正常对照组(C)、高尿酸血症模型组(M)、别嘌呤醇(AP)治疗组和发酵乳杆菌干预组(FM-LF-SR6),每组8只。除正常对照组外,其他三组小鼠每天按30mg/100g标准腹腔注射氧嗪酸钾液,并按照0.18g/mL标准进行酵母膏灌胃,此外FM-LF-SR6组小鼠按照0.2mL/d(2.01*108cfu/mL标准灌胃,AP组小鼠按照5.2mg/100g·d标准灌胃别嘌呤醇(AP)混悬液,持续14d。实验结束时采用一次性眼眶取血0.4mL,4000R/min离心10min,取血清,采用尿酸试剂盒测定血清中尿酸含量。实验数据如表2所示。从表2数据可以看出,连续腹腔注射氧嗪酸钾并佐以高嘌呤饮食14d后,M组、FM-LF-SR6组和AP组小鼠血尿酸含量较正常组小鼠均显著升高,而通过发酵乳杆菌(FM-LF-SR6)的干预,小鼠血尿酸水平与M组小鼠相比显著降低,并接近AP组小鼠血尿酸水平,说明发酵乳杆菌(FM-LF-SR6)具有降低小鼠血尿酸的作用。
表2 14d时各组小鼠血尿酸(SUA)含量(mg/L)
8、FM-LF-SR6在发酵酸奶中的应用
配制含有10%的脱脂牛奶粉和8%的蔗糖的溶液,105℃灭菌10min,冷却至37℃左右,分别商业的酸奶发酵剂接种、商业酸奶发酵剂和FM-LF-SR6混合接种,其中商业酸奶发酵剂和FM-LF-SR6混合接种处理的两者之间的菌体量比例为1∶1。接种之后置于37℃条件下发酵,凝乳后,90℃处理10min,离心取上清,水相膜过滤后测定上清液降解肌苷、鸟苷和XOD酶活抑制能力,并组织专业技术人员10名对发酵酸奶感观评定;具体结果见表2。从表中可以看出,FM-LF-SR6和商业发酵剂混合发酵酸奶,能显著提高酸奶的降解肌苷、鸟苷能力和XOD酶活抑制能力。感官评分显示,FM-LF-SR6 的加入可以提高酸奶的感官评分。可见FM-LF-SR6能显著提高酸奶的降尿酸能力。
表3 FM-LF-SR6和商业酸奶发酵剂接种发酵酸奶的比较
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例所公开的内容。
Claims (8)
1.一株来源于传统贵州酸肉的具有较好的降尿酸功能的发酵乳杆菌新菌株,命名为FM-LF-SR6,经鉴定该菌为发酵乳杆菌(Lactobacillus fermentum),该菌株于2022年1月17在中国微生物菌种保藏管理委员会普通微生物中心进行保藏,保藏号是CGMCC NO.24329。
2.权利要求1所述的发酵乳杆菌(Lactobacillus fermentum)FM-LF-SR6,其特性在于具有较高的降尿酸能力,在菌体密度为5.01×108cfu/ml左右,菌体的对肌苷、鸟苷的降解能力都在98%以上。
3.根据权利要求1所述的发酵乳杆菌(Lactobacillus fermentum)FM-LF-SR6,其特性在于其胞内提取物具有较强的黄嘌呤氧化酶(xanthine oxidase,XOD)的抑制活性,抑制率在50%以上。
4.根据权利要求1所述的发酵乳杆菌(Lactobacillus fermentum)FM-LF-SR6,其特性在于其具有很强的耐酸和耐胆盐能力,在pH 2.0环境下,其4h后存活率为76.51%;在饱腹状态下的人工肠液模拟消化4h后其活菌数仍能达到6.85×106CFU/mL。
5.根据权利要求1所述的发酵乳杆菌(Lactobacillus fermentum)FM-LF-SR6,其特性在于:生长快速,产酸能力强,发酵性能优良,粘附能力强;在30-37℃条件下,12~14h进入稳定期,菌体密度达到109cfu/ml,PH达到4.4以下;对Caco-2细胞的黏附数为60/cells以上。
6.根据权利要求1所述的发酵乳杆菌FM-LF-SR6与乳制品商业发酵剂(保加利亚乳杆菌和嗜热链球菌)具有较好的共培养特性,与商业发酵剂以3∶1~1∶3的比例混合发酵制备酸奶,可以提高发酵乳制品的降尿酸功能,混合发酵奶对肌苷、鸟苷的降解能力和黄嘌呤氧化酶(xanthine oxidase,XOD)的抑制活性提高1.2倍以上。
7.如权利要求1-6项中任意一项所述发酵乳杆菌FM-LF-SR6在制备具有缓解高尿酸功能食品/保健食品方面的应用。
8.根据权利要求7所述的应用,其特征在于:所述功能食品和保健品中的发酵乳杆菌(Lactobacillus fermentum)FM-LF-SR6为活菌株、干菌株或菌株代谢物。
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CN118006491A (zh) * | 2024-02-01 | 2024-05-10 | 乾生(宁波)科技有限公司 | 一种发酵粘液乳杆菌及其应用 |
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CN116024130B (zh) * | 2022-12-13 | 2023-11-24 | 广西爱生生命科技有限公司 | 一株降血尿酸发酵乳杆菌a21215及其应用 |
CN118006491A (zh) * | 2024-02-01 | 2024-05-10 | 乾生(宁波)科技有限公司 | 一种发酵粘液乳杆菌及其应用 |
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