CN116376770B - 一种鼠李糖乳酪杆菌rh0121在制备降血糖制品中的应用 - Google Patents
一种鼠李糖乳酪杆菌rh0121在制备降血糖制品中的应用 Download PDFInfo
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- CN116376770B CN116376770B CN202310354572.9A CN202310354572A CN116376770B CN 116376770 B CN116376770 B CN 116376770B CN 202310354572 A CN202310354572 A CN 202310354572A CN 116376770 B CN116376770 B CN 116376770B
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Abstract
本发明提供了鼠李糖乳酪杆菌(Lacticaseibacillus rhamnosus)RH0121,已于2023年01月09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(CGMCC),保藏编号为:CGMCC NO.26429。RH0121体外α‑葡萄糖苷酶抑制率高达39.22%,并且对消化道逆环境耐受能力强,能够有效改善小鼠的血糖水平。RH0121分离自健康男婴粪便,菌株安全无毒,可广泛应用于降血糖药品和食品中。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种鼠李糖乳酪杆菌RH0121在制备降血糖制品中的应用。
背景技术
糖尿病是由遗传和环境因素相互作用而引起的常见病,临床以高血糖为主要标志,常见症状有多饮、多尿、多食以及消瘦等。糖尿病可引起身体多系统的损害,引起胰岛素绝对或相对分泌不足以及靶组织细胞对胰岛素敏感性降低,引起蛋白质、脂肪、水和电解质等一系列代谢紊乱综合征。血糖显著升高可以出现糖尿病酮症酸中毒、高渗性昏迷等急性并发症,久病还可引起血管与神经病变,从而造成心、脑、肾、眼、神经、皮肤等多脏器组织损害,影响患者的生活质量,甚至导致寿命减短,应积极预防和治疗。
Ⅱ型糖尿病(T2DM)是一种发病机制较复杂的慢性代谢紊乱性疾病,以胰岛素抵抗和胰岛细胞损伤导致的慢性高血糖为主要特征。传统治疗糖尿病药物有双胍类、磺酰脲类、噻唑烷二酮类等。α葡萄糖苷酶抑制剂是20世纪90年代开发的降糖药物,是目前临床治疗T2DM的一线口服药物。α葡萄糖苷酶抑制剂主要作用于位于肠系膜刷状缘的α葡萄糖苷酶,通过抑制α葡萄糖苷酶分解多糖为葡萄糖的作用,从而减缓葡萄糖的吸收速度和降低血糖水平,α葡萄糖苷酶抑制剂具有作用温和持久、无毒或副作用小等优点,受到国内外很多研究者的关注,而它的来源也成为了研究亮点。目前常用的α葡萄糖苷酶抑制剂有主要有阿卡波糖(Acarbose)、伏格列波糖(Voglibose)和米格列醇。阿卡波糖最初是从游动放线菌的次级代谢物中分离而来,因此许多α葡萄糖苷酶抑制剂的微生物源研究都是从土壤中的放线菌入手,目前,已用于临床治疗的α葡萄糖苷酶抑制剂也主要来源于放线菌(井冈霉素产生菌和野尻霉素产生菌)。随着研究的深入,α葡萄糖苷酶抑制剂的来源已深入周围环境的各个领域。其中,乳酸菌来源的α葡萄糖苷酶抑制也成了益生菌降糖功能筛选的重要指标。
乳酸菌被公认是安全的食品级微生物,广泛应用于发酵食品生产和食品保藏方面,已经有非常悠久的历史,对人体具有多种益生功能,常见的有乳酸杆菌、双歧杆菌和芽孢杆菌等。由于乳酸菌的天然、安全和益生特性,近年来已成为防治糖尿病的研究热点。专利CN109055278A公开了一种鼠李糖乳杆菌JYLR-005及其降血糖制品和应用;鼠李糖乳杆菌JYLR-005,在中国普通微生物菌种保藏管理中心(简称CGMCC)保藏,其保藏编号为:CGMCCNo.16103;该发明利用保藏菌株鼠李糖乳杆菌JYLR-005,制成内服产品使用;主要解决高血糖人群的降糖问题,避免用药对身体造成不适症状,降低了高血糖病症复发的风险;该菌种有较强的胃肠道存活率和肠道定植能力,能够影响肠道中多种短链脂肪酸的产生,抑制α-淀粉酶的活性。专利CN113293118A公开了一种鼠李糖乳杆菌菌株LR3001及其应用,已于2020年11月9日保藏于中国典型培养物保藏中心,保藏编号为:CCTCCNO:M2020705,地址为中国武汉武汉大学;该发明还提供了所述鼠李糖乳杆菌LR3001在制备降血糖产品和减肥减脂产品中的应用;该发明提供的鼠李糖乳杆菌分离于西藏林芝地区的雪莲菌,具有抑制α葡萄糖苷酶和α淀粉酶活性的能力。
因此,开发出具有更高α葡萄糖苷酶抑制效果、消化道逆环境耐受能力更强、同时具有较高安全性、具有降血糖和更多功能的益生菌将具有重要意义。
发明内容
为了解决上述问题,本发明提供了鼠李糖乳酪杆菌(Lacticaseibacillusrhamnosus)RH0121,已于2023年01月09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(CGMCC),保藏编号为:CGMCC NO.26429。RH0121体外α-葡萄糖苷酶抑制率高达39.22%,并且对消化道逆环境耐受能力强,能够有效改善小鼠的血糖水平。RH0121分离自健康男婴粪便,菌株安全无毒,可广泛应用于降血糖药品和食品中。
一方面本发明提供了一种鼠李糖乳酪杆菌(Lacticaseibacillus rhamnosus)RH0121,已于2023年01月09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(CGMCC),保藏编号为:CGMCC NO.26429。
另一方面本发明还提供了鼠李糖乳酪杆菌RH0121的培养方法,其特征在于,将鼠李糖乳酪杆菌RH0121接种于改良的MRS固体培养基中。
具体地,所述的改良MRS固体培养基配方为:蛋白胨8-10g/L,牛肉粉2-3g/L,酵母粉3-4g/L,磷酸氢二钾1-2g/L,柠檬酸1-2g/L,乙酸钠4-5g/L,葡萄糖16-20g/L,七水合硫酸镁0.48-0.58g/L,四水合硫酸锰0.19-0.25g/L,吐温-800.5-0.6g/L,碳酸钙8-10g/L,中性红0.04-0.05g,番茄汁8-10mL/L,琼脂粉16-20g/L。
所述的番茄汁是将番茄进行榨汁,然后过滤弃去滤渣。
优选地,所述的改良MRS固体培养基配方为:蛋白胨10g/L,牛肉粉3g/L,酵母粉4g/L,磷酸氢二钾2g/L,柠檬酸2g/L,乙酸钠5g/L,葡萄糖20g/L,七水合硫酸镁0.58g/L,四水合硫酸锰0.25g/L,吐温-80 0.6g/L,碳酸钙10g/L,中性红0.05g,番茄汁10mL/L,琼脂粉20g/L。
具体地,所述的改良MRS固体培养基pH为5-6。
优选地,所述的改良MRS固体培养基pH为5.5。
另一方面本发明提供了鼠李糖乳酪杆菌RH0121在制备降血糖药物中的应用。
具体地,所述的药物可以是鼠李糖乳酪杆菌RH0121的发酵液、发酵液上清、发酵液沉淀、活菌、死菌中的一种或多种。
具体地,所述的药物还包括其他药学上可以接受的辅料。
进一步具体地,所述的药学上可接受的辅料包括但不限于淀粉、糊精、蔗糖、乳糖、微晶纤维素中的一种或多种。
具体地,所述的药物的剂型可以是片剂、液体剂、胶囊剂、散剂、栓剂和颗粒剂。
又一方面本发明提供了一种药物,所述的药物包括鼠李糖乳酪杆菌RH0121。
具体地,所述的鼠李糖乳酪杆菌RH0121可以是发酵液、发酵液上清、发酵液沉淀、活菌、死菌中的一种或多种。
具体地,所述的药物还包括其他药学上可以接受的辅料。
进一步具体地,所述的药学上可接受的辅料包括但不限于淀粉、糊精、蔗糖、乳糖、微晶纤维素中的一种或多种。
具体地,所述的药物的剂型可以是片剂、液体剂、胶囊剂、散剂、栓剂和颗粒剂。
再一方面本发明提供了鼠李糖乳酪杆菌RH0121在食品或保健品中的应用。
又一方面本发明提供了包含鼠李糖乳酪杆菌RH0121的食品或保健品。
具体地,所述的保健品为控制血糖的保健品。
本发明所取得的的技术效果:鼠李糖乳酪杆菌RH0121体外α-葡萄糖苷酶抑制率高达39.22%,并且对消化道逆环境耐受能力强。鼠李糖乳酪杆菌RH0121对酸及胆盐环境的耐受能力、对α-葡萄糖苷酶的抑制能力和对小鼠的血糖调节能力均优于鼠李糖乳杆菌JYLR-005和鼠李糖杆菌LR3001。RH0121分离自健康男婴粪便,菌株安全无毒,可广泛应用于降血糖药品和食品中。
附图说明
图1为鼠李糖乳酪杆菌RH0121菌落形态。
图2为鼠李糖乳酪杆菌RH0121革兰氏染色结果。
图3为鼠李糖乳酪杆菌RH0121冻干粉对小鼠体质量的影响(n=10)。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1鼠李糖乳酪杆菌RH0121菌株的筛选与鉴定
1.1初筛
将500μL健康男婴粪便样品加入到4.5mL改良MRS(pH至5.5)液体培养基中稀释,以10倍稀释法稀释至10-6,从10-3-10-6稀释度分别取50μL样液涂布到改良MRS固体培养基平板上,每个梯度两个平行,将平板放入自封袋中于37℃条件下正常培养24-48h。
1.2纯化
挑取具有目的菌株典型特征(观察形态、大小、色泽、及其透明度等)、菌落较大、活性较强的单菌落,于改良MRS固体培养基平板进行划线纯化培养,37℃培养24-48h,如此反复2-3次,直到划线平皿中菌落特征一致(图1)。
改良MRS培养基:蛋白胨10g/L,牛肉粉3g/L,酵母粉4g/L,磷酸氢二钾2g/L,柠檬酸2g/L,乙酸钠5g/L,葡萄糖20g/L,七水合硫酸镁0.58g/L,四水合硫酸锰0.25g/L,吐温-800.6g/L,碳酸钙10g/L,中性红0.05g(1%浓度5mL),番茄汁10mL/L(v/v),琼脂粉20g/L,调pH至5.5;115℃,30min灭菌。
番茄汁配制:将番茄称量后碾碎,放入榨汁机中榨汁,然后进行过滤,弃去滤渣,称重,根据之前称得重量,用蒸馏水补充至原有的质量,最后分装至10mL离心管中,每管5mL,放入-20℃冰箱中待用。
1.3菌株的鉴定
将1.2纯化后的纯培养物通过划线、涂片镜检,进一步确定其为纯培养物后进行菌种鉴定,包括革兰氏染色试验(图2)、接触酶试验及16S rDNA全序列(SEQ ID NO.1)测序鉴定。最终鉴定分离得到的菌株为一株鼠李糖乳酪杆菌,命名为鼠李糖乳酪杆菌(Lacticaseibacillus rhamnosus)RH0121。鼠李糖乳酪杆菌RH0121菌体呈杆状,0.5-0.7μm×1.2-2.3μm,单个或成对排列,革兰氏阳性,生理生化特征如表1所示。
表1鼠李糖乳酪杆菌RH0121生理生化特征(Physiological andBiochemicalCharacteristic)
符号说明:“+”,阳性;“w”,弱阳性;“-”,阴性。
实施例2鼠李糖乳酪杆菌RH0121耐消化道逆环境实验
2.1培养基配方
MRS培养基配方:酵母蛋白胨10g/L,牛肉粉3g/L,酵母浸出物4g/L,磷酸二氢钾2g/L,一水柠檬酸2g/L,乙酸钠5g/L,无水葡萄糖20g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,吐温80 0.6g/L,番茄汁10mL/L(v/v),pH 6.5。
耐酸试验培养基:用1mol/L盐酸将MRS培养基pH值调成3.0和2.0。
耐胆盐试验培养基:在MRS培养基中添加胆盐,浓度为0.3%、0.5%、1.0%和1.5%W/V。
2.2耐酸实验
将RH0121菌株划线得到的单菌落接种到5mL MRS液体培养基中,37℃培养20h;将5mL菌液全部接种于100mL MRS液体培养基中,37℃培养17h,作为试验种子液。
将MRS液体培养基活化三代的RH0121菌株以一定比例(菌液:培养基=1mL:4mL)接种到pH3.0和pH2.0的MRS培养基中,37℃保持2h后,按2%的接种量接种在新鲜pH6.5的MRS液体培养基中,通过多功能酶标仪每隔10min测定一次OD600值,直到OD600值增长0.3个单位时,停止测定。
将MRS液体培养基活化三代的菌株以一定比例(菌液:培养基=1mL:4mL)接种到新鲜pH6.5的MRS液体培养基中,37℃保持2h后,通过多功能酶标仪每隔10min测定一次OD600值,直到OD600值增长0.3个单位时,停止测定。
计算菌株在pH3.0、pH2.0和正常培养基中生长OD600值增加0.3个单位所需时间,二者之差被称为延迟时间。
耐酸实验结果如表2所示,在pH2.0条件下的延迟时间为3.16h,在pH3.0条件下的延迟时间为2.5h,菌株RH0121对酸有耐受能力。
表2 RH0121耐酸实验数据表
处理方式 | OD600增加0.3个单位所用时间(h) | 延迟时间(h) |
NA酸 | 3.67 | —— |
pH 3.0 | 6.17 | 2.50 |
pH 2.0 | 6.83 | 3.16 |
2.2耐胆盐实验
将MRS液体培养基活化三代的菌株按2%的接种量分别接种于含0.3%、0.5%、1.0%、1.5%胆盐的MRS液体培养基中和不含胆盐的MRS培养基中,通过多功能酶标仪每隔10min测定一次OD600值,直到OD600值增加0.3个单位以上停止测定,计算菌株在含0.3%、0.5%、1.0%、1.5%胆盐和不含胆盐的MRS培养基中生长OD600值增加0.3个单位所需时间,二者之差被称为延迟时间。
耐胆盐实验结果如表3所示,菌株RH0121的延迟时间均在0.16h-0.83h之间,且在0.3%胆盐浓度条件下的延迟时间为0.16h,表现出较强的胆盐耐受能力。
表3 RH0121耐胆盐实验数据表
处理方式 | OD600增加0.3个单位所用时间(h) | 延迟时间(h) |
NA胆盐 | 3.67 | —— |
0.3%胆盐 | 3.83 | 0.16 |
0.5%胆盐 | 4.00 | 0.33 |
1.0%胆盐 | 4.17 | 0.50 |
1.5%胆盐 | 4.50 | 0.83 |
实施例3鼠李糖乳酪杆菌RH0121体外降血糖功能实验
3.1实验菌株制备
(1)一级培养:将保藏于-80℃冰箱的RH0121菌株冻存管取出,室温解冻,混匀,取1环菌液于MRS固体平板划线,37℃静置培养48h;
(2)二级培养:挑单菌落至5mL MRS液体培养基中,37℃培养20h;
(3)三级培养:5mL菌液接种至100mL MRS液体培养基中,37℃培养24h,得菌悬液。
3.2待测样品制备
a:取培养好的菌悬液于4500g,4℃离心10min后取发酵上清液,4℃保存备用;
b:取培养好的菌悬液于4500g,4℃离心10min后,吸取上清液用0.22μm滤膜过滤得到菌株无细胞上清液,4℃保存备用;
c:取培养好的菌悬液70℃条件下水浴30min,得灭活的菌悬液,4℃保存备用;
d:取c处理的灭活菌悬液于4500g,4℃离心10min后取灭活的发酵上清液,4℃保存备用;
e:取c处理的灭活菌悬液于4500g,4℃离心10min后,吸取上清液用0.22μm滤膜过滤得到灭活的菌株无细胞提取物,4℃保存备用。
3.3α-葡萄糖苷酶活性抑制率实验
按照表4中的添加量分别在酶标板孔中加入酶、样品和PBS缓冲液,混匀后37℃,10min。加入底物,37℃反应20min后,加入Na2CO3终止反应,于405nm波长下测定其吸光值。
表4实验反应体系及反应条件
3.4实验结果
实验结果计算方法:
其中,A405(A):含有α-葡萄糖苷酶溶液,不含待测样品的吸光值;A405(B):不含α-葡萄糖苷酶溶液,不含待测样品的吸光值;A405(C):含有α-葡萄糖苷酶溶液,含待测样品的吸光值;A405(D):不含α-葡萄糖苷酶溶液,含待测样品的吸光值。
表5鼠李糖乳酪杆菌RH0121的α-葡萄糖苷酶抑制率
实施例4降低血糖功能评价
4.1Ⅱ型糖尿病小鼠模型建立及分组
选用SPF级健康雄性小鼠30只,4周龄,体质量在16-18g,分为模型组、空白组和样品组。喂养饲料分为正常饲料(5%脂肪,53%碳水,23%蛋白质)、高脂饲料(22%脂肪,48%碳水,20%蛋白质)。具体分组和给药见表6所示。
表6小鼠分组和灌胃方法
4.2一般体征试验
记录小鼠第10周初始及第11-14周的体质量。
如图3所示,空白组小鼠体重变化幅度小,模型组的小鼠体重下降较为明显,这是由于长期血糖值偏高导致体内的脂肪和蛋白质被利用,使得小鼠体重下降幅度较大。样品组的小鼠在第12周之前体重下降较为明显,第12周以后体重缓慢上升,说明食用RH0121冻干粉后小鼠血糖值有一定的影响,使小鼠体重有所回升。
4.3口服葡萄糖耐量的测定
样品灌胃结束后,禁食不禁水12h后进行灌胃葡萄糖溶液,灌胃量按照2g/kg,分别在灌胃0、30、60、90、120min后测量小鼠的血糖值。
结果如表7所示,在30min时血糖浓度达到最高值。样品组的血糖浓度显著(P<0.05)低于模型组,说明小鼠服用鼠李糖乳酪杆菌(Lacticaseibacillus rhamnosus)RH0121冻干粉改善了血糖水平。超过30min后血糖水平随着时间延长而降低,说明此时的葡萄糖正在被机体利用。灌胃120min后,样品组的小鼠血糖浓度为10.3moL/L,低于灌胃0min时的血糖11.1moL/L,说明小鼠服用样品后可以辅助机体调节血糖的浓度,改善血糖水平。
表7鼠李糖乳酪杆菌RH0121冻干粉对小鼠血糖含量的影响(n=10)
注:*表示样品组与模型组相比差异显著,P<0.05;**表示样品组与模型组相比差异极显著,P<0.01。
对比例1
参照实施例2的方法设置对比例并进行实验,对比例设置方式和实验结果如下:
表8耐酸实验数据表
表9耐胆盐实验数据表
综合分析表2、表3、表8和表9结果表明鼠李糖乳酪杆菌RH0121对酸及胆盐环境的耐受能力均优于鼠李糖乳杆菌JYLR-005和鼠李糖杆菌LR3001。
对比例2
参照实施例3的方法,对鼠李糖杆菌JYLR-005和鼠李糖杆菌LR3001分别进行体外降血糖功能实验。结果(表10)表明本发明鼠李糖乳酪杆菌RH0121对α-葡萄糖苷酶的抑制能力高于鼠李糖乳杆菌JYLR-005和鼠李糖杆菌LR3001。
表10对比菌株的α-葡萄糖苷酶抑制率
对比例3
参照实施例4的方法,对鼠李糖杆菌JYLR-005和鼠李糖杆菌LR3001分别进行降低血糖功能评价,结果(表11)口服葡萄糖耐量的测定显示从0-120min的小鼠血糖含量均高于鼠李糖乳酪杆菌RH0121样品组,说明鼠李糖乳酪杆菌RH0121对小鼠的血糖调节能力高于鼠李糖杆菌JYLR-005和鼠李糖杆菌LR3001。
表11对小鼠血糖含量的影响(n=10)
Claims (10)
1.一种鼠李糖乳酪杆菌(Lacticaseibacillus rhamnosus)RH0121,其特征在于,已于2023年01月09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,保藏编号为:CGMCC NO.26429。
2.权利要求1所述的鼠李糖乳酪杆菌RH0121的培养方法,其特征在于,包括将鼠李糖乳酪杆菌RH0121接种于改良的MRS固体培养基中。
3.根据权利要求2所述的培养方法,其特征在于,所述的改良MRS固体培养基配方为:蛋白胨8-10g/L,牛肉粉2-3g/L,酵母粉3-4g/L,磷酸氢二钾1-2g/L,柠檬酸1-2g/L,乙酸钠4-5g/L,葡萄糖16-20g/L,七水合硫酸镁0.48-0.58g/L,四水合硫酸锰0.19-0.25g/L,吐温-800.5-0.6g/L,碳酸钙8-10g/L,中性红0.04-0.05g,番茄汁8-10mL/L,琼脂粉16-20g/L。
4.权利要求1所述的鼠李糖乳酪杆菌RH0121在制备降血糖药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述的药物包括鼠李糖乳酪杆菌RH0121的发酵液、发酵液上清、发酵液沉淀、活菌中的一种或多种。
6.一种药物,其特征在于,所述的药物包括鼠李糖乳酪杆菌RH0121。
7.根据权利要求6所述的药物,其特征在于,所述的鼠李糖乳酪杆菌RH0121是发酵液、发酵液上清、发酵液沉淀、活菌中的一种或多种。
8.权利要求1所述的鼠李糖乳酪杆菌RH0121在制备降血糖的食品或保健品中的应用。
9.包含权利要求1所述的鼠李糖乳酪杆菌RH0121的食品或保健品。
10.根据权利要求9所述的食品或保健品,其特征在于,所述的保健品为控制血糖的保健品。
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