CN117025492B - 一种减轻宠物肥胖的嗜热链球菌jyst-81及其应用 - Google Patents
一种减轻宠物肥胖的嗜热链球菌jyst-81及其应用 Download PDFInfo
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Abstract
本发明提供一种减轻宠物肥胖的嗜热链球菌JYST‑81及其应用,属于微生物技术领域,所述嗜热链球菌JYST‑81于2023年7月24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.27993,分类命名为嗜热链球菌Streptococcus thermophilus,所述嗜热链球菌JYST‑81在制备减轻宠物肥胖产品中的应用。本发明所述嗜热链球菌JYST‑81在体外模拟肠胃环境下对油脂具有较好的吸附作用;所述嗜热链球菌JYST‑81能够改善肠道菌群,减轻宠物体重;所述嗜热链球菌JYST‑81能够降低血清中TG、TC、LDL含量,改善血脂代谢紊乱。
Description
技术领域
本发明涉及一种减轻宠物肥胖的嗜热链球菌JYST-81及其应用,属于微生物技术领域。
背景技术
宠物超重和肥胖是宠物行业面临的最大问题之一,宠物们身上的多余体重势必会损害它们的整体健康,增加它们患上糖尿病、脂肪肝、甲状腺功能减退、癌症、高血压、髋关节发育不良等诸多疾病的风险。
现有技术中减轻宠物肥胖的方法主要有以下几种:
饮食控制:通过减少宠物每天的食物摄入量或者使用低热量的食物来实现。缺点是宠物可能会感到饥饿,出现情绪暴躁或者出现营养不良的问题。
加强宠物运动:增加宠物的运动量,例如多散步、玩耍或参加户外活动等增强宠物的代谢率和体力。缺点是某些宠物可能由于年龄、健康状况或行动不便而无法进行高强度的活动,且猫狗等宠物汗腺较少,排汗能力较差,夏季容易出现中暑现象。
宠物减肥药:一些减肥药含有药物成分或化学物质,宠物的身体对药物的反应与人类不同,可能会导致不良反应或副作用,对宠物的健康有潜在风险,甚至危及宠物的生命。某些减肥药通过减少食欲或影响食物吸收来减轻宠物的体重,会导致宠物营养不均衡,对宠物的健康造成负面影响,且一旦停止使用减肥药,宠物会恢复原有的饮食习惯和体重,无法长期维持减肥效果。
目前针对宠物减肥的产品和研究较少。
发明内容
针对现有技术存在的不足,本发明提供一种减轻宠物肥胖的嗜热链球菌JYST-81及其应用,实现以下发明目的:
吸附油脂,减轻宠物体重,降低血清中血脂、胆固醇的含量。
为解决上述技术问题,本发明采取以下技术方案:
一种减轻宠物肥胖的嗜热链球菌JYST-81,所述嗜热链球菌JYST-81于2023年7月24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CGMCCNo.27993,分类命名为嗜热链球菌Streptococcus thermophilus。
所述嗜热链球菌JYST-81经制备得到菌剂。
所述菌剂中,嗜热链球菌JYST-81的数量为1.0×1010cfu/g。
所述菌剂的制备方法为将嗜热链球菌JYST-81经过活化、培养得到菌液,离心后收集菌体,重悬于复原脱脂乳中,冷冻干燥后得到菌粉,再与葡萄糖混合,得到嗜热链球菌的菌剂。
所述嗜热链球菌JYST-81在制备减轻宠物肥胖产品中的应用。
所述嗜热链球菌JYST-81,于2023年7月24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,邮编:100101,保藏编号:CGMCCNo.27993;该菌株具有对宠物减肥的功能。
与现有技术相比,本发明取得以下有益效果:
本发明所述嗜热链球菌JYST-81在体外模拟肠胃环境下对油脂具有较好的吸附作用;所述嗜热链球菌JYST-81能够改善肠道菌群,增加能量和脂肪的代谢,减轻宠物体重;所述嗜热链球菌JYST-81能够降低血清中TG、TC、LDL含量,改善血脂代谢紊乱。
附图说明
图1为本发明嗜热链球菌JYST-81在模拟肠胃消化液中吸油量的折线图;
图2为本发明嗜热链球菌JYST-81对肥胖宠物犬血清生化指标影响的柱状图;
其中图2A为本发明嗜热链球菌JYST-81对肥胖宠物犬血清中TG含量影响的柱状图;图2B为本发明嗜热链球菌JYST-81对肥胖宠物犬血清中TC含量影响的柱状图;图2C为本发明嗜热链球菌JYST-81对肥胖宠物犬血清中LDL含量影响的柱状图;图2D为本发明嗜热链球菌JYST-81对肥胖宠物犬血清中HDL含量影响的柱状图。*表示P<0.05,**表示P<0.01,***表示P<0.001。
具体实施方式
实施例1菌种的分离鉴定
1、菌株的分离纯化
(1)菌株的分离:将2022年1月23日在重庆市铜梁区采集的自制酸菜切成小块,取1g加入到10mL无菌生理盐水中振荡混匀,然后用无菌生理盐水进行梯度稀释,从10-1至10-7共稀释7个梯度,分别取10-5、10-6、10-7三个梯度的稀释液100μL涂布到MRS固体培养基平板上,置于厌氧培养箱中37℃恒温静置培养48h。
所述MRS固体培养基的制备方法为:取蛋白胨10g、牛肉膏10g、酵母浸粉5g、葡萄糖20g、醋酸钠5g、柠檬酸三铵2g、磷酸氢二钾2g、硫酸镁0.58g、硫酸锰0.25g、吐温-80 1mL、琼脂粉15g,用去离子水溶解,定容至1L,调整pH为6.3,115℃高压蒸汽灭菌20min,倒入平皿中,冷却成固体。
(2)菌株的纯化:根据菌落直径1~2mm、边缘整齐、白色不透明的菌落特征,挑取单菌落在MRS固体培养基平板上进行三区划线,划线后在37℃、厌氧条件下培养48h,重复上述操作,共经过3次划线,纯化得到纯菌株,将纯菌株加入到甘油管中于-80℃保藏。
2、菌株鉴定
将上一步分离纯化所得单菌落送至生工生物工程(上海)股份有限公司进行16SrDNA测序,使用的引物如下:
27F:5'-AGAGTTTGATCCTGGCTCAG-3'(如序列表中的SEQ ID NO.1所示);
1492R:5'-CTACGGCTACCTTGTTACGA-3'(如序列表中的SEQ ID NO.2所示)。
该菌株的16S rDNA核苷酸序列如序列表中的SEQ ID NO.3所示。
将所得的16S rDNA与GenBank网站中已知菌株的序列进行BLAST分析,用MEGA-X软件构建系统发育树。
经系统发育树分析该菌株鉴定为嗜热链球菌(Streptococcus thermophilus),命名为嗜热链球菌JYST-81。
将上述嗜热链球菌JYST-81送至中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏信息如下:
嗜热链球菌JYST-81,分类命名为嗜热链球菌Streptococcus thermophilus,于2023年7月24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,保藏地址:北京市朝阳区北辰西路1号院3号,邮编:100101,保藏编号为CGMCC No.27993。
实施例2嗜热链球菌JYST-81菌剂的制备
将-80℃保藏的嗜热链球菌JYST-81在实施例1所述的MRS固体培养基平板上活化,挑取活化后的1个单菌落转接到100mL 的MRS液体培养基中,37℃恒温静置培养12h制得种子液,将种子液按照1%(v/v)接种量接种于MRS液体培养基中,然后在厌氧条件下37℃恒温静置培养48h,获得菌液。
将菌液离心,收集菌体,用无菌生理盐水洗涤后,重悬于15%(w/w)的复原脱脂乳中,获得浓度为1.0×1010cfu/mL的菌悬液,将菌悬液冷冻干燥后,获得菌粉;
将菌粉与葡萄糖混合,制成嗜热链球菌JYST-81菌剂;葡萄糖购自山东西王糖业有限公司;在本实施例中,制备的嗜热链球菌JYST-81菌剂中菌体数量为1.0×1010cfu/g。
所述MRS液体培养基是在实施例1的MRS固体培养基的配方中省略琼脂的加入。
实施例3嗜热链球菌JYST-81在模拟肠胃消化液中对油脂的吸附作用
(1)模拟胃液的制备:用9.5%(w/v)盐酸加蒸馏水稀释至pH值为2,每100mL稀释后的盐酸溶液中加入1.0g胃蛋白酶,混匀后用0.22μm无菌滤膜过滤,得到模拟胃液,现配现用。
(2)模拟肠液的制备:将6.8g的KH2PO4溶于500mL蒸馏水中,加入3g胆盐(购自上海阿拉丁生化科技股份有限公)和10g胰蛋白酶,用浓度为4g/L的NaOH溶液调节溶液pH值至6.8,用蒸馏水定容至1L后,混匀后用0.22μm无菌滤膜过滤,得到模拟肠液,现配现用。
(3)在模拟肠胃消化液中对油脂的吸附作用:
实验组:在90mL模拟胃液中加入10g食用花生油,然后将实施例2的嗜热链球菌JYST-81菌剂按照4%、8%、12%、16%、20%、24%(w/w)的添加量添加到含花生油的模拟胃液中,37℃、80rpm恒温振荡处理2h,抽滤得到菌体油脂复合物;然后将复合物加入到90mL模拟肠液中,同时加入10g食用花生油,37℃、80rpm恒温振荡处理2h,抽滤得到最终的菌体油脂复合物,将菌体油脂复合物烘干,测量得到实验组菌体油脂复合物干重A2。
对照组:和实验组的区别为菌剂添加到不含食用花生油的模拟肠胃消化液中,其他处理方法同实验组,得到菌体干重A1,每个处理三个平行。按照下列公式计算嗜热链球菌JYST-81在模拟肠胃消化液中对油脂的吸附量:
;
嗜热链球菌JYST-81在模拟肠胃消化液中对油脂的吸附情况如图1所示,随着菌剂添加量的增加,嗜热链球菌JYST-81对油脂的吸附作用先增强后减弱,在添加量为16%(w/w)时,嗜热链球菌JYST-81对油脂的吸附作用最好,吸油量为0.74g/g,嗜热链球菌JYST-81在体外模拟肠胃环境下对油脂具有较好的吸附作用。
嗜热链球菌JYST-81对油脂的吸附作用先增强后减弱,可能是因为嗜热链球菌JYST-81自身会产生脂肪酶对油脂进行分解,添加量少时产生的脂肪酶较少对油脂的分解能力弱;添加量多时产生的脂肪酶较多,菌体吸附的油脂被大量分解,导致菌体油脂复合物减少。
实施例4嗜热链球菌JYST-81对肥胖宠物犬体重的影响
与宠物医院进行合作,由一位专业的宠物营养师根据9分制BCS(Body ConditionScore)体况评分系统挑选5只健康犬、20只肥胖犬,健康犬为空白对照组(NC),肥胖犬随机分为4组,分别为肥胖对照组(OC)、低剂量组(LD)、中剂量组(MD)和高剂量组(HD),试验开始前记录宠物犬初始空腹体重,按照下列方式饲喂8周,每两周记录一次空腹体重数据,结果见表1。
空白对照组(NC):健康犬,饲喂普通饲料。
肥胖对照组(OC):肥胖犬,饲喂普通饲料。
低剂量组(LD):肥胖犬,按照犬的体重添加菌剂2×108cfu/kg/d,与普通饲料混匀饲喂。
中剂量组(MD):肥胖犬,按照犬的体重添加菌剂1×109cfu/kg/d,与普通饲料混匀饲喂。
高剂量组(HD):肥胖犬,按照犬的体重添加菌剂5×109cfu/kg/d,与普通饲料混匀饲喂。
表1嗜热链球菌JYST-81对肥胖宠物犬体重的影响
注:与空白对照组相比,#表示P<0.05,##表示P<0.01,###表示P<0.001。与肥胖对照组相比,*表示P<0.05,**表示P<0.01,***表示P<0.001。
由表1可知,肥胖对照组体重显著高于空白对照组(P<0.001),低剂量、中剂量、高剂量三组体重试验8周后体重均显著降低(P<0.05),其中中剂量组试验6周后体重开始降低(P<0.05),高剂量组试验4周后体重开始降低(P<0.05),说明嗜热链球菌JYST-81菌剂具有显著的减肥效果。
实施例5宠物犬血清生化指标的检测
实施例4中的宠物犬试验结束时由宠物医生进行静脉抽血,收集血液,3000rpm离心15min得到血清。用购自生工生物工程(上海)股份有限公司的试剂盒检测血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)含量。
由图2可知,肥胖对照组的TG、TC、LDL水平显著高于空白对照组(P<0.001),HDL水平显著低于空白对照组(P<0.001),说明肥胖宠物犬血脂代谢紊乱。与肥胖对照组相比,低剂量组、中剂量组、高剂量组的TG、TC、LDL水平均显著降低(P<0.05),HDL水平显著升高(P<0.05)。高剂量组在TG、TC、LDL及HDL水平与肥胖对照组差异极显著(P<0.001),说明嗜热链球菌JYST-81菌剂能够降低血清中TG、TC、LDL含量,改善血脂代谢紊乱。
实施例6嗜热链球菌JYST-81对宠物身体状况的评价
实施例4试验结束后,由一位专业的宠物营养师根据9分制BCS评分系统对25只宠物犬进行身体状况评价,评分结果如下:
表2宠物犬的体况评分
注:*表示P<0.05,**表示P<0.01,***表示P<0.001。
通过BCS体况评分可知,饲喂嗜热链球菌JYST-81菌剂的肥胖宠物犬的体型、脂肪堆积情况均得到明显改善(P<0.05),说明嗜热链球菌JYST-81菌剂能够有效减轻宠物肥胖情况。
Claims (4)
1.一种嗜热链球菌(Streptococcus thermophilus)JYST-81在制备减轻宠物肥胖产品中的应用,其特征在于:所述嗜热链球菌JYST-81于2023年7月24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.27993。
2.根据权利要求1所述的应用,其特征在于:所述嗜热链球菌JYST-81经制备得到菌剂。
3.根据权利要求2所述的应用,其特征在于:所述菌剂中,嗜热链球菌JYST-81的数量为1.0×1010cfu/g。
4.根据权利要求2所述的应用,其特征在于:所述菌剂的制备方法为将嗜热链球菌JYST-81经过活化、培养得到菌液,离心后收集菌体,重悬于复原脱脂乳中,冷冻干燥后得到菌粉,再与葡萄糖混合,得到嗜热链球菌的菌剂。
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