CN115227808A - 德氏乳杆菌保加利亚亚种sf-l-18发酵制品及其制备方法和缓解便秘的应用 - Google Patents
德氏乳杆菌保加利亚亚种sf-l-18发酵制品及其制备方法和缓解便秘的应用 Download PDFInfo
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- CN115227808A CN115227808A CN202211140068.0A CN202211140068A CN115227808A CN 115227808 A CN115227808 A CN 115227808A CN 202211140068 A CN202211140068 A CN 202211140068A CN 115227808 A CN115227808 A CN 115227808A
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Abstract
本发明涉及一种德氏乳杆菌保加利亚亚种SF‑L‑18发酵制品及其制备方法和缓解便秘的应用，属于微生物的技术领域。包括重量份的以下原料：山药1‑5份、茯苓1‑3份、桃花0.5‑2份、当归1‑5份、蜂蜜1‑5份、泽泻0.5‑2份、黄芪0.5‑2份，弹性胶原蛋白肽0.1‑0.5份，胡萝卜浓缩汁3‑8份、益生元糖浆2‑5份和沙棘果浓缩汁2‑5份。本发明是利用德氏乳杆菌保加利亚亚种SF‑L‑18发酵中草药，成品中含有乳酸的代谢产物、菌体多糖以及一些益生元，可以从根本上增加肠道水分；增加大便水分，促进肠道蠕动，从而促进排便，改善便秘。
Description
技术领域
本发明涉及微生物的技术领域，具体涉及一种德氏乳杆菌保加利亚亚种SF-L-18发酵制品及其制备方法和缓解便秘的应用。
背景技术
便秘随着人们膳食结构的改变、生活节奏的加快，以及受社会心理因素的影响，发病率呈逐年上升趋势。据统计，目前我国便秘的发病率约为8.2%，且女性高于男性，尤其是城市女性患病率（15.2%）明显高于男性（4%）。便秘病程较长，病情易反复，严重影响患者的生活品质，且易引发其他疾病，对身体健康危害极大。便秘易造成肠道菌群紊乱，重金属、有害毒素蓄积及过度吸收，继而引发其他疾病。慢性便秘患者发生结直肠癌的风险是正常人的1.78倍，而发生良性结直肠肿瘤的风险是正常人的2.70倍。治疗便秘的药物很多种，大的种类大约分以下几种，最常见的是一种叫做容积性泻药，如纤维素类，多糖类，还有聚乙二醇4000这类的，它是单纯增加大便水分的，增加大便容量的。第二种是渗透性泻药，比如乳果糖、甘露醇；就是把肠道的水分，从肠壁上吸出来渗透性的泻药。第三种叫做刺激性泻药：番泻叶，三黄片等含有蒽醌类成分刺激排泄，还有润滑型泻药如：莫沙必利、阿片类抗结剂，促进胃肠道蠕动。容积性泻药和渗透性的泻药是相对安全的，可以长期服用，但起效慢，作用相对较弱；刺激性泻药作用强，可以短期服用，不能长期服用，长期服用会引起结肠黑便病，加重便秘。
发明内容
针对现有技术存在的非刺激性药物见效慢，刺激性药物副作用大问题，本发明提供一种德氏乳杆菌保加利亚亚种SF-L-18发酵制品及其制备方法和缓解便秘的应用，以解决上述技术问题。本发明是利用德氏乳杆菌保加利亚亚种SF-L-18发酵中草药，成品中含有乳酸的代谢产物、菌体多糖以及一些益生元，可以从根本上增加肠道水分；增加大便水分，促进肠道蠕动，从而促进排便，改善便秘，无副作用，见效快。
本发明的技术方案为：
一种德氏乳杆菌保加利亚亚种SF-L-18发酵制品，包括重量份的以下原料：山药1-5份、茯苓1-3份、桃花0.5-2份、当归1-5份、蜂蜜1-5份、泽泻0.5-2份、黄芪0.5-2份，弹性胶原蛋白肽0.1-0.5份，胡萝卜浓缩汁3-8份、益生元糖浆2-5份和沙棘果浓缩汁2-5份。
所述德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii subsp. bulgaricus)SF-L-18，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC NO.20928，保藏日期为2020年10月20日，保藏机构地址：北京市朝阳区北辰西路1号院3号。
优选的，包括重量份的以下原料：山药2份、茯苓2份、桃花1.5份、当归1.5份、蜂蜜3份、泽泻1份、黄芪1份、弹性胶原蛋白肽0.15份、胡萝卜浓缩汁6份、益生元糖浆3份和沙棘果浓缩汁3份。
上述植物发酵制品的制备方法，包括以下步骤：
（1）将山药、茯苓、桃花、当归、蜂蜜、泽泻、黄芪进行混合，添加6倍水，熬煮四遍，经过200目过滤得混合液。
（2）将混合液添加弹性胶原蛋白肽121℃高压灭菌30分钟，冷却至30~40℃，得到灭菌液；
（3）将德氏乳杆菌保加利亚亚种SF-L-18，接种到MRS液体培养基中，在37℃下培养制备接种剂；
（4）将接种剂接种到灭菌液中，37℃发酵24~48h，得发酵液；
（5）发酵液添加胡萝卜浓缩汁、益生元糖浆和沙棘果浓缩汁调味后，经破壁、巴氏灭菌，得益生菌植物制品。所制备的植物发酵制品为褐色液体，可直接饮用。
优选的，所述MRS液体培养基包括以下组分：牛肉膏10.0g，酵母膏5.0g，蛋白胨10.0g，葡萄20.0g，K2HPO4 2.0g，醋酸钠5.0g，MgSO4•7H2O 0.2g，MnSO4•4H2O 0.05g，吐温801.0g，柠檬酸三铵2.0g，加蒸馏水至1000mL。
优选的，所述步骤（3）中，接种量为1%。
优选的，所述步骤（4）中，接种量为500~1000万cfu/mL。
优选的，所述破壁压力为20~60Mpa。
本发明使用德氏乳杆菌保加利亚亚种发酵的制品用于制备缓解便秘的饮品。
本发明的有益效果为：
本发明是利用德氏乳杆菌保加利亚亚种SF-L-18发酵中草药，成品中含有乳酸的代谢产物、菌体多糖以及一些益生元，可以从根本上增加肠道水分；增加大便水分，促进肠道蠕动，从而促进排便，改善便秘。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，对于本领域普通技术人员而言，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是不同组别小鼠粪便中短链脂肪酸的含量对比图。
图2是不同组别小鼠粪便中5种菌群菌落数对比图。
图3是服用实施例1制备的植物饮品前后粪便中短链脂肪酸含量变化图。
图4是服用实施例1制备的植物饮品前后粪便5种细菌的变化图。
具体实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
实施例中使用的MRS液体培养基包括以下组分：牛肉膏10.0g，酵母膏5.0g，蛋白胨10.0g，葡萄20.0g，K2HPO4 2.0g，醋酸钠5.0g，MgSO4•7H2O 0.2g，MnSO4•4H2O 0.05g，吐温801.0g，柠檬酸三铵2.0g，加蒸馏水至1000mL。
实施例1
一种植物发酵饮品的制备方法，包括以下步骤：
（1）将山药2份、茯苓2份、桃花1.5份、当归1.5份、蜂蜜3份、泽泻1份、黄芪1份进行混合，添加6倍水，熬煮四遍，经过200目过滤得混合液。
（2）将混合液添加0.15份的弹性胶原蛋白肽121℃高压灭菌30分钟，冷却至30~40℃，得到灭菌液；
（3）将德氏乳杆菌保加利亚亚种SF-L-18，以1%的接种量接种到MRS液体培养基中，在37℃下培养制备接种剂；
（4）将接种剂接种到灭菌液中，接种量为800万cfu/mL，37℃发酵36h，得发酵液；
（5）发酵液添加胡萝卜浓缩汁6份、益生元糖浆3份和沙棘果浓缩汁3份调味后经破壁，破壁压力为40Mpa、巴氏灭菌，得植物发酵饮品。
实施例2
一种植物发酵饮品的制备方法，包括以下步骤：
（1）将山药1份、茯苓1份、桃花0.5份、当归1份、蜂蜜1份、泽泻0.5份、黄芪0.5份进行混合，添加6倍水，熬煮四遍，经过200目过滤得混合液。
（2）将混合液添加0.1份的弹性胶原蛋白肽121℃高压灭菌30分钟，冷却至30~40℃，得到灭菌液；
（3）将德氏乳杆菌保加利亚亚种SF-L-18，以1%的接种量接种到MRS液体培养基中，在37℃下培养制备接种剂；
（4）将接种剂接种到灭菌液中，接种量为500万cfu/mL，37℃发酵48h，得发酵液；
（5）发酵液添加胡萝卜浓缩汁3份、益生元糖浆2份和沙棘果浓缩汁2份调味后经破壁，破壁压力为20Mpa、巴氏灭菌，得植物发酵饮品。
实施例3
一种植物发酵饮品的制备方法，包括以下步骤：
（1）将山药5份、茯苓3份、桃花2份、当归5份、蜂蜜5份、泽泻2份、黄芪2份进行混合，添加6倍水，熬煮四遍，经过200目过滤得混合液。
（2）将混合液添加0.5份的弹性胶原蛋白肽121℃高压灭菌30分钟，冷却至30~40℃，得到灭菌液；
（3）将德氏乳杆菌保加利亚亚种SF-L-18，以1%的接种量接种到MRS液体培养基中，接种量为1000万cfu/mL，在37℃下培养制备接种剂；
（4）将接种剂接种到灭菌液中，接种量为1000万cfu/mL，37℃发酵24h，得发酵液；
（5）发酵液添加胡萝卜浓缩汁8份、益生元糖浆5份和沙棘果浓缩汁5份调味后经破壁，破壁压力为60Mpa、巴氏灭菌，得植物发酵饮品。
测试例1
植物饮品的小鼠便秘模型实验
购买160只雄性ICR小鼠，随机选择140只建立小鼠便秘模型，依据便秘模型复制方法（陈奇《中药药理实验方法学》），让小鼠进行12h的禁食，但不禁水，然后按照20mL/kg的剂量为其灌胃复方地芬诺酯混悬液（2.5mg/mL），判定小鼠造模成功的标准为给药后小鼠具有蓬乱的皮毛、明显较少的排便粒数、干粉状粪便等。
将山药2份、茯苓2份、桃花1.5份、当归1.5份、蜂蜜3份、泽泻1份、黄芪1份进行混合，添加水6倍水熬煮四遍得中草药汁作为中草药组给药；实施例1所得植物发酵饮品作为植物饮品组给药（1mL/kg）；模型组和正常组饲喂相同剂量的无菌水。
1. 测定便秘小鼠的大肠水分
将140只上述便秘模型小鼠，随机分为模型组、植物饮品低剂量组（0.75mL/kg）、植物饮品中剂量组（1mL/kg）、植物饮品高剂量组（1.25mL/kg）、中草药低剂量组（0.75mL/kg）、中草药中剂量组（1mL/kg）、中草药高剂量组（1.25mL/kg）7组，每组20只，剩余的20只为正常组；依据实验方案给予8组小鼠9d的连续给药，小鼠脱颈处死后将其腹部皮肤及腹膜切开，将肠管暴露出来。之后用眼科剪刀将肠系膜切开，谨慎结扎直肠末端，将肠管剪断，将大肠取出来。对8组小鼠的大肠湿重进行称量，然后在80℃的干燥箱中放置烘干，将其取出后对其干重进行称量，计算大肠内水分含量，计算方法为湿重与干重之差与干重的百分比。实验结果如表1所示。
表1-植物饮品和中草药对小鼠大肠水分含量的影响
便秘的发生一方面受到结肠动力异常的影响，另一方面也受到肠道内水分多少的影响，以此认为便秘发生的一个极为重要的病理学机制就是水分过度吸收。因此，在对便秘症状进行缓解的过程中，对肠道水分吸收功能进行有效改善，或促进肠道水分含量的有效增加能够收到有效的效果。由表1可以看出，跟模型组相比植物饮品和中草药能够显著增加小鼠大肠水分含量，对大肠吸收水分的现象进行抑制，能够缓解便秘；还可以看出植物饮品中剂量组效果更佳。
2. 测定小鼠肠组织中五羟色胺（5-HT）含量
小鼠脱颈处死后将一段小肠从幽门一端小肠后2/5处长度为2cm左右取出来，立刻冻存在液氮中。对5-HT样品进行测量处理，称量小肠组织重量后在匀浆器中放置，将10%生理盐水加入其中，进行肠组织匀浆。将8组小鼠的肠段组织液取出来，留取其肠组织匀浆标本。4℃的温度下以3000r/min离心10min，将上清液取出并保存。运用双抗体两步夹心酶联免疫吸附法（ELISA法）严格实际试剂盒说明书对小鼠肠组织中5-HT含量进行测定。具体测试结果如表2所示。
表2-植物饮品和中草药对小鼠5-HT含量的影响
临床研究发现五羟色胺（5-HT）是一种神经递质，参与调节胃肠道运动及分泌功能，通过肠道黏膜层的嗜铬细胞将5-HT释放了出来，进而激发肠蠕动及分泌反射，肠道平滑肌收缩及胃肠蠕动随着5-HT含量的降低而降低，从而造成粪便积滞在肠道，引发便秘。因此，临床日益广泛地关注了肠道正常动力及分泌受到5-HT的调控作用。相关医学研究表明，5-HT受体激动剂能够促进兴奋性神经通路传导的增强，在便秘的治疗中发挥着积极的作用。同时，5-HT还能够促进便秘症状的有效减轻，途径为有效恢复肠壁的顺应性。由表2可以看出中草药高剂量组、植物饮品中剂量组和植物饮品高剂量组与模型组相比可以显著的提高5-HT的含量，其中植物饮品中剂量组的效果最佳。
我们还研究了不同组别的小鼠粪便中短链脂肪酸的含量以及5种菌群的菌落数。具体结果如图1和图2所示。通过图1显示可以看出，与模型组相比其他7组粪便中短链脂肪酸总酸含量多（乙酸、丙酸、丁酸含量的总和），其中植物饮品中剂量组比模型组多26%，正常组和中草药高剂量组都多23%，结果说明中草药和植物饮品可以增加肠道中短链脂肪酸的含量，从而改善便秘，植物饮品中剂量组的效果更佳。由图2可以看出，植物饮品中剂量组、植物饮品高剂量组、中草药高剂量组和正常组与模型组相比粪便中的乳杆菌的数量显著增加（P<0.05），有害菌肠杆菌的数量显著减少（P<0.05），其他细菌的数量没有显著差异（P>0.05）；植物饮品中剂量组比中草药高剂量组的有益菌多，有害菌少，发酵植物饮品比直接熬煮的草药效果要好些。
通过以上试验结果可以看出实施例1所得植物发酵饮品的中剂量组和中草药高剂量组都可以增加便秘模型小鼠的肠道水分，减少肠道吸水，缓解便秘；可以增加肠道中短链脂肪酸的含量，可以改善肠道菌群，相对而言植物饮品的效果更佳。
测试例2
1.受试者筛选
1.1筛选标准
（1）年龄18~60岁，男女不限；(2)便秘症状符合罗马IV标准；(3)患者对本研究知情同意并自愿参与。
1.2排除标准
（1）胃肠道梗阻、出血者；（2）严重肝肾功能不全者；（3）严重心脑血管疾病者；（4）严重呼吸系统疾病者；（5）严重的水电解质紊乱者；（6）血糖控制不住的糖尿病患者；（7）近1年有胃肠手术史者；（8）妊娠及哺乳期妇女；（9）对肠道准备药物过敏的患者。
2.产品的服用试验结果
（1）本次完成试服的人数共60人，早晚各一次，每次30mL。
（2）测试结果如下表3所示。
表3-试服疗效统计表
如表3所示，根据试食者不同病情分别进行疗效分析，本发明的德氏乳杆菌保加利亚亚种SF-L-18植物饮品在治疗和缓解便秘方面，效果较好，痊愈率53.33%，有效40%，总有效率93.33%。
我们对受试者服用实施例1制备的发酵饮品前后粪便中短链脂肪酸含量变化进行了统计，具体结果如图3所示。乙酸、丙酸、丁酸等短链脂肪酸是肠道微生物的最终代谢产物；可以通过降低肠道的pH值抑制致病菌的繁殖，促进有益菌的增殖，从而改善肠道微环境；短链脂肪酸为肠黏膜细胞提供能量来源，促进肠上皮细胞增殖，有利于维护肠屏障，抵抗炎症，从而缓解便秘。有研究表明乙酸、丙酸等有机酸可直接刺激肠壁神经，加快肠道蠕动；乙酸是微生物的主要代谢产物，其浓度增加使得肠管内渗透压增大，肠道内水分吸收增加，从而使粪便质量和润滑度增加，更易运输与排出。因此乙酸、丙酸、丁酸等短链脂肪酸的含量可作为缓解便秘效果的重要评价指标。服用实施例1制备的发酵饮品后，由于饮品中含有大量的益生菌代谢产物，可以促进益生菌的增值，从而改善便秘。如图3所示，服用实施例1制备的发酵饮品后，粪便中乙酸含量增加20%，丙酸增加43%，丁酸增加18%，总酸（乙酸、丙酸、丁酸含量的总和）增加23%，但试食前后无显著性差异（P>0.05）。以上结果说明食用本品可以增加肠道中短链脂肪酸的含量。
我们对受试者服用实施例1制备的发酵饮品前后粪便中5种细菌的变化进行了统计，具体结果如图4所示。由图4可以看出，受试者服用本品后，粪便中益生菌双歧杆菌和乳酸杆菌的数量与服用前相比显著增加（P<0.05），有害菌肠杆菌的数量显著减少（P<0.05），其他细菌的数量与服用前相比没有显著差异（P>0.05）。
3.结果分析
本次试验完成试服人数共60人，除4人服用后症状没有得到有效改善外，其余56人均有效果。
受试者粪便中短链脂肪酸的含量服用后增加23%，益生菌双歧杆菌和乳酸杆菌显著增加（P<0.05），有害菌肠杆菌的数量显著减少（P<0.05）。说明此植物饮品对轻中度痔疮患者可以起到显著的缓解和辅助治疗作用。
尽管通过参考附图并结合优选实施例的方式对本发明进行了详细描述，但本发明并不限于此。在不脱离本发明的精神和实质的前提下，本领域普通技术人员可以对本发明的实施例进行各种等效的修改或替换，而这些修改或替换都应在本发明的涵盖范围内/任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以权利要求所述的保护范围为准。
Claims (8)
1.一种德氏乳杆菌保加利亚亚种SF-L-18发酵制品，其特征在于，包括重量份的以下原料：山药1-5份、茯苓1-3份、桃花0.5-2份、当归1-5份、蜂蜜1-5份、泽泻0.5-2份、黄芪0.5-2份，弹性胶原蛋白肽0.1-0.5份，胡萝卜浓缩汁3-8份、益生元糖浆2-5份和沙棘果浓缩汁2-5份；
所述德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii subsp. bulgaricus)SF-L-18，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCCNO.20928，保藏日期为2020年10月20日，保藏机构地址：北京市朝阳区北辰西路1号院3号。
2.如权利要求1所述的发酵制品，其特征在于，包括重量份的以下原料：山药2份、茯苓2份、桃花1.5份、当归1.5份、蜂蜜3份、泽泻1份、黄芪1份、弹性胶原蛋白肽0.15份、胡萝卜浓缩汁6份、益生元糖浆3份和沙棘果浓缩汁3份。
3.一种如权利要求1所述的发酵制品的制备方法，其特征在于，包括以下步骤：
（1）将山药、茯苓、桃花、当归、蜂蜜、泽泻、黄芪进行混合，添加6倍水，熬煮四遍，经过200目过滤得混合液；
（2）将混合液添加弹性胶原蛋白肽121℃高压灭菌30分钟，冷却至30~40℃，得到灭菌液；
（3）将德氏乳杆菌保加利亚亚种SF-L-18接种到MRS液体培养基中，在37℃下培养制备接种剂；
（4）将接种剂接种到灭菌液中，37℃发酵24~48h，得发酵液；
（5）发酵液添加胡萝卜浓缩汁、益生元糖浆和沙棘果浓缩汁调味后，经破壁、巴氏灭菌，得益生菌植物饮品。
4.如权利要求3所述的制备方法，其特征在于，所述MRS液体培养基包括以下组分：牛肉膏10.0g，酵母膏5.0g，蛋白胨10.0g，葡萄糖20.0g，K2HPO4 2.0g，醋酸钠5.0g，MgSO4•7H2O0.2g，MnSO4•4H2O 0.05g，吐温80 1.0g，柠檬酸三铵2.0g，琼脂15.0g，加蒸馏水至1000mL。
5.如权利要求3所述的制备方法，其特征在于，所述破壁压力为20~60Mpa。
6.如权利要求3所述的制备方法，其特征在于，所述步骤（3）中，接种量为1%。
7.如权利要求3所述的制备方法，其特征在于，所述步骤（4）中，接种量为500~1000万cfu/mL。
8.一种如权利要求1所述的德氏乳杆菌保加利亚亚种SF-L-18发酵制品在制备缓解便秘饮品中的应用。
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