CN113412060A - 用于预防或治疗幽门螺杆菌相关疾病的泡菜 - Google Patents
用于预防或治疗幽门螺杆菌相关疾病的泡菜 Download PDFInfo
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Abstract
本申请涉及能够有效地用于预防和治疗幽门螺杆菌相关疾病的含有乳酸菌的泡菜。
Description
技术领域
本申请涉及能够有效地用于预防和治疗幽门螺杆菌(Helicobacter pylori)相关疾病的含有乳酸菌的泡菜。
背景技术
1983年,澳大利亚微生物学家Marchall和Warren首次在B型胃炎患者的胃粘膜活检组织中发现了弯曲细菌,然后培养所述细菌并鉴定它是与弯曲杆菌属(Campylobactergenus)相关的新种细菌。此后,对幽门螺杆菌与诸如胃炎和消化性溃疡等的上消化道疾病之间的相关性进行了积极的研究。幽门螺杆菌是一种革兰氏阴性杆菌,已知其具有被薄膜包围的鞭毛,能释放大量的脲酶,通过粪-口途径传播,并且在1994年由世界卫生组织(WHO)确定为致癌物,因此成为在全世界范围内受到关注的病原体。
目前,由幽门螺杆菌引起的疾病的致病机理已知如下:与食物一起进入胃腔的细菌通过具有强运动性的鞭毛穿透胃粘液层,并且定殖在胃粘液层内的胃上皮细胞层上部与细胞间的联结部,并且分泌出大量的脲酶以将粘液层改变为碱性,从而异常过量地刺激胃泌素对胃酸的分泌,并由此导致炎症和溃疡。此外,根据最近的研究报告,幽门螺杆菌是胃腺癌致病机理的重要危险因素,世界卫生组织将幽门螺杆菌指定为I类致癌物。
特别是,据报道幽门螺杆菌感染在包括韩国在内的发展中国家中发生频率高,并且根据对无上消化道症状的5732例患者进行的韩国流行病学调查,幽门螺杆菌感染率为46.6%,16岁以上成人感染率为69.4%,尤其是在40岁以上人群确认到78.5%的高感染率。此外,根据对1031例消化性溃疡患者进行的幽门螺杆菌感染率的调查发现,胃溃疡患者中的66%和十二指肠溃疡患者中的79%感染有幽门螺杆菌,而同时患有胃和十二指肠溃疡患者中的71%感染有幽门螺杆菌。
作为用于治疗由如上所述的幽门螺杆菌感染引起的疾病的方法,目前主要实施利用抗生素的化疗。然而,这种方法的广泛使用在短期内可能是有效的,但是这种方法存在着由于抗生素的持续使用而导致抗生素抗性菌株出现的风险问题。此外,已有报道称持续使用抗菌疗法会引起诸如腹泻、反流性食管炎、肠中正常菌群的抑制、抗性菌株出现等各种副作用。
因此,需要开发能够在替代在人体内引起如上所述的副作用和抗性的药物治疗剂而同时抑制幽门螺杆菌的适当的抗菌物质或食品。
另外,作为传统发酵食品的泡菜的发酵乳酸菌是人类长期与发酵食品一同摄入的GRAS(公认安全(Generally Recognized As Safe))级细菌,由于这种乳酸菌的功能性和安全性的优点,利用乳酸菌的功能性食品或药品的开发受到了关注。例如,本申请人已经报道过气体产生量低的肠膜明串珠菌CJLM119菌株和利用其制备泡菜的方法、植物乳杆菌CJLP133菌株和包含其的用于治疗肠道疾病和增强免疫力的组合物(专利文献1至专利文献3)。然而,对通过如泡菜的经常食用的发酵食品预防或治疗由幽门螺杆菌感染引起的疾病的方法的研究尚未完成,并且仍在进行中。
现有技术文献
(专利文献1)韩国注册专利公告第1,807,995号
(专利文献2)韩国注册专利公告第1,486,999号
(专利文献3)韩国注册专利公告第1,406,168号
发明内容
要解决的技术问题
本申请涉及包含对幽门螺杆菌具有抑制效果的乳酸菌作为有效成分的泡菜、所述泡菜或其提取物用于预防和治疗幽门螺杆菌相关疾病的用途。
技术解决方案
为了解决所述技术问题,本申请的一方面提供包含作为源自泡菜的乳酸菌的肠膜明串珠菌(Leuconostoc mesenteroides)CJLM119和植物乳杆菌(Lactobacillusplantarum)CJLP133的泡菜及泡菜制备方法。
本申请的另一方面提供包含所述泡菜或其提取物的用于改善幽门螺杆菌相关疾病的组合物。
本申请的另一方面提供利用所述泡菜或其提取物预防、治疗或改善幽门螺杆菌相关疾病的方法。
发明效果
根据本申请的包含作为源自泡菜的乳酸菌的肠膜明串珠菌CJLM119和植物乳杆菌CJLP133的泡菜或其提取物具有对幽门螺杆菌的抑制活性,并且在长时间施用时,可预防、治疗或延迟由幽门螺杆菌引起的萎缩性胃炎和胃溃疡的进展,并且也可有用地用于预防或治疗由幽门螺杆菌引起的胃癌。
然而,本申请的效果不限于上述效果,并且本发明所属技术领域的技术人员能从以下描述中明确地理解未提及的其它效果。
附图说明
图1是示出本申请的泡菜对幽门螺杆菌细菌的抑制效果的图表。
图2是示出本申请的泡菜对各种血管生长因子的表达产生的影响的电泳照片。
图3是示出本申请的泡菜对各种炎性因子的表达产生的影响的电泳照片。
图4是示出本申请的泡菜在幽门螺杆菌感染模型中对细胞的紧密连接产生的影响的图表。
图5是示出本申请的泡菜对胃癌细胞系的生长产生的影响的图表。
图6是示出本申请的泡菜对胃癌细胞系的细胞凋亡产生的影响的图表。
图7是示出本申请的泡菜对胃癌细胞系的各种细胞凋亡因子的表达产生的影响的电泳照片。
图8是示出本申请的泡菜对胃癌细胞系的细胞周期相关因子的表达产生的影响的电泳照片。
图9示出了用于确认本申请的泡菜对幽门螺杆菌相关疾病产生的影响的动物实验的设计。
图10是示出本申请的泡菜在感染幽门螺杆菌的动物模型中对总体病变总评分(Gross Lesion Index Score)产生的影响的图表。
图11是示出本申请的泡菜在感染幽门螺杆菌的动物模型中对胃癌的发生产生的影响的照片。
具体实施方式
以下,对本申请的内容进行具体说明。
1.包含肠膜明串珠菌CJLM119和植物乳杆菌CJLP133的泡菜及其制备方法
本申请提供包含肠膜明串珠菌CJLM119和植物乳杆菌CJLP133的泡菜及其制备方法。
肠膜明串珠菌CJLM119是以保藏号KCTC 13043BP保藏在韩国生物科学和生物技术研究所基因银行(Korea Research Institute of Bioscience and Biotechnology GeneBank)的微生物菌株,并且所述菌株的分离和鉴定方法具体地公开在韩国注册专利公告第1,807,995号中。此外,植物乳杆菌CJLP133是以保藏号KCTC 11403BP保藏在韩国生物科学和生物技术研究所基因银行的微生物菌株,并且所述菌株的分离和鉴定方法具体地公开在韩国注册专利公告第1,486,999号中。
在本申请中,“泡菜”包括但不限于用盐腌制蔬菜之后混入调味料并发酵而成的食品。在本申请的一个实施方案中,蔬菜可使用白菜、萝卜、葱、芥菜叶、黄瓜等,并且具体地,可使用白菜,但是除此以外的任意蔬菜均可用作成为本申请的主题的泡菜的原料。在本申请的一个实施方案中,虽然所述蔬菜可为番茄红素含量高的蔬菜,但并不局限于此。在本申请的其它实施方案中,每100g的蔬菜的番茄红素的含量可为0.05至5mg,并且例如可为0.1至3mg、0.2至2mg、0.3至1mg、0.5至0.8mg,但并不局限于此。
番茄红素含量高的蔬菜可为白菜,并且具体地可为利用韩1号(品种申请公开号:2015-688,2015年12月15日公开)的白菜品种的白菜。在利用肠膜明串珠菌CJLM119和植物乳杆菌CJLP133对番茄红素含量高的蔬菜进行发酵的情况下,可防止番茄红素含量的降低,从而可保持番茄红素的癌症预防功能。
在本申请的一个实施方案中,所述调味料是混合辣椒粉、蒜、姜、咸鱼等材料而制备的,并且除了这些调味料以外,构成调味料的材料可根据个人喜好、发酵方法、发酵周期等而在需要时适当地添加或排除任何其它的材料。
在本申请的一个实施方案中,本申请的泡菜可通过常规的泡菜的制备方法来制备,即,在用盐腌制的蔬菜(例如,用盐腌制的白菜)上抹上调味料之后进行发酵的方法。虽然作为蔬菜使用番茄红素含量高的蔬菜可为更加适当的,但并不局限于此,并且常规的蔬菜可不受限制地用于本申请的泡菜的制备。
在本申请的其它实施方案中,本申请的泡菜可另外地包含作为食品可接受的已知的添加剂,并且作为所述添加剂,本申请的泡菜中可不受限制地包含有梅子香、柠檬香、菠萝香、香草香等的各种天然香料,天然果汁、叶绿酸、类黄酮等的各种天然色素,诸如果糖、蜂蜜、糖醇、白糖的甜味成分,或者诸如柠檬酸、柠檬酸钠的酸化剂等。
在本申请的一个实施方案中,在本申请的泡菜中,基于泡菜的整体重量,所述肠膜明串珠菌CJLM119的含量可为0.01至5重量%,具体地可为0.02至3重量%,并且更具体地可为0.05至2.0重量%。
在本申请的其它实施方案中,在本申请的泡菜中,基于泡菜的整体重量,所述植物乳杆菌CJLP133的含量可为0.01至5重量%,具体地可为0.02至3重量%,并且更具体地可为0.05至2.0重量%。
在本申请的一个实施方案中,本申请的泡菜可通过在用盐腌制的蔬菜(例如,用盐腌制的番茄红素含量高的蔬菜)上添加0.01至5重量%(具体地为0.02至3重量%,并且更具体地为0.05至2.0重量%)的所述肠膜明串珠菌CJLM119,并且添加0.01至5重量%(具体地为0.02至3重量%,并且更具体地为0.05至2.0重量%)的所述植物乳杆菌CJLP133之后,在预定温度下在预定周期期间进行发酵来制备。
发酵时的温度不受特别限定,但是例如可在1℃至30℃,具体地在3℃至15℃,或者在5℃至10℃下实施发酵。
发酵周期不受特别限定,但是例如可在1日至365日期间,具体地在5日至180日、10日至90日、15日至60日或者20日至40日期间执行。
在本申请的一个实施方案中,可利用肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株自身,但是也可利用包括所述菌株的培养物。在本申请的一个实施方案中,肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株可用作活菌、死菌或其混合物。
在本申请中,术语“培养物”表示将肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株接种到培养基来培养的产物。例如,所述培养物可包括在培养基中培养肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株的培养物自身(即,可包括肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株、培养基或菌株的代谢物)、过滤或离心所述培养物而去除菌株的滤液(例如,离心的上清液)等。此外,本申请的培养物也可不受限制地包括对其进行干燥(例如,冷冻干燥)之后粉末化的形态。
在本申请的一个实施方案中,菌株的培养可在10℃至30℃下在6小时至48小时期间,具体地在20℃至30℃下在12小时至36小时或者在18小时至30小时期间实施,但并不局限于此。
只要是已知的用于乳酸菌的培养基,所述培养基可不受限制地使用。所述培养基可包括碳源和氮源,所述碳源可包括蔗糖、葡萄糖、果糖等中的一种或多种,并且所述氮源可包括酵母提取物、蛋白胨、牛肉提取物、麦芽提取物、玉米浆、柠檬酸铵、硫酸铵、氯化铵、磷酸铵、碳酸铵、硝酸铵等中的一种或多种,但并不局限于此。此外,所述培养基可另外地包含吐温80、柠檬酸钠、磷酸钾、乙酸钠、硫酸锰、硫酸镁、蒸馏水等中的一种或多种。
2.包含本申请的泡菜或其提取物的用于改善幽门螺杆菌相关疾病的组合物
本申请提供用于改善幽门螺杆菌相关疾病的组合物,所述组合物包括包含有肠膜明串珠菌CJLM119和植物乳杆菌CJLP133的泡菜或其提取物。
本申请的泡菜及其制备方法与所述“1.包含肠膜明串珠菌CJLM119和植物乳杆菌 CJLP133的泡菜及其制备方法”中提及的相同,因此省略其记载。
在本申请中,作为幽门螺杆菌相关疾病,可列举出胃炎、胃溃疡、胃癌等,但并不局限于此。
在本申请中,“泡菜提取物”表示通过任意方法从作为原料的泡菜提取的物质,并且不受限制地包括这样提取的提取物、可从提取物获得的浓缩物、所述浓缩物的干燥产物和粉末等的含义来使用。
当从作为原料的泡菜提取并获得所述提取物时,作为提取方法,可不受限制地使用诸如溶剂提取法、超声波提取法、过滤法和回流提取法等的已知的常规、常用提取方法,并且具体地,可通过利用溶剂提取法或回流提取法来制备,但并不局限于此。所述提取过程可反复几次,并且之后可另外地经历浓缩或冷冻干燥等步骤。具体地,可对获得的泡菜提取物进行减压浓缩以获得浓缩物,并且在对所述浓缩物进行冷冻干燥之后利用粉碎机来制备高浓度的泡菜提取物粉末。
在本申请的一个实施方案中,所述泡菜提取物以不受限制地包括利用水、有机溶剂或其混合溶剂提取泡菜而获得的提取物、所述提取物的浓缩物或粉末等的含义来使用。所述有机溶剂可选自醇(具体地为C1-C4低级醇,例如,甲醇、乙醇、丙醇或丁醇)、己烷(正己烷)、醚、甘油、丙二醇、丁二醇、乙酸乙酯、乙酸甲酯、二氯甲烷、氯仿、苯及它们的混合溶剂中的任一种。具体地,提取溶剂可选自水、C1-C4醇和C1-C4醇与水的混合溶剂中的任一种,例如,提取溶剂可为甲醇水溶液或乙醇水溶液。甲醇或乙醇水溶液可为10%(v/v)至99%(v/v)的水溶液,具体地,可为20%(v/v)至70%(v/v)的水溶液,并且更具体地,可为30%(v/v)至60%(v/v)的水溶液,但并不局限于此。
在本申请的一个实施方案中,所述提取可在5℃至100℃的温度下实施,具体地可在20℃至95℃下实施,并且更具体地可在50℃至90℃下实施,但并不局限于此。此外,所述提取可在1小时至20小时期间实施,具体地可在2小时至16小时期间实施,并且更具体地可在3小时至12小时期间实施,但并不局限于此。
在本申请的一个实施方案中,所述提取可实施1次至8次,具体地可实施1次至6次,并且更具体地可实施1次至5次,但并不局限于此。所述提取物可为在各次提取中获得的单独提取物或者在各次提取中获得的提取物的混合提取物,并且具体地可为在各次提取中获得的提取物的混合提取物。
在本申请中,术语“改善”以不受限制地表示因本申请的泡菜或其提取物的摄入而引起的至少降低与治疗相关的参数(例如,症状程度)的所有行为的含义来使用。本申请的幽门螺杆菌相关疾病的改善可为肠膜明串珠菌CJLM119和植物乳杆菌CJLP133自身的效果,并且可为所述菌株的因蔬菜发酵而引起的效果,但机制不限于此。
在本申请中,所述组合物可为健康功能食品组合物。
健康功能食品(Functional Food)作为与特定保健食品(Food for SpecialHealth Use,FoSHU)相同的术语,其表示被加工为除了营养供给以外还有效地发挥出机体调节功能的医学和医疗效果高的食品。此处,术语“功能”表示对人体的结构和功能进行营养素调节或者对诸如生理学作用等的保健用途时获得有益的效果。
健康食品组合物能够通过本技术领域中常用的方法制备,并且在制备时可添加本技术领域通常添加的原料和成分来制备。此外,健康食品组合物的制剂可不受限制地包括只要是认可为食品的制剂。健康食品组合物可制备成多种形态的制剂,并且与一般药品不同,因为将食品作为原料,因此不会发生可能因长期摄入药品而发生的副作用等的优点,并且因为携带方便,因此健康食品组合物能够作为用于促进幽门螺杆菌相关疾病症状的改善效果的辅助剂来摄取。
健康食品表示具有比一般食品积极的健康保持或促进效果的食品,并且健康辅助食品表示健康辅助目的的食品。根据情况,健康功能食品、健康食品、健康辅助食品的术语可互换使用。
在本申请中,本申请的组合物可另外地包括生理学上可接受的载体。载体的类型不受特别限制,并且只要是本技术领域中常用的载体,则任意载体可不受限制地使用。此外,本申请的组合物可包括能够通常用于食品组合物中以改善气味、味道、视觉外观等的额外成分。例如,本申请的组合物可包括维生素A、维生素C、维生素D、维生素E、维生素B1、维生素B2、维生素B6、维生素B12、烟酸、生物素、叶酸、泛酸等。此外,本申请的组合物可包括锌(Zn)、铁(Fe)、钙(Ca)、铬(Cr)、镁(Mg)、锰(Mn)、铜(Cu)、铬(Cr)等的矿物质、以及赖氨酸、色氨酸、半胱氨酸、缬氨酸等氨基酸。
此外,本申请的组合物可包括防腐剂、杀菌剂、抗氧化剂、着色剂、固色剂、漂白剂、调味料、甜味剂、香料、溶胀剂、增强剂、乳化剂、增稠剂、包衣、泡沫抑制剂、溶剂、改良剂等的食品添加剂。添加剂可根据食品的类型选择并以适当的量使用。添加剂的比例并不是关键的,但是在100重量份的本申请的组合物中通常会选择在0.01至5重量份的范围内。
包含在本申请的组合物中的泡菜或其提取物的含量可根据使用目的来适当地确定。在本申请的一个实施方案中,相对于组合物的总重量,包含在本申请的组合物中的泡菜或其提取物的含量可例如为0.0001至20.0重量%,并且具体地可为0.001至1.0重量%,但并不局限于此。在所述泡菜或其提取物的含量为上述含量的情况下,可以经济的量获得对幽门螺杆菌相关疾病症状的改善效果。然而,在为了健康和卫生的目的或者为了健康调节的目的而长期摄入的情况下,所述量可为上述范围以下,并且在安全性方面,由于没有任何问题,因此所述泡菜或其提取物也可以所述范围以上的量来使用。
除了本申请的泡菜或其提取物以外,本申请的组合物还可另外地包括在食品制备时通常添加的成分,例如,蛋白质、碳水化合物、脂肪、营养素、调味料、调味剂等。碳水化合物的实例可为例如葡萄糖、果糖等的单糖、例如麦芽糖、蔗糖、寡糖等的二糖、以及例如糊精、环糊精和糖醇等的多糖和例如木糖醇、山梨醇、赤藓糖醇等的糖醇。作为所述调味剂,可使用诸如索马甜、甜菊提取物的天然调味剂和合成调味剂。例如,在本申请的健康食品组合物制备为饮料时,除了本申请的泡菜提取物外,还可另外地包括柠檬酸、果糖液、白糖、葡萄糖、乙酸、苹果酸、果汁、天然提取液等。
组合物的类型不受特别限制。可添加本申请的泡菜或其提取物的食品的实例可为肉类、香肠、面包、巧克力、糖果、小吃类、糖果类、零食类、饼干类、披萨、拉面、其它面类、口香糖类、包括冰淇淋类的乳制品、各种汤、饮料、茶、饮料、酒精饮料和维生素复合剂等,并且可包括在通常的含义上的所有健康食品。
本申请的泡菜或其提取物中包括有肠膜明串珠菌CJLM119和植物乳杆菌CJLP133,这两种乳酸菌具有高度抑制幽门螺杆菌的活性的特征,因此包括本申请的泡菜或其提取物的组合物可有用地使用于由幽门螺杆菌引起的相关疾病的预防或治疗中。
3.利用本申请的泡菜或其提取物的幽门螺杆菌相关疾病的预防、治疗或改善方法
本申请提供了预防、治疗或改善幽门螺杆菌相关疾病的方法,包括向患有幽门螺杆菌相关疾病或者可能患上幽门螺杆菌相关疾病的个体施用所述泡菜或其提取物的步骤。
本申请提供了所述泡菜或其提取物用于预防、治疗或改善幽门螺杆菌相关疾病的用途。
本申请提供了所述泡菜或其提取物在用于制备预防、治疗或改善幽门螺杆菌相关疾病的组合物的用途。
本申请的泡菜或其提取物及其制备方法与在“1.包含肠膜明串珠菌CJLM119和植 物乳杆菌CJLP133的泡菜及其制备方法”和“2.包含本申请的泡菜或其提取物的用于改善幽 门螺杆菌相关疾病的组合物”中提及的相同,因此省略其记载。
在本申请中,术语“预防”以包括通过本申请的泡菜或其提取物的施用而抑制或延缓幽门螺杆菌相关疾病的症状的所有行为的含义来使用。
此外,在本申请中,术语“治疗”以不受限制地包括通过本申请的泡菜或其提取物的施用而使幽门螺杆菌相关疾病的症状好转或有利地变化的行为的含义来使用。
此外,在本申请中,术语“施用”以通过适当的方法将预定物质引入个体的含义来使用。
此外,在本申请中,术语“个体”以包括人类在内的大鼠、小鼠和家畜等的所有动物的含义来使用。作为具体的实例,所述个体可为包括人类在内的哺乳动物。作为另一实例,所述个体可为患有幽门螺杆菌相关疾病或可能患上幽门螺杆菌相关疾病的个体。
在本申请的方法中,本申请的泡菜或其提取物的施用途径和施用方式不受特别限制,并且只要能够使包括本申请的泡菜或其提取物的组合物到达期望的部位,则可根据任意的施用途径和施用方式。具体地,所述泡菜或其提取物可通过口服或非口服的多种途径施用,但通常为通过口服施用。
在本申请的一个实施方案中,作为用于对幽门螺杆菌的感染进行膳食调整的食品组合物,新研究出了预防癌症的泡菜的组分。在本申请的其它实施方案中,通过利用鼠模型的预防癌症的泡菜的膳食调整,代表性地假设了能够预防、改善或治疗诸如胃炎、胃溃疡、胃癌等的幽门螺杆菌相关疾病。在常规的泡菜的制备方法中,预防癌症的泡菜可通过添加番茄红素含量高的蔬菜、肠膜明串珠菌CJLM119菌株和植物乳杆菌CJLP133菌株来制备,但并不局限于此。在本申请的一个实施方案中,根据本申请的泡菜或其提取物对幽门螺杆菌相关疾病展现出了优秀的预防和治疗效果,尤其是在胃炎、胃溃疡和胃癌中展现出了优秀的效果。
根据本申请的一个实施方案,作为在本申请的泡菜的制备中使用的乳酸菌的肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株与属于相同物种的其它菌株相比对幽门螺杆菌展现出显著的抑制活性。
根据本申请的另一个实施方案,包括肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株的泡菜在预定浓度中展现出对于幽门螺杆菌的抑制效果(参见图1)。
根据本申请的另一个实施方案,本申请的泡菜可显著抑制正常胃细胞中血管生长因子和炎性因子的表达,并且可恢复细胞的紧密连接(参见图2至图4)。
根据本申请的另一个实施方案,本申请的泡菜可显著抑制胃癌细胞的生长,诱导胃癌细胞的细胞凋亡(参见图5和图6),并且可抑制幽门螺杆菌感染的动物模型中胃癌的形成(参见图10)。
如上所述,本申请的包括肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株的泡菜展现出对幽门螺杆菌的显著抑制活性,并且由此可有用地使用于可从幽门螺杆菌引发的多种疾病(例如,胃炎、胃溃疡和胃癌)的预防、治疗或改善中。
在下文中,将通过实施例对本申请进行详细说明。
然而,以下实施例仅仅是具体地示例出了本申请,本申请的内容并不受以下实施例限定。
首先,对本申请的泡菜的制备过程、实验方法和统计等进行说明。统计分析利用GraphPad Prism(GraphPad软件,美国加利福尼亚州拉霍亚)和SPSS软件(12.0版本,SPSS公司,美国伊利诺伊州芝加哥)来执行,各组之间的统计学显著性通过曼-惠特尼U检验(Mann-Whitney U test)来确定,并且统计学显著性在p<0.05中得到认可。
实施例1:幽门螺杆菌细菌生长抑制能力高的菌株分离
<1-1>菌株分离和鉴定
在本申请的泡菜的制备中使用的作为源自泡菜的乳酸菌的肠膜明串珠菌CJLM119是以保藏号KCTC 13043BP保藏在韩国生物科学和生物技术研究所基因银行中的微生物菌株,并且所述菌株的分离和鉴定方法具体地公开在韩国注册专利公告第10-1807995号中。此外,在本申请的泡菜的制备中使用的作为源自泡菜的乳酸菌的植物乳杆菌CJLP133是以保藏号KCTC 11403BP保藏在韩国生物科学和生物技术研究所基因银行中的微生物菌株,并且所述菌株的分离和鉴定方法具体地公开在韩国注册专利公告第10-1486999号中。因此,省略其具体记载。
在本申请中,利用肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株通过常规制备方法制备了泡菜。
<1-2>对幽门螺杆菌细菌的抑制能力高的菌株筛选
利用在实施例<1-1>中分离并鉴定的肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株、以及用作对照组的肠膜明串珠菌KCTC 3100/KCTC 3722、植物乳杆菌KCTC 3108/DSM17853对幽门螺杆菌抑制活性进行测量。
结果显示,与用作对照组的相同物种的其它菌株相比,肠膜明串珠菌CJLM119和植物乳杆菌CJLP133菌株展现出了对幽门螺杆菌细菌的显著的抑制活性。
实施例2:泡菜的制备
通过以下方式制备了作为对照组的标准泡菜(在下文中存在着表示为“sKimchi”或“SK”的情况)和根据本申请的预防癌症的泡菜(在下文中存在着表示为“CpKimchi”或“CPK”的情况)。
<2-1>标准泡菜(sKimchi)的制备
标准泡菜是基于世界泡菜研究所的标准化泡菜配方在用盐腌制的白菜上抹上包括辣椒粉、蒜、姜、凤尾鱼酱、萝卜汁、葱、糯米糊、白糖的调味料之后在低温中发酵而制备的。
<2-2>预防癌症的泡菜(CpKimchi)的制备
预防癌症的泡菜将番茄红素含量高的白菜作为主原料,购买在一般白菜中没有的作为番茄红素含量高的白菜品种的“韩1号”(品种申请公开号:2015-688,2015年12月15日公开)种子,并通过与白菜种植户签订种植合同来确保了白菜,并且收获的白菜是腌制得符合泡菜制备工序后被使用。除了作为白菜使用所述番茄红素含量高的白菜并且添加在实施例<1-1>中分离和鉴定的源自泡菜的肠膜明串珠菌CJLM119、植物乳杆菌CJLP133的2种乳酸菌以外,通过与所述标准泡菜的制备相同的方式制备了预防癌症的泡菜。
<2-3>泡菜提取物的浓缩物的制备
在对所述实施例<2-1>和<2-2>中制备的泡菜进行冷冻干燥之后进行粉碎并制备成粉末。在向粉末加入20倍重量的甲醇之后,利用搅拌机提取12小时。提取的上清液通过滤纸过滤,并且剩余的沉淀物再次通过添加甲醇来提取12小时。用浓缩机在约40℃的温度中对所述提取物进行加热浓缩而制备浓缩物之后,在4℃下将其储存并使用在进一步实验中。
实施例3:测定幽门螺杆菌的生长抑制效果
<3-1>幽门螺杆菌菌株的培养
幽门螺杆菌菌株ATCC 43504(美国典型培养物保藏中心,cagA+和vacA s1-m1型菌株)与5%的羊血一同在BBL的胰蛋白酶大豆(TS)琼脂培养基(TSAII;BD生物科学,新泽西州富兰克林湖)中在微需氧条件(BD GasPaK EZ气体发生***,BD生物科学)下在37℃下培养3天。在TS培养基中收集幽门螺杆菌菌株,并且以3000×g离心5分钟,并且最终以浓度为109菌落形成单位(CFU)/ml再悬浮在培养基中。所有实验均使用了在TS琼脂培养基中生长72小时的培养菌。
<3-2>确认幽门螺杆菌的抑制能力
为了确认本申请的泡菜的幽门螺杆菌的抑制效果,在接种有实施例<3-1>的幽门螺杆菌菌株的培养基中按照浓度用实施例<2-3>中制备的泡菜提取物的浓缩物进行处理并培养之后,对抑制环的大小进行测量。
结果表明,确认本申请的泡菜提取物的浓缩物在500mg/ml以上的浓度中具有对幽门螺杆菌细菌的抑制效果(参见图1)。
实施例4:测定对于正常胃细胞系(RGM1;正常胃粘膜细胞)中的幽门螺杆菌相关疾病的效果
<4-1>测定血管生成因子的抑制能力
为了确认本申请的泡菜对血管生长因子产生的影响,在对正常胃细胞系的RGM1细胞以50MOI进行幽门螺杆菌处理之后,以5mg/ml的浓度用实施例<2-3>中制备的泡菜提取物的浓缩物处理6小时。在处理后,去除培养基成分,并且用PBS(Dulbecco)对细胞洗涤2次。随后为了执行RT-PCR,将RiboEX(500μl,GeneAll,首尔,韩国)在4℃下添加到平板中,培养10分钟。回收RiboEX并置于1.5ml的管中之后,加入100μl的氯仿并缓慢地进行混合。在冰中孵育10分钟之后,将样品以10,000g离心30分钟。提取上清液并与200μl的异丙醇混合,并且将混合物在4℃下孵育1小时。在以13,000g离心30分钟之后,用70%(vol/vol)乙醇对沉淀物进行洗涤。在使乙醇完全蒸发之后,将颗粒溶解在100μl DEPC处理的水(英杰生命技术,加利福尼亚州卡尔斯巴德)中,且随后执行PCR并确认相关基因的变化。
结果表明,确认了作为因幽门螺杆菌的处理而增加的血管生成因子的指标的VEGF和bFGF的表达在预防癌症的泡菜处理组(CPK)中降低(图2)。
<4-2>抗炎效果的比较
为了确认本申请的泡菜的抗炎效果,在RGM1细胞中以50MOI进行幽幽门螺杆菌处理之后,以5mg/ml的浓度用实施例<2-3>中制备的泡菜提取物的浓缩物进行处理,根据实施例<4-1>的RT-PCR方法测定相关基因的变化。
结果表明,确认了作为通过幽门螺杆菌处理而增加的炎症的指标的COX-2和iNOS的表达在预防癌症的泡菜处理组(CPK)中降低(图3)。
<4-3>细胞连接效果的比较
为了确认本申请的泡菜对细胞的紧密连接指示物产生的影响,在RGM1细胞中以100MOI进行幽门螺杆菌处理之后,以5g/ml和10g/ml的浓度用实施例<2-3>中制备的泡菜提取物的浓缩物处理6小时。此时,在用0.5ml的冷D-PBS(-)对因细胞的增殖而成为融合状态的单细胞层的上层部洗涤3次之后,填充0.5ml的冷HBSS并转移到装有1.5ml的冷HBSS的新孔,且然后放置30分钟之后测量上层部与下层部之间的跨上皮电阻(TERR%)。
结果表明,确认了在幽门螺杆菌感染模型中以高浓度的本申请的预防癌症的泡菜处理时细胞的紧密连接得到恢复(图4)。
实施例5:测定对于胃癌细胞系(MKN28;胃癌细胞)中的幽门螺杆菌相关疾病的效果
<5-1>测定胃癌细胞系的生长抑制效果
为了确认本申请的泡菜对胃癌细胞系的生长抑制效果,在MKN28细胞中用通过实施例<2-3>中的方法制备标准泡菜和预防癌症的泡菜而获得的泡菜提取物的浓缩物分别以10、20、50mg/ml的浓度处理6小时后,对各个处理组的细胞活力进行比较分析。
结果表明,确认了本申请的预防癌症的泡菜能够以浓度依赖方式比标准泡菜上更加显著地抑制胃癌细胞系的生长(图5)。
<5-2>测定胃癌细胞系的细胞凋亡诱导效果
为了针对具有没有细胞凋亡特性的癌细胞确认在预防癌症的泡菜处理的细胞凋亡诱导效果,通过实施例<2-3>的方法制备标准泡菜和预防癌症的泡菜而获得的泡菜提取物的浓缩物以对各个细胞以适当的浓度处理48小时之后,用添加1%的FBS的1X PBS回收。按照浓度来回收的各个细胞与膜联蛋白V和死细胞试剂(AnnexinV&Dead Cell Reagent)以1:1混合,在常温避光条件下进行反应20分钟,并且对诱导细胞凋亡的细胞比例进行测量和分析。
结果表明,确认了在对MKN28胃癌细胞系进行本申请的预防癌症的泡菜处理时,在细胞的70%以上中诱导了细胞的凋亡(图6)。
<5-3>测定胃癌细胞系细胞凋亡调节因子
为了确认本申请的泡菜对胃癌细胞系的凋亡调节因子产生的影响,在MKN28胃癌细胞系中以20mg/ml的浓度用实施例<2-3>中制备的泡菜提取物的浓缩物实施处理,然后去除培养基成分,并且用PBS(Dulbecco)溶液洗涤细胞2次。用含有1mM PMSF(苯甲基磺酰氟,西格玛奥德里奇,密苏里州圣路易斯)的冰冷细胞裂解缓冲液(细胞信号技术公司,马萨诸塞州丹佛)裂解细胞。在反应20分钟后,将各个样品以10,000×g离心10分钟。接着,获得上清液。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离裂解物的蛋白质,并转移到聚偏二氟乙烯(PVDF)膜上,与一抗进行反应之后进行洗涤,并且与过氧化物酶缀合的二抗进行反应后进行再次洗涤,然后通过ECL***(通用电气医疗,英国白金汉郡)进行确认。
结果表明,确认了与标准泡菜处理组(SK)相比,参与细胞的凋亡的切割的半胱天冬酶3、切割的半胱天冬酶8和切割的PARP的表达在本申请的预防癌症的泡菜(CPK)处理组中显著地增加(图7)。
<5-4>测定胃癌细胞系的细胞周期相关因子
为了确认本申请的泡菜对胃癌细胞系的细胞周期相关因子产生的影响,在MKN28细胞系中以20mg/ml的浓度用实施例<2-3>中制备的泡菜提取物的浓缩物进行处理,然后去除培养基成分,并且用PBS(Dulbecco)溶液洗涤细胞2次。用含有1mM PMSF(西格玛奥德里奇,密苏里州圣路易斯)的冰冷细胞裂解缓冲液(细胞信号技术公司,马萨诸塞州丹佛)裂解细胞。在反应20分钟后,将各个样品以10,000×g离心10分钟。接着,获得上清液。用SDS-PAGE分离裂解物的蛋白质,并转移到PVDF膜上并与一抗进行反应之后进行洗涤,并且与过氧化物酶缀合的二抗进行反应后进行再次洗涤,然后通过ECL***(通用电气医疗,英国白金汉郡)确认细胞周期相关蛋白质的表达变化。
结果表明,确认了与标准泡菜(SK)处理组相比,参与细胞周期的细胞周期蛋白D1、CDK4、CDK2、细胞周期蛋白E和细胞周期蛋白A的表达在本申请的预防癌症的泡菜(CPK)处理组中显著地减少,并由此发现,胃癌细胞系的凋亡机制依赖于细胞周期调节(图8)。
实施例6:测定对于感染幽门螺杆菌的长期动物模型中的幽门螺杆菌相关疾病的效果
为了确认本申请的泡菜在感染幽门螺杆菌的长期动物模型中抑制病理学指标的效果,向5周龄的雌性C57BL/6鼠(查尔斯河公司,日本)随意喂食无菌商业颗粒饮食(Biogenomics,韩国)和无菌水,并饲养在调节为24℃的温度的室中。在适应1周之后,注射3次20mg/kg的泮托拉唑(pantoprazole)来降低胃的酸度以促进幽门螺杆菌定殖。然后,使用胃肠插管针向各个动物的胃内分别注射0.3ml的干净的TS培养基和幽门螺杆菌悬浮液(含有109CFU/ml)。对照组注射TS培养基,并且所有实验组在一周内总共注射4次幽门螺杆菌。给鼠喂食基于含有7.5%NaCl的AIN76的特殊颗粒饲料以引发胃炎长达4周。
然后,将幽门螺杆菌阳性鼠随机分为4组(n=10)。施用含有7.5%NaCl的颗粒饲料AIN76长达24周,以在所有动物中促进由幽门螺杆菌引起的致癌过程。对照组和实验组分别在图9中进行说明。分别以组1:对照组、组2:幽门螺杆菌感染组、组3:幽门螺杆菌感染以及预防癌症的泡菜低浓度饮食组、组4:幽门螺杆菌感染以及预防癌症的泡菜高浓度饮食组、组5:幽门螺杆菌感染以及标准泡菜饮食组进行实验。
结果表明,在作为对照组的G2中总体病变总评分显著增加,然而在作为预防癌症的泡菜处理组的G3和G4中表现出有意义的降低(p<0.01)。此外,在作为幽门螺杆菌感染对照组的G2中形成了明显大小的胃癌病灶,然而在作为预防癌症的泡菜处理组的G3和G4中确认了胃癌的形成被减少(图10和图11)。
上文中虽然示例性地对本申请的优选实施方案进行了说明,但是本申请的范围并不仅限定于如上所述的特定实施方案,并且所属技术领域的普通技术人员能够在本申请的权利要求书中记载的范畴内进行适当的改变。
Claims (12)
1.一种泡菜,所述泡菜用盐腌制蔬菜之后混入调味料并用乳酸菌发酵而成,
所述乳酸菌包括肠膜明串珠菌(Leuconostoc mesenteroides)CJLM119(登记号:KCTC11403BP)和植物乳杆菌(Lactobacillus plantarum)CJLP133(登记号:KCTC 13043BP)。
2.如权利要求1所述的泡菜,其中所述蔬菜选自白菜、萝卜、葱、芥菜叶、黄瓜和它们的组合。
3.如权利要求2所述的泡菜,其中所述蔬菜为白菜。
4.如权利要求3所述的泡菜,其中所述白菜为番茄红素含量高的白菜。
5.如权利要求4所述的泡菜,其中每100g的白菜的番茄红素的含量为0.05至5mg。
6.如权利要求1所述的泡菜,其中基于所述泡菜的整体重量,所述肠膜明串珠菌CJLM119的含量为0.01至5重量%,并且基于所述泡菜的整体重量,所述植物乳杆菌CJLP133的含量为0.01至5重量%。
7.一种用于改善幽门螺杆菌(Helicobacter pylori)相关疾病的组合物,所述组合物包含权利要求1至6中的任一项所述的泡菜或其提取物。
8.如权利要求7所述的组合物,其中所述泡菜提取物为利用水、有机溶剂或其混合溶剂提取所述泡菜而获得的提取物。
9.如权利要求8所述的组合物,其中所述有机溶剂选自醇、己烷、醚、甘油、丙二醇、丁二醇、乙酸乙酯、乙酸甲酯、二氯甲烷、氯仿、苯及它们的混合溶剂。
10.如权利要求8所述的组合物,其中所述混合溶剂选自水、C1-C4醇和C1-C4醇与水的混合溶剂。
11.如权利要求10所述的组合物,其中所述混合溶剂为甲醇水溶液或乙醇水溶液。
12.如权利要求7所述的组合物,其中所述泡菜提取物选自提取物、提取物的浓缩物、浓缩物的干燥产物和粉末。
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US20220095636A1 (en) | 2022-03-31 |
EP3903589A2 (en) | 2021-11-03 |
TW202038747A (zh) | 2020-11-01 |
TWI734322B (zh) | 2021-07-21 |
SG11202107002QA (en) | 2021-07-29 |
JP7196314B2 (ja) | 2022-12-26 |
MY197563A (en) | 2023-06-23 |
CA3125104A1 (en) | 2020-07-02 |
AU2019416609A1 (en) | 2021-07-22 |
JP2022516116A (ja) | 2022-02-24 |
KR102651296B1 (ko) | 2024-03-26 |
AU2019416609B2 (en) | 2022-12-08 |
WO2020139020A3 (ko) | 2021-04-22 |
EP3903589A4 (en) | 2022-08-31 |
WO2020139020A2 (ko) | 2020-07-02 |
KR20200081878A (ko) | 2020-07-08 |
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