CN114921389A - 一种具有女性肠私处护理和乳腺抗炎功效的益生菌组合物及其应用 - Google Patents
一种具有女性肠私处护理和乳腺抗炎功效的益生菌组合物及其应用 Download PDFInfo
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Abstract
本发明涉及益生菌发酵物技术领域,具体公开了一种具有女性肠私处护理和乳腺抗炎功效的益生菌组合物及其应用。益生菌组合物包括长双歧杆菌W68、两歧双歧杆菌Y22、嗜酸乳杆菌K43、干酪乳杆菌K35和罗伊氏乳杆菌K07。本发明提供的益生菌发酵制品引用清法和补法两种治疗原则,通过清法从根本上祛除女性肠道、私处以及其他五脏六腑的热气和毒素,散结解毒,有效减轻体内炎症,保持乳腺健康,补法可配合清法起到稳固体内正气的效果。两法搭配可协同益生菌抑制有害菌的生长繁殖,促进益生菌的定殖和增殖,调节肠道和***菌群正向平衡。本发明还结合了“君、臣、佐、使”的用药原则,从根源处解决女性私处微环境失衡,缓解女性私处不适,减轻女性乳腺炎症。
Description
技术领域
本发明涉及益生菌发酵物技术领域,尤其是一种具有女性肠私处护理和乳腺抗炎功效的益生菌组合物及其应用。
背景技术
当代社会生活压力大,与之带给女性的疾病问题接踵而至,如:肠道不适、私处不适、乳腺炎症等。女性私处有***微生态***,***微生态失衡即是***菌群失调,可以引发需氧菌性***炎(AV)、细菌性***病(BV)、细胞溶解性***病(CV)、外阴***假丝酵母菌病(VVC)、滴虫***炎(TV)、性传播感染、***等女性生殖泌尿道感染性疾病,女性***内乳酸菌数量的减少或缺失,与子***和***性疾病的高风险相关。当女性***的pH异常时,容易滋生大量的有害菌,导致益生菌群比重降低,进而产生瘙痒、白带异常、异味以及各种炎症等。此外,由于压力太大导致的内分泌失调、卵巢功能紊乱等,进而引发非哺乳期乳腺炎症,非哺乳期乳腺炎以病理组织学分类,常见类型主要包括浆细胞性乳腺炎(PCM)和肉芽肿性乳腺炎(GM)。
妇科炎症不仅会影响日常生活和工作,也会对身心造成负面影响,严重者还可能导致***。目前对于妇科炎症主要通过抗生素(如:克霉唑栓、硝酸咪康唑栓、伊曲康唑、甲硝唑等)治疗,而抗生素的副作用非常大,且随着抗生素的广泛和不规范应用,临床上常出现抗生素耐药问题。非哺乳期乳腺炎症虽为乳腺良性炎症性疾病,但缺乏规范的药物治疗方法,外科手术切除复发率高,部分人经多种药物和手术治疗反复复发而迁延不愈,严重影响女性的正常生活和工作。非哺乳期乳腺炎症是临床上治疗方法均未解决的难治乳腺良性疾病之一,且其发病率呈增高趋势,已引起临床医生的重视。针对妇科炎症和非哺乳期乳腺炎症,积极寻找副作用较少、疗效较好的产品至关重要。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种具有女性肠私处护理和乳腺抗炎功效的益生菌组合物及其应用。本发明采用益生菌组合物发酵中药组合物获得的益生菌发酵制品具有女性肠私处护理、乳腺抗炎的功效,同时所得的益生菌制品在活菌型或无菌型时均有显著效果。
为实现上述目的,本发明采取的技术方案为:
第一目的,本发明提供了一种益生菌组合物,所述益生菌组合物包括长双歧杆菌W68、两歧双歧杆菌Y22、嗜酸乳杆菌K43、干酪乳杆菌K35和罗伊氏乳杆菌K07。
本申请的发明人经过大量创造性劳动,筛选出一组益生菌组合物,上述益生菌对常见肠道、***病原菌均有抑制生长的作用;并且上述筛选的益生菌产酸能力较佳,pH值均在5以上。经过β-谷甾醇含量测定,本发明的益生菌组合物可以提高发酵底物中的β-谷甾醇含量,进而增加抗炎、抗氧化、免疫调节等功效,有利于肠私处护理和乳腺抗炎。
并且经过动物实验知,益生菌组合物可以降低TNF-α、IL-6的含量,减轻乳腺炎的炎症效果较佳。
作为本发明所述益生菌组合物的优选实施方式,以重量份数计,所述益生菌组合物包括长双歧杆菌W68 10-40份、两歧双歧杆菌Y22 10-40份、嗜酸乳杆菌K43 15-40份、干酪乳杆菌K35 15-40份和罗伊氏乳杆菌K07 15-40份。
当上述益生菌采用上述重量份数时,对常见肠道、***病原菌抑制生长的作用更佳,有利于提高肠私处护理和乳腺抗炎的效果。
第二目的,本发明提供了一种益生菌发酵制品--益生菌发酵膏方,所述益生菌制品包括上述益生菌组合物和中药组合物;
所述中药组合物包括低聚果糖、水苏糖、抗性糊精、阿胶、鳖甲胶、姜黄、蒲公英、香附、土茯苓、白花蛇舌草、漏芦、牛膝、枸杞子、黄连、芍药、金银花、车前草、蜂蜜、冬瓜皮、玉米须、金钱草、猪苓、木通、桑根白皮、关木通、小通草、当归和山楂;使用上述益生菌组合物发酵中药组合物制得。
本发明制备的益生菌发酵制品可以提高β-谷甾醇含量,通过嗜酸性粒细胞和分泌黏性因子更好地减少氧自由基和炎症,降低TNF-α、IL-6含量,有利于肠私处护理和乳腺抗炎。
作为本发明所述益生菌发酵制品的优选实施方式,以重量份数计,所述中药组合物包括以下组分:
低聚果糖3.4-30份、水苏糖2-20.6份、抗性糊精1-8.2份、阿胶3-6份、鳖甲胶1.8-3.6份、姜黄3.6-7.2份、蒲公英4.8-9.6份、香附1.2-2.4份、土茯苓2.4-4.8份、白花蛇舌草2.4-4.8份、漏芦1.2-2.4份、牛膝1.2-2.4份、枸杞子1.8-3.6份、黄连1.2-2.4份、芍药1.2-2.4份、金银花1.2-2.4份、车前草1.2-2.4份、蜂蜜1.2-2.4份、冬瓜皮3-6份、玉米须4.8-9.6份、金钱草1.2-2.4份、猪苓0.6-1.2份、木通1.2-2.4份、桑根白皮1.2-2.4份、关木通0.6-1.2份、小通草2.4-4.8份、当归1.2-2.4份和山楂1.2-2.4份。
其中,低聚果糖能快速增殖肠道乳杆菌属和双歧杆菌属,正向调节肠道微生态,抑制有害菌的生长繁殖;水苏糖是“天然超强双歧因子”,无法被胃肠消化液分解,可直接被肠道微生物利用,双歧杆菌在肠道中会优先分解利用非消化性的低聚糖,还可以促进肠道蠕动、减少毒素在肠道的停滞时间,加速有毒有害物质排出。
抗性糊精是水溶性膳食纤维,可通过选择性刺激大肠菌群中双歧杆菌和其他短链脂肪酸产生菌的生长来降低 pH 值,增加有益微生物、降低有害微生物的产生,以改善有益微生物群的繁殖环境;
阿胶具有滋阴养血的功效,主治血虚、虚劳等病症;鳖甲胶具有养阴清热、平肝息风、软坚散结的功效,主治阴虚风动、劳疟、疟母等病症;姜黄具有破血行气、通经止痛的功效,主治胸肋刺痛、闭经、症瘕、跌扑肿痛等病症;蒲公英具有清热解毒、利尿散结的功效,主治急性乳腺炎、***等病症;香附具有理气、调经的功效,主治胁肋胀痛、***胀痛、疝气疼痛,***等病症;土茯苓具有清热除湿、泄浊解毒的功效,主治梅毒、淋浊、泄泻、瘿瘤等病症;白花蛇舌草具有清热利湿、解毒消痈的功效,主治痢疾、尿道炎等病症;漏芦性寒,味苦、咸,归胃、大肠经,具有清热解毒、消肿排脓、通筋脉等功效,主治***胀痛、皮肤瘙痒、阴疹等病症;牛膝具有散瘀消肿的功效,主治淋病、尿血、腰膝酸痛等病症;枸杞子具有养肝滋肾、益精明目、抗肿瘤、抗突变、提高免疫力的功效,主治肝肾阴亏、虚劳精亏等病症;黄连性寒,味苦,归心、脾、胃、肝、胆、大肠经,具有清热泻火、燥湿、解毒的功效,主治热病邪入心经之高热、烦躁、湿热胸痞等病症;芍药具有散淤、活血、止痛、泻肝火的功效,主治***、痰滞腹痛、关节肿痛、胸痛、肋痛等病症;金银花具有清热解毒的功效,主治热毒血痢、痈疡、肿毒、瘰疬、痔瘘等病症;车前草具有清热利尿,解毒的功效,主治热淋、血淋、尿血、白浊、带下等病症;蜂蜜具有润燥、止痛、解毒的功效,主治肠燥便秘、胃脘疼痛、发炎等病症;冬瓜皮性微寒,味甘,归肺、大肠经,具有清热利水、消肿利尿的功效,主治周身水肿、小便不利、泄泻、尿赤热痛等病症;玉米须性平,味甘,归膀胱、肝、胆经,主治***、乳汁不通、白浊、消渴等病症;金钱草具有清热利湿、通淋排石、解毒的功效,主治热淋、肾炎水肿,肝、胆及泌尿系结石等;猪苓性平,味甘、淡,归肾、膀胱经,具有利水渗湿的效果,主治小便不利、泄泻、淋浊等病症;木通具有泻火行水、通利血脉的功效,主治小便赤涩、胸中烦热等病症;桑根白皮具有行气消肿的功效,主治水肿、吐血等病症;小通草性平,味淡,无毒,归肺、胃经,具有利尿肾湿的功效,主治热病小便赤黄或尿闭,湿热瘙、淋等病症;当归性温,味甘、苦、辛,归心、肝、脾经,具有补血和血、润燥滑肠的功效,主治***、经闭腹痛、痛经、***、血瘀腹痛、便秘等病症;山楂性温,味酸、甘,归脾、胃、肝经,具有消食健胃、行气散淤的功效,主治饮食积滞、脘腹胀痛、泄泻痢疾、便秘、食欲不佳、淤血经闭等病症。
中药组合物引用清法和补法两种治疗原则,结合“君、臣、佐、使”的用药原则,从根源处解决女性私处微环境失衡,缓解女性私处不适,减轻女性乳腺炎症,全方位守护女性健康。清法是通过清热、泻火、解毒、凉血等作用,以清楚里热之邪的一类治法。该法适用于里热证、火证、热毒证以及虚热证等里热病症。由于里热证有热在气分、营分、血分、热壅成毒以及热在某一脏腑之分,因而在清法之中,又有清气分热、清营凉血、清热解毒、清脏腑热等不同。热证最易伤阴,大热又易耗气,所以清热剂中常配伍生津、益气之品。若温病后期,热灼阴伤,或久病阴虚而热伏于里的,又当清法与滋阴并用,更不可纯用苦寒直折之法,热必不除。补法是通过补益人体气血阴阳,以主治各种虚弱症候的一类治法。补法的目的,在于通过药物的补益,使人体气血阴阳虚弱或脏腑之间的失调状态得以纠正,复归于平衡。此外,在正虚不能祛邪外出时,也可以补法扶助正气,并配合其他治法,达到助正祛邪的目的。补法的具体内容甚多,既有补益气、血、阴、阳的不同,又有分补五脏之侧重,但较常用的治法分类仍以补气、补血、补阴、补阳为主。
本发明的益生菌发酵膏方通过清法从根本上祛除女性肠道、私处以及其他五脏六腑的热气和毒素,散结解毒,有效减轻体内炎症,保持乳腺健康,补法可配合清法起到稳固体内正气的效果,此外,两法搭配可协同益生菌抑制有害菌的生长繁殖,促进益生菌的定殖和增殖,调节肠道和***菌群正向平衡。在用药原则中,君药组合为“玉米须,姜黄,蜂蜜,芍药,山楂,香附,当归,木通,桑根白皮”,能强健脾胃、破血行气、利尿消炎、通淋镇痛。臣药组合为“蒲公英,土茯苓,白花蛇舌草,漏芦,黄连,金银花,车前草,金钱草”,可协助君药进行清热解毒,如去心火、解梅毒、缓解尿道炎等,强化君药调节女性肠私处健康、乳腺抗炎的功效。佐药组合为“冬瓜皮,牛膝,猪苓,关木通,小通草”,可作为正佐药从利水方向协助君药排出体内毒素,辅助治疗热毒、疮毒、丹毒、肿毒等。使药组合为“阿胶,鳖甲胶,枸杞子”,通气血,滋补肝肾,调经暖宫,作为调和药调和方中诸药,强化组方保持女性肠私处健康、乳腺抗炎的功效。本发明所提供的益生菌发酵膏方仅从中药组合物配方(不包括益生菌)上来说,已经非常具有创新性,其虽作为中药配方,但药效温和,可以达到肠私处护理和乳腺抗炎的效果。
第三目的,本发明提供了一种上述益生菌发酵制品的制备方法,包括以下步骤:
S1、熬胶:将阿胶、鳖甲胶粉碎,加入黄酒浸泡,炖煮溶解,得基料A;
S2、浸泡:将玉米须、姜黄、香附、当归、木通、桑根白皮、蒲公英、土茯苓、白花蛇舌草、漏芦、黄连、牛膝、猪苓、关木通、金银花、车前草、金钱草、冬瓜皮粉碎,加8-12倍量水超声,浸泡,得基料B;
S3、煎煮:将基料B煎煮,第一次过滤得基料C;取第一次过滤的滤渣加入6-10倍量水煎煮,第二次过滤得基料D;取第二次过滤的滤渣加入4-6倍量水煎煮,过滤得基料E;将芍药、山楂、小通草、枸杞子加入4-6倍量的水,炖煮,过滤得基料F;
S4、混合:将基料C、基料D、基料E、基料F倒入锅中搅拌混匀,再加入1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精、蜂蜜混合搅拌溶解,得基料G;
S5、灭菌:将基料G以70-75℃、1h灭菌,冷却至30-32℃,得基料H;
S6、菌种复活:在无菌操作下,将上述益生菌组合物的混合菌粉接种到灭菌的10%葡萄糖水溶液中,于21-26℃下培养16-19h,得基料I;
S7、发酵:将1/4重量份的基料I和基料H混合,转入发酵罐中发酵,将品温控制在29-35℃,进行密闭发酵10h,得一级发酵物;一级发酵物按照步骤S5灭菌后,再补充1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精,再重复上述发酵操作即得二级发酵物;二级发酵物按照步骤S5灭菌后,再补充1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精,再重复上述发酵操作即得三级发酵物;三级发酵物按照步骤S5灭菌后,再补充1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精,再重复上述发酵操作即得四级发酵物;将四级发酵物浓缩,收膏,得液体膏方,然后预冻,冻干,得益生菌发酵制品。
作为本发明所述益生菌发酵制品的制备方法的优选实施方式,所述液体膏方的相对密度为1.12-1.20。
作为本发明所述益生菌发酵制品的制备方法的优选实施方式,将四级发酵物置于减压0.08Mpa,72℃的条件下运行60分钟,得清膏。
作为本发明所述益生菌发酵制品的制备方法的优选实施方式,所述步骤S3中,所述过滤的目数为100-150目。
作为本发明所述益生菌发酵制品的制备方法的优选实施方式,所述冻干步骤包括:
预冻步骤:将上述液体膏方倒入方格模具(3.0cm*3.0cm),在-40℃超低温冰箱里预冻处理,冻藏时间为12-24h,得预冻后的膏方;
冻干步骤:将预冻后的膏方置于干燥温度为-40℃,真空度为10 pa -40pa,干燥时间为10-12h的条件下真空冷冻干燥,然后置于功率160-220W,作用时间10-15s条件下震动;再将膏方置于干燥温度为- 20℃~- 40℃,真空度为10-40pa,干燥时间为10-12h的条件下真空冷冻干燥,制得益生菌发酵制品。
第四目的,本发明提供了上述益生菌组合物或益生菌发酵制品在制备女性肠道、私处护理或乳腺抗炎药物中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明提供的益生菌发酵制品引用清法和补法两种治疗原则,通过清法从根本上祛除女性肠道、私处以及其他五脏六腑的热气和毒素,散结解毒,有效减轻体内炎症,保持乳腺健康,补法可配合清法起到稳固体内正气的效果,此外,两法搭配可协同益生菌抑制有害菌的生长繁殖,促进益生菌的定殖和增殖,调节肠道和***菌群正向平衡。本发明还结合了“君、臣、佐、使”的用药原则,从根源处解决女性肠私处微环境失衡,缓解女性肠道及私处不适,减轻女性乳腺炎症,全方位守护女性健康。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。
在以下实施例中,所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、筛选益生菌
实验益生菌菌株:动物双歧杆菌乳亚种Y6、鼠李糖乳杆菌YGG、长双歧杆菌W68、副干酪乳杆菌K9、两歧双歧杆菌Y22、植物乳杆菌FEED8、嗜酸乳杆菌K43、干酪乳杆菌K35、发酵乳杆菌FM-12、罗伊氏乳杆菌K07,分别编号A-J。所用菌株均来源于“广东益可维生物技术有限公司”。
培养基:
LB 培养基 (g/L):胰蛋白胨 10.0 g/L,酵母提取物 5.0 g/L,NaCl 10.0,pH 7.0~7.2,配制固体培养基时需添加 20 g琼脂。
MRS培养基: 葡萄糖20g、蛋白胨10g、牛肉膏10g、酵母膏5g、乙酸钠5g、磷酸氢二钾2g、柠檬酸氢二铵2g、硫酸镁0. 58g和硫酸锰 0. 25 g,吐温80 1mL和蒸馏水 1 000 mL。用1 mol /L的氢氧化钠溶液调节pH 至7. 2,115℃灭菌30 min。固体培养基在液体培养基的基础上加入1. 6%的琼脂。
一、病原菌拮抗实验:
1、实验病原菌菌株:
选取 9种泌尿生殖道感染常见细菌和真菌:金黄色葡萄球菌(26111)、表皮葡萄球菌(26069)、大肠杆菌(24752)、粪链球菌(32221)、大肠杆菌(25922)、变形杆菌(49027)、白假丝酵母菌、光滑假丝酵母菌、热带假丝酵母菌,分别编号1-9号,编号为1-6号的菌株来源于中国药品生物制品检定所;编号为7-9号的菌株来源于宁夏医科大学附属医院。
2、益生菌培养菌液:
分别称取1 g益生菌菌粉样品(A-J)于 9 mL 无菌生理盐水中,充分混匀,80℃水浴保温30 min 后,按照 3%的接种量接种于MRS培养基中,37℃培养 24 h;然后,取菌液依次稀释至 10-6,得益生菌的培养菌液,分别编号(A1-J1)。
研究方法:牛津杯法。在LB液体培养基中分别接种各类病原菌(编号1-9号),37℃过夜培养,然后分别取 1 mL 病原菌菌液(标定为108CFU/mL)加入 100 mL 牛肉膏蛋白胨琼脂溶液中,充分混匀,倒入加有牛津杯的平板中,冷却后,取出牛津杯,将A1-J1培养菌液(100 μL)经 0.25 μL 滤膜过滤后分别加入到平板上用牛津杯打出的直径为6 mm的孔中,37℃孵育24 h后,测量抑菌圈直径。结果如表1所示。
表1. 10株益生菌对常见肠道、***病原菌的拮抗作用
由表1可得上述10益生菌对常见肠道、***病原菌均有抑制生长的作用,其中长双歧杆菌W68、两歧双歧杆菌Y22、嗜酸乳杆菌K43、干酪乳杆菌K35、发酵乳杆菌FM-12、罗伊氏乳杆菌K07的抑菌圈直径较大,均在20+以上,因此初步筛选出这6株菌进行进一步地筛选。
二、益生菌产酸筛选:
无菌操作下,将上述6株益生菌的菌粉样品(长双歧杆菌W68、两歧双歧杆菌Y22、嗜酸乳杆菌K43、干酪乳杆菌K35、发酵乳杆菌FM-12、罗伊氏乳杆菌K07)分别用生理盐水溶解后,在MRS平板上分离纯化,37 ℃培养48 h 以上。从平板上挑取单菌落,接种至50 mLMRS液体培养基中,37 ℃,180 r /min 震荡培养20~22 h,测定pH,以判断乳酸菌产酸能力。结果如表2所示。
表2. 6株乳酸菌产酸试验结果
益生菌 | C | E | G | H | I | J |
pH | 5.41 | 5.16 | 5.73 | 5.25 | 3.56 | 5.42 |
由表2可得上述6株益生菌均有产酸能力,其中长双歧杆菌W68、两歧双歧杆菌Y22、嗜酸乳杆菌K43、干酪乳杆菌K35、罗伊氏乳杆菌K07产酸能力较好,pH值均在5以上,因此筛选出这5株菌作为益生菌组合物的菌株种类进行配方研究。
三、益生菌组合物配方筛选:
益生菌组合物配方如表3所示。
表3. 益生菌组合物配方
益生菌 | 组合1 | 组合2 | 组合3 | 组合4 | 组合5 | 组合6 | 组合7 | 组合8 | 组合9 | 组合10 |
长双歧杆菌W68 | 20 | 40 | 15 | 15 | 15 | 15 | 10 | 15 | 15 | 10 |
两歧双歧杆菌Y22 | 20 | 15 | 40 | 15 | 15 | 15 | 10 | 50 | 15 | 10 |
嗜酸乳杆菌K43 | 20 | 15 | 15 | 40 | 15 | 15 | 30 | 15 | 15 | 30 |
干酪乳杆菌K35 | 20 | 15 | 15 | 15 | 40 | 15 | 20 | 15 | 50 | 30 |
罗伊氏乳杆菌K07 | 20 | 15 | 15 | 15 | 15 | 40 | 30 | 15 | 15 | 30 |
实施例2-6、一种中药组合物及其制备方法
本实施例的中药组合物的配方如表4所示。
表4. 中药组合物的配方
上述中药组合物的制备方法,包括以下步骤:
1、按表4的重量份数分别称取阿胶、鳖甲胶,用粉碎机粉碎成200目细粉,加入适量黄酒浸泡12小时,炖煮使之溶解,得基料A;
2、将玉米须、姜黄、香附、当归、木通、桑根白皮、蒲公英、土茯苓、白花蛇舌草、漏芦、黄连、牛膝、猪苓、关木通、金银花、车前草、金钱草、冬瓜皮按表1重量份称样,用粉碎机粉碎成60目细粉,加 10 倍量水超声1~2h,再浸泡3~4 h,得基料B;
3、将基料B倒入中药煎煮机中煎煮1.5小时,100目滤网过滤得基料C;剩余滤渣加入8倍量水煎煮1小时,100目滤网过滤得基料D;剩余滤渣加入6倍量水煎煮30分钟,100目滤网过滤得基料E;
4、将芍药、山楂、小通草、枸杞子按表4重量份称样,加入6倍量的水,并置于锅中炖煮2小时,100目过滤网过滤得基料F;
5、混合步骤:将基料C、基料D、基料E、基料F倒入锅中搅拌混匀,再加入1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精、蜂蜜于800r/min的条件下搅拌10min至溶解,得基料G;
6、浓缩步骤:将基料G加入液体真空浓缩煎药机中,减压0.08Mpa 72℃的条件下运行60分钟,得清膏H;
7、收膏步骤:将清膏H用文火加热,把基料A加入清膏内,不断用铲搅拌,搅拌至稠膏用细棒挑起夏天挂旗,冬天挂丝为度,相对密度为1.12-1.20之间,待稠膏冷却后,装入清洗、干燥、消毒后的容器,即得液体膏方(分别获得实施例2-6的中药组合物)。
取适量液体膏方(实施例2-6的中药组合物)分别加入70%的水,搅匀溶解后制成膏方饮品。招募20名患有肠私处不适、乳腺炎症等亚健康病症的女性,随机分成5组,每组4人,分别服用实施例2-6的中药组合物的膏方饮品,坚持60天,每日2杯,每杯300ml。测试者需自我评估排便质量、私处炎症、乳腺炎症改善情况,评价指标分别见下表5-7。评价结果取该组4名志愿者的平均值,结果见下表8。
表5. 排便质量标准
分数A/分 | A≤2 | 2<A≤4 | 4<A≤6 | 6<A≤8 | 8<A≤10 |
排便质量 | 无规律 | 腹泻/便秘 | 粪便较硬 | 有规律 | 香蕉便 |
表6. 私处炎症改善情况标准
分数A/分 | A≤2 | 2<A≤4 | 4<A≤6 | 6<A≤8 | 8<A≤10 |
改善情况 | 没有 | 轻微 | 有 | 明显 | 强烈 |
表7. 乳腺炎症改善情况标准
分数A/分 | A≤2 | 2<A≤4 | 4<A≤6 | 6<A≤8 | 8<A≤10 |
改善情况 | 没有 | 轻微 | 有 | 明显 | 强烈 |
表8. 中药膏方评价结果
实施例2 | 实施例3 | 实施例4 | 实施例5 | 实施例6 | |
排便质量 | 7.2 | 6.0 | 7.3 | 5.8 | 5.7 |
私处炎症 | 2.6 | 4.2 | 6.1 | 6.2 | 6.1 |
乳腺炎症 | 2.2 | 4.2 | 6.5 | 6.3 | 6.4 |
由表8可得,服用实施例4的膏方的志愿者均反馈排便有规律,没有出现便秘或者腹泻情况,且私处炎症以及乳腺炎症有所减轻,因此,选择实施例4的中药组合物进行益生菌发酵研究。
实施例7、一种益生菌发酵制品及其制备方法
以下中药组合物的重量份数选择实施例4的组合物的重量份数。
本实施例的益生菌发酵制品的制备方法,包括以下步骤:
1、将长双歧杆菌W68菌粉、两歧双歧杆菌Y22菌粉、嗜酸乳杆菌K43菌粉、干酪乳杆菌K35菌粉、罗伊氏乳杆菌K07菌粉分别按表3(组合1-10)进行称样,制成益生菌组合物。
2、将阿胶、鳖甲胶用粉碎机粉碎成200目细粉,加入适量黄酒浸泡12小时,炖煮使之溶解,得基料A;
3、将玉米须、姜黄、香附、当归、木通、桑根白皮、蒲公英、土茯苓、白花蛇舌草、漏芦、黄连、牛膝、猪苓、关木通、金银花、车前草、金钱草、冬瓜皮用粉碎机粉碎成60目细粉,加10 倍量水超声1~2h,再浸泡3~4 h,得基料B;
4、将基料B倒入中药煎煮机中煎煮1.5小时,100目滤网过滤得基料C;剩余滤渣加入8倍量水煎煮1小时,100目滤网过滤得基料D;剩余滤渣加入6倍量水煎煮30分钟,100目滤网过滤得基料E;
5、将芍药、山楂、小通草、枸杞子加入6倍量的水,并置于锅中炖煮2小时。100目过滤网过滤得基料F;
6、混合步骤:将基料C、基料D、基料E、基料F倒入锅中搅拌混匀,再加入1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精、蜂蜜于800r/min的条件下搅拌10min至溶解,得基料G;
7、灭菌步骤:将基料G以70-75℃、1h灭菌,冷却至30-32℃,得基料H;
8、菌种复活:在无菌操作下,将益生菌组合物(例如组合7)接种到灭菌的10%葡萄糖水溶液中,于21-26℃下培养16-19h,得基料I;
9、发酵步骤:将1/4重量份的基料I和基料H转入发酵罐中,将品温控制在29-35℃,进行密闭发酵10h,得一级发酵物J;一级发酵物J按照步骤7灭菌后,再补充1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精,再重复上述发酵操作即得到二级发酵物K;二级发酵物K按照步骤7灭菌后,再补充1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精,再重复上述发酵操作即得到三级发酵物L;三级发酵物L按照步骤7灭菌后,再补充1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精,再重复上述发酵操作即得到四级发酵物M;
10、浓缩步骤:将四级发酵物M加入液体真空浓缩煎药机中,减压0.08Mpa 72℃的条件下运行60分钟,得清膏N;
11、收膏步骤:将清膏N用文火加热,把基料A加入清膏内,不断用铲搅拌,搅拌至稠膏用细棒挑起夏天挂旗,冬天挂丝为度,相对密度为1.12-1.20之间,待稠膏冷却后,装入清洗、干燥、消毒后的容器即得液体膏方O;
12、预冻步骤:将液体膏方O倒入方格模具(3.0cm*3.0cm),在-40℃超低温冰箱里预冻处理,冻藏时间为12-24h,得基料P;
13、冻干步骤:将基料P放入真空冷冻干燥机中,启动真空冷冻干燥机,干燥温度-40℃,抽真空至10-40pa,干燥时间10-12h,然后放到小型超声波发生器震动网格上,功率160-220W,作用时间10-15s;第二次干燥温度- 20℃~- 40℃,抽真空至10-40pa,干燥时间10-12h。物料冷却后,即得益生菌发酵制品7。其余益生菌组合物(例如组合1-6、组合8-10)也按照上述相同方法制备益生菌发酵制品,分别标注为益生菌发酵制品1-6、8-10。
试验例一、β-谷甾醇含量测定
将实施例7制备的益生菌发酵制品1-10作为实验组;除不加益生菌外,其余步骤按照实施例7中的步骤2~13的操作进行,得空白组。
采用 β-谷甾醇含量测定方法对益生菌发酵制品1-10的β-谷甾醇进行含量检测,结果如表9。
β-谷甾醇是植物甾醇类成分之一,属于四环三萜类化合物,是真核生物细胞膜的主要组成成分,具有抗肿瘤、抗炎、抗氧化、免疫调节等功效。β-谷甾醇能渗入细胞膜中并改变膜流动性,抑制对雄性激素敏感的人***癌细胞增殖,诱导癌细胞凋亡且提高凋亡速率;通过嗜酸性粒细胞和分泌黏性因子减少氧自由基和炎症。此外,β -谷甾醇及其糖苷可以增加T -淋巴细胞增殖,增加自然杀伤细胞对癌细胞的裂解能力,增加Th-1型淋巴细胞,Th-2型淋巴细胞被抑制或没有变化。
β-谷甾醇含量测定方法包括:
1、精密称取 18mgβ―谷甾醇标准样品,用氯仿溶解后定容到 10mL(浓度为1.8mg/ml)。分别配制浓度为0 mg/ml、0.108 mg/ml、0.18 mg/ml、0.36mg/ml、0.43mg/ml、0.54mg/ml、0.72mg/ml、0.9mg/ml 甾醇标准液,在 243nm 处测定吸光度,建立标准曲线;
2、分别称取益生菌发酵制品1-10、空白组样品各1g加入85%乙醇,液固比为42.1(ml/g),搅拌后分别在48.8℃提取87.1min,得到乙醇提取物质,过滤石油醚萃取后,挥干溶剂,氯仿溶解,得样品测试液,在 243nm 处测定吸光度。
表9. 益生菌发酵制品的β-谷甾醇含量
由表9可知,从制品1-制品10的β-谷甾醇含量均明显高于空白组,即本发明的益生菌组合物在不同比例均可提高发酵底物中的β-谷甾醇含量,其中益生菌发酵制品7的β-谷甾醇含量最高。
试验例二、乳腺炎动物模型实验
1、实验动物:ICR雌性哺乳期小鼠140只,购自上海市实验动物质量监督检查站,哺乳3-7d,体重约18-25g。动物腺饲养温度控制在18-25℃,相对湿度控制在50%-60%,通风良好,小鼠自由饮水进食。
2、实验菌株:标准金黄色葡萄球菌菌株(编号ATCC25923),购自中国工业微生物菌种。
3、菌悬液制备:金黄色葡萄球菌接种于LB液体培养基,37℃培养12h,将菌液10倍倍比稀释,测定OD值并接种于LB固体平板上,37℃培养箱中培养24h后,采用平板菌落计数法进行细菌计数。对菌液进行稀释,用PBS清洗,根据计数结果,用PBS调整细菌浓度为107CFU/mL细菌悬液。
4、供试样品制备:将上述空白组、益生菌发酵制品1-10加水溶解,配制成浓度为0.43g/ml的原药溶液,4℃冰箱保存,用前水浴加热至37℃。
5、动物分组及实验处理:将小鼠随机分为14组,每组动物10只,即空白对照组:乳腺注射无菌PBS 50μL,0.1mL/10g蒸馏水灌胃7d;模型组:乳腺注射菌悬液50μL,0.1mL/10g蒸馏水灌胃7d;阳性药(头孢噻肟)组:乳腺注射菌悬液50μL,0.1mL/10g头孢噻肟钠1次/日;空白样品组:乳腺注射菌悬液50μL,0.1mL/10g空白样灌胃7d;组合A:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品1灌胃7d;组合B:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品2灌胃7d;组合C:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品3样品灌胃7d;组合D:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品4样品灌胃7d;组合E:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品5样品灌胃7d;组合F:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品6灌胃7d;组合G:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品7灌胃7d。组合H:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品8灌胃7d。组合I:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品9灌胃7d。组合J:乳腺注射菌悬液50μL,0.1mL/10g的益生菌发酵制品10灌胃7d。
给药结束后,小鼠禁食12h,摘眼球取血,室温静置1h,然后于4000rpm/min、4℃离心15min分离血清,-20℃保存备用。取血后处死小鼠,取出小鼠第4对乳腺、胸腺和脾,并用灭菌PBS冲洗。对胸腺、脾进行称重测定,乳腺分为两份,一份放入4%多聚甲醛固定液固定,一份-80℃冰箱中保存待用。
乳腺中TNF-α、IL-6的检测:严格按照ELISA试剂盒说明书进行操作,检测小鼠乳腺血清中TNF-α、IL-6的含量,结果如表10,具体步骤如下:
血浆中TNF-α、 IL-6含量测定釆用ELISA法,用纯化的抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入样本,再和HRP标记的羊抗鼠抗体结合,形成抗体-抗原-酶标抗体复合物。经过彻底洗涤后加入底物,底物在HRP酶的催化下转化成蓝色,加入酸终止反应,在酸的作用下最终变成黄色,颜色的深浅和样品中的含量呈正比。用酶标仪在450nm波长下测定其OD值,通过标准曲线方程计算出样品中 TNF-α、IL-6含量,具体操作严格按照试剂盒说明书进行,简述之:
1.标准品的加样:设置标准品孔和样本孔,标准品孔各加不同浓度的标准品50μL。
2.加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余各步操作相同)、待测样品孔。在酶标包被板上待测样品孔中先加样品稀释液40μL,然后再加待测样品10μL(样品最终稀释度为5倍)。加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀。
3.加酶:每孔加入酶标试剂100μL,空白孔除外。
4.温育:用封板膜封板后置37℃温育60min。
5. 配液:将20倍浓缩洗涤液用蒸馏水20倍稀释后备用。
6. 洗搽:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30s后弃去,如此重复5次,拍干。
7.显色:每孔先加入显色剂A 50μL,再加入显色剂B 50μL,轻轻震荡混匀,37℃避光显色15min。
8.终止:每孔加终止液50μL,终止反应(此时蓝色立转黄色)。
9.测定:以空白孔调零,450nm波长依序测量各孔的吸光度(OD值)。测定应在加终止液后15min以内进行。根据吸光度及标准曲线测定样品中TNF-α、 IL-6的含量。
表10 小鼠乳腺血清中TNF-α、IL-6的含量(pg/mL)
由表10可得,组合G的TNF-α、IL-6含量最低,减轻乳腺炎的炎症效果最好,再结合表9的结果,最终选择组合7作为益生菌组合物的最佳组合。
试验例三、人群功效验证
招募16名女性志愿者,志愿者均患有乳腺炎患者、***炎患者等疾病,年龄范围22~60岁。
纳入标准:全部志愿者均符合《妇科学》(第 8 版)中相关诊断标准。患者对此次研究知情同意,签订研究意向书,研究经过医学伦理委员会审核。
排除标准:患者存在精神障碍;患者不配合研究;患者存在其他严重性胃肠道疾病;患者为妊娠期或哺乳期孕产妇;患者存在明显药物过敏史。
将志愿者分成2组,每组8人,记为A、B组,安排志愿者分别服用益生菌粉(采用本发明的益生菌组合7制成的益生菌粉)以及本发明实施例的肠私处护理、乳腺抗炎的益生菌发酵制品7,即A组服用益生菌粉,B组服用益生菌发酵制品7,并记录私处炎症治愈时间和乳腺炎症治愈时间,结果如表11。肠道舒适评价,结果如表12。
益生菌粉的制备:
1)将长双歧杆菌W68菌粉、两歧双歧杆菌Y22菌粉、嗜酸乳杆菌K43菌粉、干酪乳杆菌K35菌粉、罗伊氏乳杆菌K07菌粉分别按表3(组合7)的比例进行称样,制成益生菌组合物;
2)称取益生菌组合物10 ~ 20份,低聚果糖80 ~ 90份,混合均匀后分装成2g/袋,即得益生菌粉。
服用方式:
1)益生菌发酵制品7:将益生菌发酵制品7置于37℃、约150mL的温水中搅匀溶解后服用。饭后半小时服用,每日1-2次,每次1块。
2)益生菌粉:将益生菌粉置于37℃、约150mL的温水中搅匀溶解后服用。饭后半小时服用,每日1-2次,每次1袋。
表11. 人群病症治愈时间/d
A组 | B组 | |
私处炎症 | 15 | 10 |
乳腺炎症 | 18 | 11 |
表12. 肠道舒适度评价
A组 | B组 |
有明显改善 | 有明显改善 |
由表11和表12可得,A组和B组均对女性私处炎症和乳腺炎症均有明显改善,此外,对于肠道舒适度也有明显改善。本发明益生菌发酵制品在体外发酵制成膏方或者直接制成菌粉服用均有显著效果,且志愿者反馈非常好。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (9)
1.一种益生菌组合物,其特征在于,以重量份数计,所述益生菌组合物包括长双歧杆菌W68 10-40份、两歧双歧杆菌Y22 10-40份、嗜酸乳杆菌K43 15-40份、干酪乳杆菌K35 15-40份和罗伊氏乳杆菌K07 15-40份。
2.一种益生菌发酵制品,其特征在于,所述益生菌发酵制品包括如权利要求1所述的益生菌组合物和中药组合物;
所述中药组合物包括低聚果糖、水苏糖、抗性糊精、阿胶、鳖甲胶、姜黄、蒲公英、香附、土茯苓、白花蛇舌草、漏芦、牛膝、枸杞子、黄连、芍药、金银花、车前草、蜂蜜、冬瓜皮、玉米须、金钱草、猪苓、木通、桑根白皮、关木通、小通草、当归和山楂;使用如权利要求1所述的益生菌组合物发酵中药组合物制得。
3.如权利要求2所述的益生菌发酵制品,其特征在于,以重量份数计,所述中药组合物包括以下组分:
低聚果糖3.4-30份、水苏糖2-20.6份、抗性糊精1-8.2份、阿胶3-6份、鳖甲胶1.8-3.6份、姜黄3.6-7.2份、蒲公英4.8-9.6份、香附1.2-2.4份、土茯苓2.4-4.8份、白花蛇舌草2.4-4.8份、漏芦1.2-2.4份、牛膝1.2-2.4份、枸杞子1.8-3.6份、黄连1.2-2.4份、芍药1.2-2.4份、金银花1.2-2.4份、车前草1.2-2.4份、蜂蜜1.2-2.4份、冬瓜皮3-6份、玉米须4.8-9.6份、金钱草1.2-2.4份、猪苓0.6-1.2份、木通1.2-2.4份、桑根白皮1.2-2.4份、关木通0.6-1.2份、小通草2.4-4.8份、当归1.2-2.4份和山楂1.2-2.4份。
4.一种如权利要求2或3所述的益生菌发酵制品的制备方法,其特征在于,包括以下步骤:
S1、熬胶:将阿胶、鳖甲胶粉碎,加入黄酒浸泡,炖煮溶解,得基料A;
S2、浸泡:将玉米须、姜黄、香附、当归、木通、桑根白皮、蒲公英、土茯苓、白花蛇舌草、漏芦、黄连、牛膝、猪苓、关木通、金银花、车前草、金钱草、冬瓜皮粉碎,加8-12倍量水超声,浸泡,得基料B;
S3、煎煮:将基料B煎煮,第一次过滤得基料C;取第一次过滤的滤渣加入6-10倍量水煎煮,第二次过滤得基料D;取第二次过滤的滤渣加入4-6倍量水煎煮,过滤得基料E;将芍药、山楂、小通草、枸杞子加入4-6倍量的水,炖煮,过滤得基料F;
S4、混合:将基料C、基料D、基料E、基料F倒入锅中搅拌混匀,再加入1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精、蜂蜜混合搅拌溶解,得基料G,灭菌,得基料H;
S5、将权利要求1所述的益生菌组合物和基料H混合发酵,得一级发酵物,再补充1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精,得二级发酵物;再补充1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精,得三级发酵物;再补充1/4重量份的低聚果糖、1/4重量份的水苏糖、1/4重量份的抗性糊精,得四级发酵物;将四级发酵物浓缩,收膏,得液体膏方,然后预冻,冻干,得益生菌发酵制品。
5.如权利要求4所述的益生菌发酵制品的制备方法,其特征在于,所述液体膏方的相对密度为1.12-1.20。
6.如权利要求4所述的益生菌发酵制品的制备方法,其特征在于,将四级发酵物置于减压0.08Mpa,72℃的条件下运行60分钟,得清膏。
7.如权利要求4所述的益生菌发酵制品的制备方法,其特征在于,所述步骤S3中,所述过滤的目数为100-150目。
8.如权利要求4所述的益生菌发酵制品的制备方法,其特征在于,所述冻干步骤包括:
预冻:将上述液体膏方倒入方格模具,在-40℃超低温冰箱里预冻处理,冻藏时间为12-24h,得预冻后的膏方;
冻干:将预冻后的膏方置于干燥温度为-40℃,真空度为10 pa -40pa,干燥时间为10-12h的条件下真空冷冻干燥,然后置于功率160-220W,作用时间10-15s条件下震动;再将膏方置于干燥温度为- 20℃~- 40℃,真空度为10-40pa,干燥时间为10-12h的条件下真空冷冻干燥,制得益生菌发酵制品。
9.如权利要求1所述的益生菌组合物、如权利要求2或3所述的益生菌发酵制品在制备女性肠道、私处护理或乳腺抗炎药物中的应用。
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