CN108159162A - 含厚朴原料药的发酵物及其制备方法和用途 - Google Patents
含厚朴原料药的发酵物及其制备方法和用途 Download PDFInfo
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/57—Magnoliaceae (Magnolia family)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
本发明公开了一种含厚朴原料药的发酵物及其制备方法和用途。该制备方法包括如下步骤:(1)将由厚朴、陈皮、山楂、马兰和甘草组成的原料药粉碎,分别取20~40重量份厚朴、20~40重量份陈皮、15~35重量份山楂、15~35重量份马兰、5~15重量份甘草与水混合得到悬浮液;(2)采用胰蛋白酶、纤维素酶和木聚糖酶对悬浮液进行酶解,得到酶解液;(3)将营养物质加入酶解液,灭菌,得到发酵培养基;(4)向发酵培养基中接种酵母菌进行厌氧发酵培养8~18h,然后接种乳酸菌进行静置发酵培养25~100h,得到培养产物;(5)将培养产物进行固液分离,得到发酵物。本发明的方法可以减少发酵时间,所得发酵物的消食化积作用显著。
Description
技术领域
本发明涉及一种发酵物及其制备方法和用途,尤其是一种含厚朴的原料药的发酵组合物及其制备方法和用途。
背景技术
胃动力障碍性疾病作为一种常见易发的胃肠道疾病,危害着人类健康。胃动力表示胃部肌肉的收缩蠕动力,包括胃部肌肉收缩的力量和频率。胃动力不足,也称为“消化不良”。胃动力障碍是造成非溃疡性消化不良的主要原因。造成胃动力障碍因素包括精神情绪变化、胃分泌功能紊乱、功能性消化不良等。当胃动力出现障碍时,会发生上腹胀满、易饱、饭后腹胀、恶心、呕吐等消化不良症状。由于生活节奏的加快,生活压力的增大以及饮食的不规律,使得越来越多的人患上由于胃动力不足引起的功能性消化不良疾病。
目前,治疗此类疾病多采用化学药物。第一代为贝胆碱、第二代为胃复安和吗丁琳、第三代为普瑞博思。它们都是激动M胆碱受体,促进乙酰胆碱释放而加强胃肠蠕动,协调胃肠运动,增加食物较低位置括约肌张力,防止食物的滞留与反流,发挥胃肠促动力。
这些化学药物虽然疗效显著,但也具有一定副作用。例如,吗丁啉有拮抗多巴胺的作用,使催乳素分泌增加;影响卵巢激素的分泌;使乙酰胆碱系统处于亢进状态。有些患者在服用吗丁啉时,可能出现女性泌乳、男性乳房发育、女子月经不调、哮喘发作、锥体外系反应、便血、大便失禁等。因此,医学界寻找一些疗效好、具有预防保健作用的植物药作为改善胃动力障碍的辅助治疗。常见的一些中成药多为普通中药提取加工品,疗效一般。
酵素是指对植物进行深层发酵,并提取得到的含生物活性物质的低盐液体。生物活性成分至少包括酶、发酵参与菌等。酵素中含有生物活性成分,可影响服用者体内的活性酶,从细胞层面调节机体的生命活动。酵素合理选择食材和发酵工艺,可分别形成一种或多种神奇功效,包括调整免疫应答、抗癌、降血压、整肠、消除便秘、抵御痛风、减肥、抗过敏等。因此,作为具有生命活力的独特保健品,酵素具有巨大的前景。
CN107296267A公开了一种中药复合养胃酵素的制备方法,原料包括白术10~20份、半夏(制)5~15份、茯苓5~15份、香附子(制)5~15份、木香2~10份、陈皮5~15份、厚朴(制)5~15份、砂仁5~15份、豆蔻4~13份、枳实(制)5~15份、广藿香5~15份、甘草2~5份;自制酵素母液100~200份,5%葡萄糖液2000~4000份。将上述原料分别烘干、粉碎,以5%的葡萄糖液和酵素母液为发酵原液,在常温下发酵2月以上,提取中药活性成分,过滤、制备得到中药养胃酵素。上述方法的中药原料药成分过于复杂,并且发酵时间太长。这样不利于工业化生产。
因此,仍然需要精简发酵原料的成分,并且缩短发酵时间,从而便于工业化生产。
发明内容
本发明的目的在于提供一种基于含厚朴的原料药的发酵物的制备方法,该方法可以精简原料药,并且缩短发酵时间。本发明的另一目的在于提供一种发酵物,具有消食化积作用。本发明的再一目的在于提供一种发酵物的用途。本发明的目的是通过如下技术方案实现的。
本发明提供一种发酵物的制备方法,包括如下步骤:
(1)将由厚朴、陈皮、山楂、马兰和甘草组成的原料药粉碎,分别取20~40重量份厚朴、20~40重量份陈皮、15~35重量份山楂、15~35重量份马兰、5~15重量份甘草与水混合得到悬浮液;
(2)采用胰蛋白酶、纤维素酶和木聚糖酶对所述悬浮液进行酶解,得到酶解液;
(3)将营养物质加入所述酶解液,灭菌,得到发酵培养基;
(4)向所述发酵培养基中接种酵母菌进行厌氧发酵培养8~18h,然后接种乳酸菌进行静置发酵培养25~100h,得到培养产物;
(5)将所述培养产物进行固液分离,得到所述发酵物。
根据本发明的制备方法,优选地,厚朴为25~30重量份、陈皮为25~30重量份、山楂为15~20重量份、马兰为20~25重量份、且甘草为5~10重量份。
根据本发明的制备方法,优选地,步骤(1)中,水的用量为原料药总重量的5~15倍。
根据本发明的制备方法,优选地,步骤(2)中,基于悬浮液的总重量,胰蛋白酶的用量为0.05~0.25wt%,纤维素酶的用量为0.03~0.25wt%,木聚糖酶的用量为0.05~0.25wt%。
根据本发明的制备方法,优选地,步骤(2)中,酶解的pH值为5.5~6.8,酶解温度为40~65℃,酶解时间为2~6h。
根据本发明的制备方法,优选地,步骤(3)中,所述营养物质包括0.1~0.5wt%酵母膏、1~5wt%铵盐、0.05~0.2wt%镁盐和0.3~0.8wt%磷酸盐;以上重量百分数均是基于所述酶解液的总重量。
根据本发明的制备方法,优选地,步骤(4)的厌氧发酵培养中,酵母菌的接种量为所述发酵培养基的0.1~1.0wt%,发酵温度为26~35℃,发酵时间为8~18h;
根据本发明的制备方法,优选地,步骤(4)的静置发酵培养中,乳酸菌的接种量为所述发酵培养基的0.1~1.0wt%,在35~40℃下发酵培养25~35h,然后在60~75℃下发酵培养18~35h。
本发明还提供上述任一项的制备方法获得的发酵物。
本发明也提供上述发酵物在制备具有消食化积作用的药物、保健品或饲料添加剂中的用途。
本发明通过采用先酶解后发酵的方式,使得发酵时间减少,从而更加有利于工业化生产。此外,本发明利用酶法进行水解,然后利用酵母菌、乳酸菌对厚朴、陈皮、山楂、马兰、甘草进行发酵处理。相对与传统中药组合的煎煮直接使用,本发明的发酵物中富含原有中药的活性成分,同时,经过发酵作用显著提升了产品的消食化积保健功效。本发明提供的发酵物与厚朴、陈皮、山楂、马兰、甘草单各药材相比,消食作用显著提升。
具体实施方式
下面结合具体实施例对本发明作进一步的说明,但本发明的保护范围并不限于此。
本发明的用于发酵的原料药由厚朴、陈皮、山楂、马兰和甘草组成。厚朴为木兰科植物厚朴Magnolia officinalis Rehd.et Wils.的干皮、根皮及枝皮;含挥发油、生物碱、皂苷、鞣质和微量烟酸等。厚朴含厚朴酚和厚朴新酚、丁香树脂酚-4’-O-β-D-吡喃葡萄糖苷、厚朴木脂体A、厚朴木脂体B、厚朴木脂体C、厚朴木脂体D、厚朴木脂体E、厚朴木脂体F、厚朴木脂体G、厚朴木脂体H、厚朴木脂体I、龙脑基厚朴酚等成分。厚朴性温,味苦、辛;归脾经、胃经、肺经、大肠经;有燥湿消痰、下气除满功效。厚朴用于治疗湿滞伤中、脘痞吐泻、食积气滞、腹胀便秘、痰饮喘咳。
陈皮为芸香科植物橘Citrus reticulata Blanco及其栽培变种的干燥成熟果皮。药材分为“陈皮”和“广陈皮”。采摘成熟果实,剥取果皮,晒干或低温干燥。陈皮含挥发油、橙皮苷、正癸醛、柠檬醛、柠檬烯、辛醇等成分。陈皮味辛、味苦,性温;归脾经、肺经;理气开胃,燥湿化痰,治脾胃病。陈皮用于治疗胸脘胀满、食少呕吐、咳嗽痰多。
山楂为蔷薇科植物山楂Crataegus pin.nati fida Bge.或山里红Crataeguspin,natiida Bge.var.majorN.E.Br.的干燥成熟果实。山楂含多种维生素、山楂酸、酒石酸、柠檬酸、苹果酸等,还含有黄酮类、内酯、糖类、蛋白质、脂肪和钙、磷、铁等矿物质。山楂所含的解脂酶能促进脂肪类食物的消化。山楂味性微温,酸、甘;归脾、胃、肝经;开胃消食、化滞消积、活血散瘀、化痰行气。山楂用于治疗肉食滞积、症瘕积聚、腹胀痞满、瘀阻腹痛、痰饮、泄泻、肠风下血等。
马兰为菊科植物马兰Kalimeris indica(L.)Sch.-Bip.[Asterindicus L.]的干燥全草或根。马兰主要含大黄素、大黄酚、香草醛、β-谷固醇、豆固醇、亚油酸、木栓酮、木栓醇、月桂酸以及多种挥发性成分如石竹烯、γ-榄香烯、十五烷等。马兰性凉,味辛;归肺经、肝经、胃经、大肠经;清热解毒,凉血止血,清热利湿,消肿,消疳。马兰用于治疗出血症、泄痢、疳积以及咳喘等。
甘草为豆科植物甘草Glycyrrhiza uralensis Fisch的根及根状茎。甘草根及根茎含甘草甜素,为甘草酸的钾、钙盐。尚含甘草甙、甘草甙元、异甘草甙、异甘草元、新甘草甙、新异甘草甙等。甘草能和中缓急,润肺,解毒,调和诸药。甘草炙用,治脾胃虚弱,食少,腹痛便溏,劳倦发热,肺痿咳嗽,心悸,惊痫;甘草生用,治咽喉肿痛,消化性溃疡,痈疽疮疡,解药毒及食物中毒。
本发明的含厚朴原料药的发酵物的制备方法包括如下步骤:(1)粉碎分散步骤;(2)酶解步骤;(3)培养基制备步骤;(4)发酵步骤:(5)分离步骤。任选地,还可以包括(6)浓缩干燥步骤。下面进行详细介绍。
在本发明的粉碎分散步骤(1)中,将由厚朴、陈皮、山楂、马兰和甘草组成的原料药粉碎,分别取20~40重量份厚朴、20~40重量份陈皮、15~35重量份山楂、15~35重量份马兰、5~15重量份甘草与水混合得到悬浮液。可以分别将各个原料药粉碎,然后称取相应比例的量,再与水混合;也可以先称取各个原料药,然后一起粉碎,再与水混合。所述原料药可以粉碎至10目以下,优选为20目以下,更优选为50目以下。本发明精简了原料药数量,从而使得后续发酵工艺更加简单,并可以减少发酵时间。在本发明中,优选地,厚朴为25~30重量份、陈皮为25~30重量份、山楂为15~20重量份、马兰为20~25重量份、且甘草为5~10重量份。根据本发明的一个实施方式,厚朴为25重量份、陈皮为25重量份、山楂为20重量份、马兰为20重量份、且甘草为10重量份。这样的组合可以使得发酵物的消食化积作用更加显著。
在本发明的粉碎分散步骤(1)中,水的用量为原料药总重量的5~15倍;优选为8~10倍。采用这样的配比,有利于后续的酶解和发酵过程。水可以为蒸馏水或去离子水等,只要不影响酶解和发酵过程即可。
在本发明的酶解步骤中,采用胰蛋白酶、纤维素酶和木聚糖酶对所述悬浮液进行酶解,得到酶解液。基于悬浮液的总重量,胰蛋白酶的用量可以为0.05~0.25wt%,优选为0.06~0.2wt%,更优选为0.1~0.15wt%。基于悬浮液的总重量,纤维素酶的用量可以为0.03~0.25wt%,优选为0.05~0.2wt%,更优选为0.1~0.15wt%。基于悬浮液的总重量,木聚糖酶的用量可以为0.05~0.25wt%,优选为0.06~0.2wt%,更优选为0.1~0.15wt%。根据本发明的一个实施方式,胰蛋白酶的用量为0.1~0.15wt%,纤维素酶的用量为0.1~0.15wt%,木聚糖酶的用量为0.1~0.15wt%。根据本发明的一个具体实施方式,胰蛋白酶的用量为0.1wt%,纤维素酶的用量为0.1wt%,且木聚糖酶的用量为0.1wt%。
在本发明中,胰蛋白酶的酶活力可以为15~55万U/g,优选为25~50万U/g,更优选为35~50万U/g。纤维素酶的酶活力可以为20~55万U/g,优选为25~55万U/g,更优选为35~50万U/g。木聚糖酶的酶活力可以为15~60万U/g,优选为25~50万U/g,更优选为35~50万U/g。根据本发明的一个具体实施方式,胰蛋白酶的酶活力为50万U/g,纤维素酶的酶活力为50万U/g,且木聚糖酶的酶活力为50万U/g。
采用上述种类和用量的生物酶时,能够将原料药充分酶解,为发酵培养提供氨基酸、单糖、寡糖等原料,从而减少发酵时间。
在本发明的酶解步骤(2)中,酶解的pH值可以为5.5~6.8,优选为6.0~6.5;酶解温度可以为40~65℃,优选为50~60℃,更优选为52~58℃;酶解时间可以为2~6h,优选为2.5~5h,更优选为2.5~4h。根据本发明的一个具体实施方式,酶解的pH值为6.0,酶解温度为55℃,酶解时间为3h。采用本发明的酶解条件,能够使上述三种生物酶对原料药进行充分酶解,从而减少发酵时间。
在本发明的培养基制备步骤(3)中,将营养物质加入所述酶解液,灭菌,得到发酵培养基。可以采用本领域已知的方法进行灭菌,这里不再赘述。基于所述酶解液的总重量,所述营养物质包括0.1~0.5wt%酵母膏、1~5wt%铵盐、0.05~0.2wt%镁盐和0.3~0.8wt%磷酸盐。可以采用市售的酵母膏。酵母膏的用量优选为0.2~0.3wt%。铵盐可以选自硫酸铵、硝酸铵或碳酸铵中的至少一种,优选为硫酸铵。铵盐的用量优选为1.5~3.5wt%,更优选为2~2.5wt%。镁盐可以选自硫酸镁、氯化镁、硝酸镁、碳酸镁中的至少一种,优选为硫酸镁。镁盐的用量优选为0.06~0.15wt%,更优选为0.08~0.1wt%。磷酸盐可以选自磷酸钠、磷酸氢二钠、磷酸二氢钠、磷酸钾、磷酸氢二钾、磷酸二氢钾、磷酸钙、磷酸镁中的至少一种,优选为磷酸二氢钾。磷酸盐的用量优选为0.35~0.65wt%,更优选为0.5~0.6wt%。酶解液中加入上述营养物质,能够有效满足酵母菌、乳酸菌的发酵培养的营养元素需求,并且可以减少发酵时间。
在本发明的培养基制备步骤(3)中,营养物质还可以含有钾盐。该钾盐与磷酸盐优选为同一物质,例如磷酸钾、磷酸氢二钾或磷酸二氢钾,优选为磷酸二氢钾。该营养物质还可以包括锌盐,锌盐可以选自硫酸锌、氯化锌、硝酸锌中的至少一种,并优选为氯化锌。基于所述酶解液的总重量,所述营养物质可以包括0.05~0.2wt%、优选为0.06~0.15wt%、更优选为0.08~0.1wt%的锌盐。
在本发明的发酵步骤(4)中,向所述发酵培养基中接种酵母菌进行厌氧发酵培养8~18h,然后接种乳酸菌进行静置发酵培养25~100h,得到培养产物。与现有技术相比,本发明的发酵时间减少很多。
在步骤(4)的厌氧发酵培养中,酵母菌的接种量为所述发酵培养基的0.1~1.0wt%,优选为0.2~0.8wt%,更优选为0.4~0.6wt%。发酵温度为26~35℃,优选为28~32℃,更优选为30℃。发酵时间为8~18h,优选为10~15h,更优选为12h。在上述条件下,可以使得酵母菌的作用充分发挥且节约时间。本发明的酵母菌的实例包括但不限于安琪酵母股份有限公司生产的高活性干酵母。本发明的乳酸菌可以选自保加利亚乳杆菌和嗜热链球菌的一种或多种;优选为保加利亚乳杆菌和嗜热链球菌的混合物。保加利亚乳杆菌和嗜热链球菌的重量比可以为1:10~10:1,优选为1:5~5:1,更优选为1:1。
在步骤(4)的静置发酵培养中,乳酸菌的接种量为所述发酵培养基的0.1~1.0wt%,优选为0.2~0.8wt%,更优选为0.4~0.6wt%。在35~40℃、优选为25~39℃、更优选为37℃下发酵培养25~35h、优选为25~30h、更优选为28h;然后在60~75℃、优选为64~72℃、更优选为60或68℃下发酵培养18~35h、优选为20~28h、更优选为24h。
在本发明的分离步骤(5)中,将所述培养产物进行固液分离,得到所述发酵物。分离得到发酵物(原液)和固体残渣。固液分离的方法可以为离心或过滤,并优选为离心。
在本发明的浓缩干燥步骤(6)中,将所述发酵物浓缩、干燥,得到发酵粉。可以采用减压的方法进行浓缩和干燥,这里不再赘述。
采用上述制备方法获得发酵物。本发明的发酵物可以为发酵液,也可以为发酵粉。发酵物有时也称之为酵素、发酵组合物等。根据本发明的一个实施方式,所述发酵粉的含水量低于5wt%,超氧化物歧化酶SOD的活力大于等于420±5U/g,乳酸菌活菌数大于等于3.3±0.5×108CFU/g。
本发明的发酵物具有消食化积作用,因而可以用在制备具有消食化积作用的药物、保健品或饲料添加剂中。保健品可以为具有保健作用的食品,例如糖果、饮料、面包、饼干等。药物可以为多种常规的固体剂型或液体剂型,不再赘述。饲料添加剂是指在饲料生产加工、使用过程中添加的少量或微量物质,在饲料中用量很少但作用显著。
以下实施例和比较例的原料说明如下:
酵母菌:安琪酵母股份有限公司生产的高活性干酵母;
乳酸菌:重量比为1:1的保加利亚乳杆菌和嗜热链球菌。
以下实施例和比较例的测试方法说明如下:
<发酵前后总酚变化>
对照品溶液的制备:取厚朴酚对照品、和厚朴酚对照品适量,精密称定,加甲醇分别制成每1m1含厚朴酚40μg、和厚朴酚24μg的溶液,即得。
发酵物样品制备:取实施例2制得的发酵粉0.45g(相当于由厚朴1.25g、陈皮1.25g、山楂1g、马兰1g、甘草0.5g原生药),置于具塞锥形瓶中,精密加入甲醇25ml,摇匀,密塞,浸渍24小时,滤过,精密量取续滤液1ml,置于25ml量瓶中,加甲醇至刻度,摇匀,即得。
水提液样品制备:取厚朴1.25g、陈皮1.25g、山楂1g、马兰1g、甘草0.5g,水浸泡30min,煎煮提取2次,每次加水量10倍量,每次提取时间2小时,过滤,滤液合并,浓缩至干粉,将其置于具塞锥形瓶中,精密加入甲醇25ml,摇匀,密塞,浸渍24小时,滤过,精密量取续滤液1ml,置25ml量瓶中,加甲醇至刻度,摇匀,即得。
测定方法:采用HPLC法。色谱条件:色谱柱为Diamonsil C18(250mm×4.6mm,5μm),以甲醇-水(78:22)为流动相;检测波长为294nm。分别精密吸取上述两种对照品溶液各4μl与供试品溶液5μl,注入液相色谱仪,测定。
表1、发酵物样品与水提液样液中厚朴酚与和厚朴酚的测定结果
<发酵前后总黄酮变化>
发酵物样品:取实施例2制得的发酵粉0.45g(相当于由厚朴1.25g、陈皮1.25g、山楂1g、马兰1g、甘草0.5g原生药),加入蒸馏水定容至50ml,即得。
水提液样品:取厚朴1.25g、陈皮1.25g、山楂1g、马兰1g、甘草0.5g,水浸泡30min,煎煮提取2次,每次加水量10倍量,每次提取时间2小时,过滤,滤液合并,浓缩,用蒸馏水定容至50ml,即得。
对照品溶液的制备:精密称取经120℃恒温干燥至恒重的芦丁对照品10mg,置50mL的量瓶中,加适量乙醇,用超声处理使其全部溶解,放冷,然后再加适量乙醇至刻度线,摇匀即得所需要的对照品溶液(每1mL中含无水芦丁0.2mg)。
对照品吸光度测定:精密量取对照品溶液6mL,将其分别置于50mL的量瓶中,然后分别加水至12mL,滴加5wt%亚硝酸钠溶液2mL,摇匀,放置6min,然后再分别滴加10wt%硝酸铝溶液2mL,摇匀,放置6min,再滴加4%氢氧化钠试液20mL,最后再加水至刻度线,摇匀,放置15min,用相应试剂作为空白对照组,然后立即照紫外-可见分光光度法,设定波长在500nm处测定吸光度为0.242。
含量测定:取样品10mL分别置于50mL的量瓶中,然后分别加水至12mL,再分别滴加5wt%亚硝酸钠溶液2mL,摇匀,放置6min,然后再分别滴加10wt%硝酸铝溶液2mL,摇匀,放置6min,再滴加4wt%氢氧化钠试液20mL,最后再加水至刻度线,摇匀,放置15min,用相应试剂作为空白对照组,然后立即照紫外-可见分光光度法,设定波长在500nm处测定各组样品溶液的吸光度(平行样品3份),记录实验所得各组吸光度。按照公式C=5×A×0.024/0.242计算样液中总黄酮含量(单位mg/mL)。根据上述曲线计算两种样液中的总黄酮含量。
表2、发酵物样品与水提液样品总黄酮含量的测定结果
实施例1-发酵物
1)分别将厚朴、陈皮、山楂、马兰和甘草粉碎,并采用药典2号筛(24目)进行筛分,得到原料药粉末;然后按照厚朴25重量份、陈皮25重量份、山楂20重量份、马兰20重量份和甘草10重量份称取原料药粉末,混合,并加入原料药10倍量(重量比)的纯净水,制得悬浮液。
2)将悬浮液与基于悬浮液总重量0.1wt%的胰蛋白酶(50万U/g)、0.1wt%的纤维素酶(50万U/g)、0.1wt%的木聚糖酶(50万U/g)混合并进行酶解,得到酶解液;酶解条件为:pH为6.0,温度为55℃,酶解时间为3h。
3)基于所述酶解液的总重量,补加如下营养物质:酵母膏0.2wt%、硫酸铵2wt%、硫酸镁0.1wt%和磷酸二氢钾0.6wt%,灭菌后得到发酵培养基。
4)向发酵培养基接种0.5wt%的酵母菌,30℃下厌氧发酵12h;然后接种0.5wt%乳酸菌,在37℃下静置发酵28h,然后在60℃下静置发酵24h,得到培养产物。
5)发酵结束后,进行离心处理,分离得到发酵原液(发酵物)和固体残渣。
实施例2-发酵粉
将实施例1的发酵原液减压浓缩,通过减压干燥得发酵粉。所得发酵粉的含水量低于5wt%;超氧化物歧化酶(SOD)的活力大于(420±5)U/g;乳酸菌活菌数大于(3.3±0.5)×108CFU/g,总酚含量大于3wt%,总黄酮含量大于3wt%。
实施例3-饮料
将实施例1的发酵原液与水按照重量比为1:10的混合得到混合物,然后基于混合物的总重量添加18wt%蔗糖、0.3wt%柠檬酸,进行均质-脱气-巴氏杀菌-灌装-冷却等步骤,得到饮料。
实施例4-发酵物
1)分别将厚朴、陈皮、山楂、马兰和甘草粉碎,并采用药典2号筛(24目)进行筛分,得到原料药粉末;然后按照厚朴25重量份、陈皮25重量份、山楂20重量份、马兰20重量份和甘草10重量份称取原料药粉末,混合,并加入原料药10倍量(重量比)的纯净水,制得悬浮液。
2)将悬浮液与基于悬浮液总重量0.1wt%的胰蛋白酶(50万U/g)、0.1wt%的纤维素酶(50万U/g)、0.1wt%的木聚糖酶(50万U/g)混合并进行酶解,得到酶解液;酶解条件为:pH为6.0,温度为55℃,酶解时间为3h。
3)基于所述酶解液的总重量,补加如下营养物质:酵母膏0.2wt%、硫酸铵2wt%、硫酸镁0.1wt%和磷酸二氢钾0.6wt%,灭菌后得到发酵培养基。
4)向发酵培养基接种0.5wt%的酵母菌,30℃下厌氧发酵12h;然后接种0.5wt%乳酸菌,在37℃下静置发酵28h,然后在63℃下静置发酵24h,得到培养产物。
5)发酵结束后,进行离心处理,分离得到发酵原液(发酵物)和固体残渣。
实施例5-发酵粉
将实施例4的发酵原液减压浓缩,通过减压干燥得发酵粉。所得发酵粉的含水量低于5wt%;超氧化物歧化酶(SOD)的活力大于(420±5)U/g;乳酸菌活菌数大于(3.3±0.5)×108CFU/g,总酚含量大于3wt%,总黄酮含量大于3wt%。
实施例6-发酵物
1)分别将厚朴、陈皮、山楂、马兰和甘草粉碎,并采用3号筛(50目)进行筛分,得到原料药粉末;然后按照厚朴25重量份、陈皮25重量份、山楂20重量份、马兰20重量份和甘草10重量份称取原料药粉末,混合,并加入原料药10倍量(重量比)的纯净水,制得悬浮液。
2)将悬浮液与基于悬浮液总重量0.1wt%的胰蛋白酶(50万U/g)、0.1wt%的纤维素酶(50万U/g)、0.1wt%的木聚糖酶(50万U/g)混合并进行酶解,得到酶解液;酶解条件为:pH为6.0,温度为55℃,酶解时间为3h。
3)基于所述酶解液的总重量,补加如下营养物质:酵母膏0.2wt%、硫酸铵2wt%、氯化锌0.1wt%、硫酸镁0.1wt%和磷酸二氢钾0.6wt%,灭菌后得到发酵培养基。
4)向发酵培养基接种0.5wt%的酵母菌,30℃下厌氧发酵12h;然后接种0.5wt%乳酸菌,在37℃下静置发酵28h,然后在68℃下静置发酵24h,得到培养产物。
5)发酵结束后,进行离心处理,分离得到发酵原液(发酵物)和固体残渣。
实施例7-发酵粉
将实施例6的发酵原液减压浓缩,通过减压干燥得发酵粉。所得发酵粉的含水量低于5wt%;超氧化物歧化酶(SOD)的活力大于(420±5)U/g;乳酸菌活菌数大于(3.3±0.5)×108CFU/g,总酚含量大于3wt%,总黄酮含量大于3wt%。
实验例1-药效实验
<药物的制备>
本发明(发酵粉组):称取实施例2的发酵粉4.5g(由厚朴12.5g、陈皮12.5g、山楂10g、马兰10g、甘草5g制得),加入蒸馏水定容至120ml,即得(按照小鼠灌胃体积20ml/kg计算体积)。
水提取液组:取厚朴12.5g、陈皮12.5g、山楂10g、马兰10g、甘草5g,水浸泡30min,煎煮提取2次,每次加水量10倍量,每次提取时间2小时,过滤,滤液合并,旋转蒸发浓缩,用蒸馏水定容至120ml,即得。
厚朴提取液组:取厚朴50g,水浸泡30min,煎煮提取2次,每次加水量10倍量,每次提取时间2小时,过滤,滤液合并,旋转蒸发浓缩,用蒸馏水定容至120ml,即得。
陈皮提取液组:取陈皮50g,水浸泡30min,煎煮提取2次,每次加水量10倍量,每次提取时间2小时,过滤,滤液合并,旋转蒸发浓缩,用蒸馏水定容至120ml,即得。
山楂提取液组:取山楂50g,水浸泡30min,煎煮提取2次,每次加水量10倍量,每次提取时间2小时,过滤,滤液合并,旋转蒸发浓缩,用蒸馏水定容至120ml,即得。
马兰提取液组:取马兰50g,水浸泡30min,煎煮提取2次,每次加水量10倍量,每次提取时间2小时,过滤,滤液合并,旋转蒸发浓缩,用蒸馏水定容至120ml,即得。
甘草提取液组:取甘草50g,水浸泡30min,煎煮提取2次,每次加水量10倍量,每次提取时间2小时,过滤,滤液合并,旋转蒸发浓缩,用蒸馏水定容至120ml,即得。
<实验方法>
取健康昆明种小鼠80只(实验前禁食不禁水20h),18~22g,雌雄各半,按性别、体重随机分为8组,每组10只,分别为生理盐水对照组、发酵粉组、水提取液组、厚朴提取液组、陈皮提取液组、山楂提取液组、马兰提取液组、甘草提取液组。各组每天灌胃给药一次,生理盐水对照组给生理盐水,容量为0.4ml/20g,连续5d。末次给药前小鼠每组按照0.8ml/只灌胃给予半固体糊剂(取10g羧甲基纤维素钠,溶于250ml生理盐水中,分别加入16g奶粉、8g糖、8g淀粉和2g活性炭,搅拌均匀,配制成300ml、约300g的黑色半固体糊状物;冰箱冷藏,用时恢复至室温),30min后再次给药,生理盐水对照组给予同体积生理盐水,30min后脱颈椎处死。处死后立即解剖小鼠腹腔,结扎胃贲门和幽门,取胃,用滤纸拭干后称全重,然后沿胃大弯剪开胃体,洗去胃内容物后拭干,称净重。计算胃残留率,参见表3。
胃残留率(%)=(胃全重-胃净重)/半固体糊重×100%
<实验结果>
由表3可知,各给药组与对照组相比,胃残留率均显著性减少(P<0.05)。与水提取液组、厚朴提取液组,陈皮提取液组、山楂提取液组、马兰提取液组、甘草提取液组相比,发酵粉组的胃残留率减少(P<0.01)。由此可见,发酵粉组的消食作用明显优于水提取液组以及其他单味药提取组。
表3、各组胃排空的影响结果
与对照组比较,*P<0.05**P<0.01;
与发酵粉组比较,△P<0.05,△△P<0.01。
本发明并不限于上述实施方式,在不背离本发明的实质内容的情况下,本领域技术人员可以想到的任何变形、改进、替换均落入本发明的范围。
Claims (10)
1.一种发酵物的制备方法,其特征在于,包括如下步骤:
(1)将由厚朴、陈皮、山楂、马兰和甘草组成的原料药粉碎,分别取20~40重量份厚朴、20~40重量份陈皮、15~35重量份山楂、15~35重量份马兰、5~15重量份甘草与水混合,得到悬浮液;
(2)采用胰蛋白酶、纤维素酶和木聚糖酶对所述悬浮液进行酶解,得到酶解液;
(3)将营养物质加入所述酶解液,灭菌,得到发酵培养基;
(4)向所述发酵培养基中接种酵母菌进行厌氧发酵培养8~18h,然后接种乳酸菌进行静置发酵培养25~100h,得到培养产物;
(5)将所述培养产物进行固液分离,得到所述发酵物。
2.根据权利要求1所述的制备方法,其特征在于,厚朴为25~30重量份、陈皮为25~30重量份、山楂为15~20重量份、马兰为20~25重量份、且甘草为5~10重量份。
3.根据权利要求1所述的制备方法,其特征在于,步骤(1)中,水的用量为原料药总重量的5~15倍。
4.根据权利要求1所述的制备方法,其特征在于,步骤(2)中,基于悬浮液的总重量,胰蛋白酶的用量为0.05~0.25wt%,纤维素酶的用量为0.03~0.2wt%,木聚糖酶的用量为0.05~0.25wt%。
5.根据权利要求4所述的制备方法,其特征在于,步骤(2)中,酶解的pH值为5.5~6.8,酶解温度为40~65℃,酶解时间为2~6h。
6.根据权利要求5所述的制备方法,其特征在于,步骤(3)中,所述营养物质包括0.1~0.5wt%酵母膏、1~5wt%铵盐、0.05~0.2wt%镁盐和0.3~0.8wt%磷酸盐;以上重量百分数均是基于所述酶解液的总重量。
7.根据权利要求6所述的制备方法,其特征在于,步骤(4)的厌氧发酵培养中,酵母菌的接种量为所述发酵培养基的0.1~1.0wt%,发酵温度为26~35℃,发酵时间为8~18h。
8.根据权利要求7所述的制备方法,其特征在于,步骤(4)的静置发酵培养中,乳酸菌的接种量为所述发酵培养基的0.1~1.0wt%,在35~40℃下发酵培养25~35h,然后在60~75℃下发酵培养18~35h。
9.根据权利要求1~8任一项所述的制备方法获得的发酵物。
10.根据权利要求9所述的发酵物在制备具有消食化积作用的药物、保健品或饲料添加剂中的用途。
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