WO2019100541A1 - 一种调理肠胃功能酵素的制备方法 - Google Patents

一种调理肠胃功能酵素的制备方法 Download PDF

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WO2019100541A1
WO2019100541A1 PCT/CN2018/000044 CN2018000044W WO2019100541A1 WO 2019100541 A1 WO2019100541 A1 WO 2019100541A1 CN 2018000044 W CN2018000044 W CN 2018000044W WO 2019100541 A1 WO2019100541 A1 WO 2019100541A1
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fermentation
parts
malt
fermentation broth
liquid
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PCT/CN2018/000044
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陈程
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陈程
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/539—Scutellaria (skullcap)

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  • the invention belongs to the technical field of health food preparation, and particularly relates to a preparation method for conditioning a gastrointestinal functional enzyme.
  • the body-derived enzymes are reduced, and the food processing causes the exogenous enzymes to be destroyed.
  • the irrational diet structure causes nutrient deficiencies, which makes the gastrointestinal function disorder.
  • the gastrointestinal microflora disorder leads to an increasing incidence of gastroenteritis and colitis and other chronic diseases of the digestive tract and blood.
  • the body is prone to enzyme imbalance or weakened effect, and the nutrition is uneven and presents a sub-health state.
  • Enzyme food can effectively adjust the body's enzyme system and nutritional balance, so that the body maintains a healthy state. Therefore, enzymes are increasingly recognized and respected as a health food. At present, it has been popular in Japan, the United States, Taiwan and other places, and the development in mainland China is relatively slow. Most of the enzyme foods are imported from the market, and the market is expensive. It is of great value for scientific research on its preparation methods.
  • the invention patent CN201210317614.3 Anshen Jianwei's longan enzyme processing method and formula are naturally fermented by processing various fruits and vegetables such as fresh fruits (especially pineapple), dried longan (or longan meat) and plant beans, and extracted from them.
  • Fruit and vegetable extract the product is rich in water-soluble fiber such as fruits, sufficient essential vitamins, amino acids, minerals and other trace elements; at the same time, the invention is designed with a comprehensive formula of diversified enzymes and probiotics.
  • the invention patent CN201610591537.9 is a multifunctional enzyme for purifying the intestines and the preparation method thereof, and the raw materials include: maca, pineapple, cherry, blackcurrant, lotus, lemon, kiwi, avocado, grapefruit, sweetsop, small ball Algae powder, red beans, mung beans, soybeans, corn, coix seed, white lentils, angelica, motherwort, mugwort, huangjing and chicory, the multi-functional enzyme produced by the invention can purify the gastrointestinal environment, remove intestinal toxins, and decompose harmful carcinogens. The substance and excess fat function to stabilize the pH of the intestines and activate the intestinal peristalsis.
  • the enzymes for regulating the stomach and intestine are obtained by fermentation of a plurality of medicinal materials combined with fruit and vegetable foods, the amount of raw materials is large, the nutrient composition is large and complicated, the pertinence is not strong, and the fermentation cycle is long because of the limitation of raw material diversity. , the loss of nutrients.
  • the present invention provides a method for preparing a gastrointestinal enzyme.
  • a preparation method for regulating gastrointestinal function enzymes is prepared by the following steps:
  • the composite bacteria I is a combination of 4 to 6% of Bacillus natto bacteria and 3 to 5% of Lactobacillus rhamnosus.
  • the composite bacteria II is a combination of 4 to 6% of Bacillus subtilis Bacillus subtilis and 2 to 4% of Aspergillus niger.
  • the precipitate obtained by the first centrifugation may be added to the double-fold amount of distilled water for washing, and then the precipitate is removed by centrifugation, and the obtained filtrate is separated from the filtrate by the first centrifugation. merge;
  • the grapefruit peel of the step (1) is dried to a moisture content of 10 to 20% and pulverized to 15 to 20 mesh.
  • the particle size affects the leaching rate of folic acid, and the folic acid can be completely and preferentially extracted and leached in 15-20 mesh.
  • the water temperature of the malt and water immersion in the step (1) is 15 to 20 ° C
  • the specific fermentation method is as follows: the mashed malt is added to the water of 1 to 1.5 times the mass and 1 to 1.2% of the mass.
  • Sodium chloride was inoculated with 3% yeast at 35 ° C for 6 h to obtain a malt fermentation broth.
  • the phytic acid in malt gradually decreases with the growth of malt, the phytic acid activity decreases at low temperature, the malt growth is inhibited, and the consumption of phytic acid is reduced.
  • the yeast fermentation changes the structure and metabolism of malt and increases the yield of phytic acid.
  • Grapefruit contains folic acid, which promotes gastrointestinal amino acid and sugar metabolism, enhances human choline synthesis, and promotes fat metabolism.
  • Folic acid also promotes the decomposition of rapeseed cake and camellia cake to release phytic acid and tea saponin; malt and rapeseed
  • the sputum is rich in phytic acid, which can promote the fermentation efficiency of the bacteria, thereby increasing the enzyme content; the phosphorus after phytic acid hydrolysis can increase the enzyme activity of the fermentation product, thereby improving the nutritional value of the enzyme; the natural bentonite can effectively adsorb the extract Protein impurities and pigments, the addition of 6g/10ml of bentonite has the greatest adsorption capacity for protein impurities and pigments, and improves the purity of malt phytic acid.
  • Tea cake contains tea saponin, which has the functions of anti-inflammatory, analgesic and stomach-strengthening.
  • tea cake and rapeseed meal are good carbon sources and nitrogen sources for microbial fermentation;
  • the bentonite described in the step (1) has a particle size of 100 to 120 mesh.
  • the composite bacteria I in the step (2) is a combination of Bacillus natto with a seed age of 12 to 14 h and Lactobacillus rhamnosus with a seed age of 14 to 16 h.
  • the baicalin in the fermentation broth of Astragalus membranaceus can increase the phytic acid dissolution rate of malt; the natto bacteria in the fermentation period will produce nattokinase, superoxide dismutase and other active enzymes during the fermentation period, which can effectively hydrolyze glycosides.
  • the glucuronic acid bond in the element converts baicalin and baicalin in Astragalus membranaceus into baicalein and wogonin; the rhamnosyl lactic acid bacteria with the age of 14-16 h have the highest ability to ferment and release rhamnolipid, which can improve
  • the bacterium's vigor enhances the metabolism of natto bacteria, thereby promoting the conversion of baicalin and baicalin to baicalein and wogonin, reducing the overall fermentation time;
  • Lactobacillus rhamnosus is one of the human flora and can improve
  • the activity of various enzymes in the reaction system and the number of natto bacteria play a role in regulating the balance of the flora and improving the environment of the organism.
  • the composite bacteria II in the step (3) is a combination of Bacillus subtilis and Aspergillus niger having an age of 12 to 14 hours.
  • the mixed bacteria fermented tea cake and rapeseed meal is better than single-fermentation fermentation.
  • the fermentation of tea cake and rapeseed meal can be carried out by using Bacillus subtilis and Aspergillus niger into the logarithmic growth phase (age 12 ⁇ 14h). It is decomposed to produce various enzymes and tea saponin and phytic acid.
  • the chitosan has a degree of deacetylation of 50 to 60% as described in the step (4).
  • Chitosan has a good enzyme adsorption capacity, can adsorb the enzyme in the fermentation broth, and increase the amount of fermentation enzyme; the adsorption of chitosan can also clarify the fermentation broth and reduce the cost of downstream engineering; the degree of deacetylation is 50 ⁇ 60% of chitosan has the best water solubility and can be effectively dissolved in the fermentation broth;
  • the invention has the beneficial effects that different enzymes and other by-products are produced by microbial fermentation of specific raw materials, thereby promoting fermentation efficiency and shortening the fermentation cycle; the active ingredients of traditional Chinese medicines and fruits and vegetables can completely retain their activities through microbial fermentation, and improve the enzymes.
  • the health care effect; the use of oil by-product cakes as a medium for fermentation and extraction of tea saponin and phytic acid promotes the growth and efficiency of the fermentation bacterium, and improves resource utilization.
  • the temperature of the water immersed in the malt is 15-20 ° C.
  • the specific fermentation method is as follows: the mashed malt is added to the water of 1 time and 1% sodium chloride, and the 3% yeast is inoculated at 35 ° C for fermentation. 6h, a malt fermentation broth was obtained.
  • the fermentation broth of Astragalus membranaceus L was obtained, and then the composite bacterium I was inoculated into the fermentation broth of Astragalus membranaceus I, and fermented at 45 ° C, 100-200 rpm for 4 days to obtain the fermentation broth of Astragalus membranaceus;
  • the composite bacterium I is a combination of Bacillus natto liquid of 4% of 12h and 12% of Lactobacillus rhamnosus of Lactobacillus rhamnosus;
  • the composite bacteria II is a combination of Bacillus subtilis Bacillus subtilis with 4% of 12h and 12% of Aspergillus niger with 12h of 12h;
  • the water temperature of the malt and water immersion is 20 ° C.
  • the specific fermentation method is as follows: the mashed malt is added to 1.5 times the mass of water and 1.2% of sodium chloride, and the 3% yeast is inoculated at 35 ° C for 6 h. A malt fermentation broth is obtained.
  • the composite bacterium I is a combination of Bacillus natto bacillus of 6% of 14 h of age and 16 h of Lactobacillus rhamnosus of Lactobacillus rhamnosus;
  • the composite bacteria II is a combination of Bacillus subtilis broth of 6% of 14h and 14% of Aspergillus niger liquid of 14h;
  • the temperature of the water immersed in the malt is 15 ° C.
  • the specific fermentation method is as follows: the mashed malt is added to the water of 1.2 times the mass and the sodium chloride of 1.1%, and the 3% yeast is inoculated at 35 ° C for 6 h. A malt fermentation broth is obtained.
  • the composite bacterium I is a combination of Bacillus natto bacillus of 5% of 13h and 4% of Lactobacillus rhamnosus of Lactobacillus rhamnosus;
  • the composite bacteria II is a combination of Bacillus subtilis broth of 5% of 13h and 13h of Aspergillus niger;
  • mice were used, weighing 18-22 g, 10 in each group. The method was to give the model drug compound diphenoxylate by oral gavage, establish a mouse constipation model, administer the ink, and determine the head of the mouse. The defecation time of the black row, the number of defecations in 5 hours and the weight of the defecation were used to reflect the defecation of the model mice.
  • Group 3 The enzyme of Example 3 is administered;
  • mice were taken twice a day, 10 ml each time, diluted with warm water and taken;
  • Group 4 model control group, the same amount of normal saline was administered
  • Group 5 A blank control group was administered with an equal amount of physiological saline.
  • mice in each group were fasted for 16 hours.
  • Group 5 blank control group was given distilled water, and groups 1 to 4 were intragastrically administered with compound diphenoxylate.
  • Groups 1 to 4 were given compound diphenoxylate for 0.5 hours.
  • Groups 1 to 5 were given to mice with ink. The animals were kept in single cages and fed with normal drinking water. The first row of each mouse was recorded from the ink filling. Black stool time, the number of black granules and weight in 5 hours. The specific results are shown in Table 1.
  • Group First blackout time Number of black granules in 5 hours Black stool weight within 5 hours (g) 1 0.80 18 2.50 ⁇ 0.51 2 0.83 16 2.20 ⁇ 0.53 3 0.78 19 2.54 ⁇ 0.43 4 4.20 5 0.88 ⁇ 0.25 5 1.05 14 2.11 ⁇ 0.44

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Abstract

一种调理肠胃功能酵素的制备方法，包括以下步骤：(1)取葡萄柚15～20份制得葡萄柚果汁和葡萄柚皮粉末；取麦芽10～20份加入酵母菌得到麦芽发酵液，将麦芽发酵液与葡萄柚果汁混匀，按总液质量的6～8％加入膨润土，调节pH至1～2，搅拌浸提1～3h后离心去除沉淀，得到提取液；(2)取黄芩根15～20份煎煮1.5～2h，离心得到黄芩提取液；按黄芩提取液的质量比加入1.0％蛋白胨，0.05％氯化钠，调节pH为7.0～8.6得到黄芩发酵液I，接种纳豆菌菌液和鼠李糖乳杆菌菌液摇床发酵4～6d，得到黄芩发酵液II；(3)取菜籽饼粕10～20份、茶饼粕10～20份混匀得到饼粕粉；将葡萄柚皮粉末、饼粕粉混匀，得到发酵液III；(4)发酵液III接种枯草芽孢杆菌菌液和黑曲霉菌液发酵6～7天后，加入提取液和黄芩发酵液II，同时加入壳聚糖溶液，混匀继续发酵6～7天后，过滤，高压灭菌，冷却后即得。

Description

一种调理肠胃功能酵素的制备方法 技术领域
本发明属于保健食品制备技术领域，特别涉及一种调理肠胃功能酵素的制备方法。
背景技术
随着生活水平的提高，生活压力增大、环境污染等因素造成人体内源性酶减少，食品精加工又使外源性酶受到破坏，不合理的饮食结构引起营养素缺乏，使得肠胃功能絮乱、肠胃微生物群混乱导致肠胃炎、结肠炎等消化道和血液慢性疾病的发生率越来越高，机体易出现酶失衡或作用减弱，营养不均衡而呈现亚健康状态。酵素食品可以有效调整机体酶系和营养平衡，使人体保持健康状态，因此，酵素作为一种养生食品越来越受到认可和推崇。目前已在日本、美国、台湾等地盛行，而在中国大陆发展相对缓慢，绝大多数酵素食品都源自进口，市场价格昂贵，对其制备方法进行科学研究具有巨大的价值。
发明专利CN201210317614.3安神健肠胃的桂圆酵素加工方法及配方，采取加工鲜水果(特别是菠萝)、桂圆干(或桂圆肉)、植物豆类等多种果蔬一起自然发酵而成，提取了其中的果蔬精华，该产品含有丰富的水果等植物水溶性纤维、充足的人体必需维生素、氨基酸、矿物质等微量元素；同时该发明设计使用多元化综合酵素、益生菌为主作用的合理配方，具有降火、润肠、通便、改善睡眠等功效，以肠胃系统为主(先养护肠道及菌相)，并辅以功能性食材，增进肠道蠕动将宿便解放出来。
发明专利CN201610591537.9一种净化肠胃的多功能酵素及其制备方法，原料包括：玛卡、凤梨、车厘子、黑加仑、莲雾、柠檬、猕猴桃、牛油果、葡萄柚、番荔枝、小球藻粉、红豆、绿豆、黄豆、玉米、薏苡仁、白扁豆、当归、益母草、艾蒿、黄精和菊苣，该发明制得的多功能酵素能净化肠胃环境，清除肠道垃圾毒素，分解有害致癌物质与多余脂肪，起到安定肠道pH值及活性化肠道蠕动的功能。
现有技术中，调理肠胃的酵素都是以多种药材结合果蔬食品进行发酵制备而得，原料用量大，营养成分多且复杂，针对性不强，同时因为原料多样性的限制导致发酵周期长，营养成分流失。
发明内容
针对现有技术制备调理肠胃酵素存在的缺陷，本发明提供一种调理肠胃酵素的制备方法。
本发明是通过以下技术方实现的：
一种调理肠胃功能酵素的制备方法，由以下步骤制备而得：
(1)按重量份数比，取葡萄柚15～20份，剥皮分离果粒和果皮，然后将果粒进行榨汁， 所得果汁中保留果肉，同时将果皮晒干并粉碎成粉末，得到葡萄柚果汁和葡萄柚皮粉末；取麦芽10～20份，加水侵泡20～24h后取出捣碎，加入酵母菌进行发酵，得到麦芽发酵液，然后将麦芽发酵液与葡萄柚果汁混合均匀，按总液质量的6～8％加入膨润土，用醋酸调节pH至1～2，室温下搅拌浸提1～3h后，离心去除膨润土沉淀，得到提取液；
(2)按重量份数比，取黄芩根15～20份，粉碎至粒度为40～50目后加入8～10倍量的蒸馏水，煎煮1.5～2h得到黄芩药液，待黄芩药液冷却至40～60℃后，于2000r/min下离心12～15分钟，取上清液，即得到黄芩提取液；按黄芩提取液的质量比加入1.0％蛋白胨，0.05％氯化钠，调节pH为7.0～8.6，高压灭菌并冷却至室温得到黄芩发酵液I，然后于黄芩发酵液I中接种复合菌I，在45～55℃、100～200r/min摇床上发酵4～6d，得到黄芩发酵液II；
按黄芩发酵液I体积比计，所述复合菌I为4～6％的纳豆菌Bacillus natto菌液和3～5％鼠李糖乳杆菌Lactobacillus rhamnosus菌液组合；
(3)按重量份数比，取菜籽饼粕10～20份、茶饼粕10～20份，粉碎至50～100目后混合均匀，得到饼粕粉；将葡萄柚皮粉末、饼粕粉混合均匀，加入10～20倍量的水，混合均匀后高压灭菌，得到发酵液III，待其冷却后，接种复合菌II于35～40℃下进行发酵；
按发酵液III体积比计，所述复合菌II为4～6％的枯草芽孢杆菌Bacillus subtilis菌液和2～4％的黑曲霉Aspergillus niger菌液组合；
(4)发酵液III发酵6～7天后，加入提取液和黄芩发酵液II，同时加入发酵液III体积比5～10％的壳聚糖溶液，混合均匀继续发酵6～7天后，将发酵液过滤，得到澄清液，将澄清液于70～80℃下高压灭菌30～50min，冷却后即可得到调理肠胃的酵素。
在步骤(1)中为了防止膨润土上吸附过多的提取物，可将第一次离心所得沉淀加入2倍量蒸馏水进行洗涤，再离心分离去除沉淀，将所得滤液与第一次离心分离所得滤液合并；
作为本发明的进一步改进，步骤(1)所述葡萄柚果皮晒干至含水量为10～20％，粉碎至15～20目。
粒度影响叶酸的浸出率，在15～20目叶酸能完整并最大化被提取浸出。
作为本发明的进一步改进，步骤(1)所述麦芽加水浸泡的水温为15～20℃，具体发酵方法如下：将捣碎后的麦芽加入其质量1～1.5倍的水和1～1.2％的氯化钠，接种3％的酵母菌于35℃下发酵6h，得到麦芽发酵液。
麦芽中的植酸随着麦芽的生长会逐渐减少，在低温下植酸活性降低，麦芽生长受到抑制，减少植酸的消耗；酵母发酵改变麦芽的结构和代谢，提高了植酸的产率。
葡萄柚中含有叶酸成分，促进肠胃氨基酸和糖代谢，提高人体胆碱合成，从而促进脂肪 代谢，叶酸还能促进菜籽饼粕和油茶饼粕分解释放植酸和茶皂素；麦芽和菜籽粕中含有丰富的植酸，能促进菌体发酵效率，从而提高酵素含量；植酸水解后的磷能提高发酵产物酶的活力，从而提高酵素的营养价值；天然膨润土能有效吸附提取液中的蛋白质杂质和色素，投加量6g/10ml的膨润土对蛋白质杂质和色素的吸附能力最大，提高了麦芽植酸的纯度。
茶饼粕中含有茶皂素，具有消炎镇痛，健胃等功效，同时茶饼粕和菜籽粕是微生物发酵的良好碳源和氮源；
作为本发明的进一步改进，步骤(1)所述的膨润土粒度为100～120目。
作为本发明的进一步改进，步骤(2)所述复合菌I是由种龄为12～14h的纳豆菌Bacillus natto和种龄为14～16h鼠李糖乳杆菌Lactobacillus rhamnosus组合。
黄芩发酵液中的黄芩素能提高麦芽植酸溶出率；种龄为12～14h纳豆菌在发酵过程中会生成纳豆激酶、超氧化物歧化酶等多种活性酶，能有效地水解苷元中的葡萄糖醛酸键，将黄芩中的黄芩苷和汉黄芩苷转化成黄芩素和汉黄芩素；种龄为14～16h的鼠李糖乳酸菌发酵分泌鼠李糖脂能力最大，能提高纳豆菌的菌活力，使纳豆菌的代谢增强，从而促进黄芩苷和汉黄芩苷转化成黄芩素和汉黄芩素，减少总体发酵时间；鼠李糖乳杆菌是人体菌群之一，能提高反应体系中各种酶的活性和纳豆菌菌的数量，起到调节菌群的平衡，改善机体环境的作用。
作为本发明的进一步改进，步骤(3)所述复合菌II为种龄为12～14h的枯草芽孢杆菌Bacillus subtilis和的黑曲霉Aspergillus niger组合。
混合菌发酵茶饼粕和菜籽粕效果优于单菌发酵，利用进入对数生长期(种龄12～14h)的枯草芽孢杆菌和黑曲霉对茶饼粕和菜籽粕进行发酵，能最大化对其进行分解，产生各种酶和茶皂素与植酸。
作为本发明的进一步改进，步骤(4)所述的壳聚糖脱乙酰度为50～60％。
壳聚糖具有很好的酶吸附力，能将发酵液中的酶进行吸附，提高发酵酶量；壳聚糖的吸附作用还能使发酵液澄清，降低下游工程的成本；脱乙酰度50～60％的壳聚糖水溶性最好，能有效的溶解于发酵液中；
本发明的有益效果：通过微生物发酵特定原料产生不同的酶和其他副产物，促进了发酵效率，缩短了发酵周期；中药和果蔬的中有效成分通过微生物发酵能完整的保留其活性，提高了酵素的保健效用；利用榨油副产物饼粕作为培养基发酵提取茶皂素和植酸促进体系发酵菌的生长和效率，提高了资源利用率。
具体实施方式
下面结合实施例对本发明进一步说明。
实施例1
(1)按重量份数比，取葡萄柚15份，剥皮分离果粒和果皮，然后将果粒进行榨汁，所得果汁中保留果肉，同时将果皮晒干至含水量为10％，并粉碎成15目的粉末，得到葡萄柚果汁和葡萄柚皮粉末；取麦芽10份，加水侵泡20h后取出捣碎，加入酵母菌进行发酵，得到麦芽发酵液，然后将麦芽发酵液与葡萄柚果汁混合均匀，按总液质量的6％加入粒度为100目的膨润土，用醋酸调节pH至1～2，室温下搅拌浸提1h后，离心去除膨润土沉淀，得到提取液；
所述麦芽加水浸泡的水温为15～20℃，具体发酵方法如下：将捣碎后的麦芽加入其质量1倍的水和1％的氯化钠，接种3％的酵母菌于35℃下发酵6h，得到麦芽发酵液。
(2)按重量份数比，取黄芩根15份，粉碎至40目后加入8倍量的蒸馏水，煎煮1.5h得到黄芩药液，待黄芩药液冷却至40℃后，于2000r/min下离心12～15分钟，取上清液，即得到黄芩提取液；按黄芩提取液的质量比加入1.0％蛋白胨，0.05％氯化钠，调节pH为7.0～8.6，高压灭菌并冷却至室温得到黄芩发酵液I，然后于黄芩发酵液I中接种复合菌I，在45℃、100～200r/min摇床上发酵4d，得到黄芩发酵液II；
按黄芩发酵液I体积比计，所述复合菌I为4％种龄为12h的纳豆菌Bacillus natto菌液和3％种龄为14h鼠李糖乳杆菌Lactobacillus rhamnosus菌液组合；
(3)按重量份数比，取菜籽饼粕10份、茶饼粕10份，粉碎至50目后混合均匀，得到饼粕粉；将葡萄柚皮粉末、饼粕粉混合均匀，加入10倍量的水，混合均匀后高压灭菌，得到发酵液III，待其冷却后，接种复合菌II于35℃下进行发酵；
按发酵液III体积比计，所述复合菌II为4％种龄为12h的枯草芽孢杆菌Bacillus subtilis菌液和2～4％种龄为12h的黑曲霉Aspergillus niger菌液组合；
(4)发酵液III发酵6天后，加入提取液和黄芩发酵液II，同时加入发酵液III体积比5％脱乙酰度为50％的壳聚糖溶液，混合均匀继续发酵7天后，将发酵液过滤，得到澄清液，将澄清液于70℃下高压灭菌30min，冷却后即可得到调理肠胃的酵素。
实施例2
(1)按重量份数比，取葡萄柚20份，剥皮分离果粒和果皮，然后将果粒进行榨汁，所得果汁中保留果肉，同时将果皮晒干至含水量为20％，并粉碎成20目的粉末，得到葡萄柚果汁和葡萄柚皮粉末；取麦芽20份，加水侵泡24h后取出捣碎，加入酵母菌进行发酵，得到麦芽发酵液，然后将麦芽发酵液与葡萄柚果汁混合均匀，按总液质量的8％加入粒度为120目的膨润土，用醋酸调节pH至1～2，室温下搅拌浸提3h后，离心去除膨润土沉淀，得到提取液；
所述麦芽加水浸泡的水温为20℃，具体发酵方法如下：将捣碎后的麦芽加入其质量1.5 倍的水和1.2％的氯化钠，接种3％的酵母菌于35℃下发酵6h，得到麦芽发酵液。
(2)按重量份数比，取黄芩根20份，粉碎至50目后加入10倍量的蒸馏水，煎煮2h得到黄芩药液，待黄芩药液冷却至60℃后，于2000r/min下离心15分钟，取上清液，即得到黄芩提取液；按黄芩提取液的质量比加入1.0％蛋白胨，0.05％氯化钠，调节pH为8.6，高压灭菌并冷却至室温得到黄芩发酵液I，然后于黄芩发酵液I中接种复合菌I，在55℃、100～200r/min摇床上发酵4～6d，得到黄芩发酵液II；
按黄芩发酵液I体积比计，所述复合菌I为6％种龄为14h的纳豆菌Bacillus natto菌液和5％种龄为16h鼠李糖乳杆菌Lactobacillus rhamnosus菌液组合；
(3)按重量份数比，取菜籽饼粕20份、茶饼粕20份，粉碎至100目后混合均匀，得到饼粕粉；将葡萄柚皮粉末、饼粕粉混合均匀，加入20倍量的水，混合均匀后高压灭菌，得到发酵液III，待其冷却后，接种复合菌II于40℃下进行发酵；
按发酵液III体积比计，所述复合菌II为6％种龄为14h的枯草芽孢杆菌Bacillus subtilis菌液和4％种龄为14h的黑曲霉Aspergillus niger菌液组合；
(4)发酵液III发酵7天后，加入提取液和黄芩发酵液II，同时加入发酵液III体积比10％脱乙酰度为60％的壳聚糖溶液，混合均匀继续发酵7天后，将发酵液过滤，得到澄清液，将澄清液于80℃下高压灭菌50min，冷却后即可得到调理肠胃的酵素。
实施例3
(1)按重量份数比，取葡萄柚18份，剥皮分离果粒和果皮，然后将果粒进行榨汁，所得果汁中保留果肉，同时将果皮晒干至含水量为15％，并粉碎成20目的粉末，得到葡萄柚果汁和葡萄柚皮粉末；取麦芽15份，加水侵泡20h后取出捣碎，加入酵母菌进行发酵，得到麦芽发酵液，然后将麦芽发酵液与葡萄柚果汁混合均匀，按总液质量的7％加入粒度为110目的膨润土，用醋酸调节pH至1～2，室温下搅拌浸提2h后，离心去除膨润土沉淀，得到提取液；
所述麦芽加水浸泡的水温为15℃，具体发酵方法如下：将捣碎后的麦芽加入其质量1.2倍的水和1.1％的氯化钠，接种3％的酵母菌于35℃下发酵6h，得到麦芽发酵液。
(2)按重量份数比，取黄芩根18份，粉碎至15目后加入9倍量的蒸馏水，煎煮2h得到黄芩药液，待黄芩药液冷却至50℃后，于2000r/min下离心13分钟，取上清液，即得到黄芩提取液；按黄芩提取液的质量比加入1.0％蛋白胨，0.05％氯化钠，调节pH为7.6，高压灭菌并冷却至室温得到黄芩发酵液I，然后于黄芩发酵液I中接种复合菌I，在50℃、100～200r/min摇床上发酵5d，得到黄芩发酵液II；
按黄芩发酵液I体积比计，所述复合菌I为5％种龄为13h的纳豆菌Bacillus natto菌液 和4％种龄为15h鼠李糖乳杆菌Lactobacillus rhamnosus菌液组合；
(3)按重量份数比，取菜籽饼粕15份、茶饼粕16份，粉碎至80目后混合均匀，得到饼粕粉；将葡萄柚皮粉末、饼粕粉混合均匀，加入15倍量的水，混合均匀后高压灭菌，得到发酵液III，待其冷却后，接种复合菌II于37℃下进行发酵；
按发酵液III体积比计，所述复合菌II为5％种龄为13h的枯草芽孢杆菌Bacillus subtilis菌液和3％种龄为13h的黑曲霉Aspergillus niger菌液组合；
(4)发酵液III发酵6天后，加入提取液和黄芩发酵液II，同时加入发酵液III体积比7％脱乙酰度为55％的壳聚糖溶液，混合均匀继续发酵7天后，将发酵液过滤，得到澄清液，将澄清液于75℃下高压灭菌40min，冷却后即可得到调理肠胃的酵素。
为保证本发明制备的酵素安全性，通过如下实验对其进行评价：
1、模拟胃液及肠液的耐受试验
取100g/L的盐酸16.4mL加蒸馏水稀释，使pH值分别为1.5、2.5和3.5，取100mL稀盐酸溶液，分别加入1g胃蛋白酶，使其充分溶解，得模拟胃液，微孔滤膜除菌(0.22μm)备用。取磷酸二氢钾6.8g，加水500mL使溶解，用0.1moL/L氢氧化钠溶液调节pH值至6.8；另取胰蛋白酶10g，加水100mL使溶解，将两液混合后，加水稀释至1000ml，得模拟肠液，微孔滤膜除菌(0.22μm)备用。
分别取1mL实施例1～3制备的酵素溶液加入到9mL的模拟胃液中(即十倍逐级稀释)，并迅速在振荡器上充分混匀，然后置于30～45℃静置培养2～4h。分别在1h、2h、3h、4h的时候取出培养液并立即计数残存活菌数，与原活菌数进行比较，结果表明，活菌存活率分别为98.6％和98.9％。然后取在人工胃液中消化不同时间的培养液各1mL，分别接种于9mL pH值为6.8的人工肠液中，置于30～45℃静置培养2～4h，并分别在0、3、6、24h取样，测定其活菌数，与原活菌数进行比较，结果表明活菌存活率均为99％。
2、依据《保健食品检与评价技术规范》的通便功能检测方法对实施例1～3获得的酵素进行评价，具体如下：
试验方法：
1.1 实验动物选用成年雌性小鼠，体重18～22g，每组10只；方法为经口灌胃给予造模药物复方地芬诺酯，建立小鼠便秘模型，灌胃墨汁，测定小鼠的首粒排黑便排便时间、5小时内排便粒数和排便重量，来反映模型小鼠的排便情况。
1.2 分组情况为：
组1：灌胃实施例1的酵素；
组2：灌胃实施例2的酵素；
组3：灌胃实施例3的酵素；
上述组别小鼠每日服用两次，每次10ml，用温开水稀释后服用；
组4：模型对照组，灌胃等量的生理盐水；
组5：空白对照组，灌胃等量的生理盐水。
1.3 模型建立
给受试样品7天后，各组小鼠禁食不禁水16小时。
组5空白对照组给蒸馏水，组1～4灌胃给予复方地芬诺酯。
1.4 指标测定的具体方法
组1～4给复方地芬诺酯0.5小时后，组1～5均给小鼠用墨汁灌胃，动物均单笼饲养，正常饮水进食，从灌墨汁开始，记录每只小鼠首粒排黑便时间，5小时内排黑便粒数及重量。具体结果如表1。
表1 各组小鼠排便结果
组别 首粒排黑便时间(h) 5小时内排黑便粒数 5小时内排黑便重量(g)
1 0.80 18 2.50±0.51
2 0.83 16 2.20±0.53
3 0.78 19 2.54±0.43
4 4.20 5 0.88±0.25
5 1.05 14 2.11±0.44
由上述结果可知，由本发明实施例1～3所得的酵素润肠通便效果较为显著，甚至优于组5空白对照组。
尽管已经描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。

Claims (7)

  1. 一种调理肠胃功能酵素的制备方法，其特征在于，包括以下步骤：
    (1)按重量份数比，取葡萄柚15～20份，剥皮分离果粒和果皮，然后将果粒进行榨汁，所得果汁中保留果肉，同时将果皮晒干并粉碎成粉末，得到葡萄柚果汁和葡萄柚皮粉末；取麦芽10～20份，加水侵泡20～24h后取出捣碎，加入酵母菌进行发酵，得到麦芽发酵液，然后将麦芽发酵液与葡萄柚果汁混合均匀，按总液质量的6～8％加入膨润土，用醋酸调节pH至1～2，室温下搅拌浸提1～3h后，离心去除膨润土沉淀，得到提取液；
    (2)按重量份数比，取黄芩根15～20份，粉碎至粒度为40～50目后加入8～10倍量的蒸馏水，煎煮1.5～2h得到黄芩药液，待黄芩药液冷却至40～60℃后，于2000r/min下离心12～15分钟，取上清液，即得到黄芩提取液；按黄芩提取液的质量比加入1.0％蛋白胨，0.05％氯化钠，调节pH为7.0～8.6，高压灭菌并冷却至室温得到黄芩发酵液I，然后于黄芩发酵液I中接种复合菌I，在45～55℃、100～200r/min摇床上发酵4～6d，得到黄芩发酵液II；
    按黄芩发酵液I体积比计，所述复合菌I为4～6％的纳豆菌Bacillus natto菌液和3～5％鼠李糖乳杆菌Lactobacillus rhamnosus菌液组合；
    (3)按重量份数比，取菜籽饼粕10～20份、茶饼粕10～20份，粉碎至50～100目后混合均匀，得到饼粕粉；将葡萄柚皮粉末、饼粕粉混合均匀，加入10～20倍量的水，混合均匀后高压灭菌，得到发酵液III，待其冷却后，接种复合菌II于35～40℃下进行发酵；
    按发酵液III体积比计，所述复合菌II为4～6％的枯草芽孢杆菌Bacillus subtilis菌液和2～4％的黑曲霉Aspergillus niger菌液组合；
    (4)发酵液III发酵6～7天后，加入提取液和黄芩发酵液II，同时加入发酵液III体积比5～10％的壳聚糖溶液，混合均匀继续发酵6～7天后，将发酵液过滤，得到澄清液，将澄清液于70～80℃下高压灭菌30～50min，冷却后即可得到调理肠胃的酵素。
  2. 根据权利要求1所述的一种调理肠胃功能酵素的制备方法，其特征在于，步骤(1)所述葡萄柚果皮晒干至含水量为10～20％，粉碎至15～20目。
  3. 根据权利要求1所述的一种调理肠胃功能酵素的制备方法，其特征在于，步骤(1)所述麦芽加水浸泡的水温为15～20℃，具体发酵方法如下：将捣碎后的麦芽加入其质量1～1.5倍的水和1～1.2％的氯化钠，接种3％的酵母菌于35℃下发酵6h，得到麦芽发酵液。
  4. 根据权利要求1所述的一种调理肠胃功能酵素的制备方法，其特征在于，步骤(1)所述的膨润土粒度为100～120目。
  5. 根据权利要求1所述的一种调理肠胃功能酵素的制备方法，其特征在于，步骤(2)所述复合菌I是由种龄为12～14h的纳豆菌Bacillus natto和种龄为14～16h鼠李糖乳杆菌Lactobacillus rhamnosus组合。
  6. 根据权利要求1所述的一种调理肠胃功能酵素的制备方法，其特征在于，步骤(3)所述复合菌II为种龄为12～14h的枯草芽孢杆菌Bacillus subtilis和的黑曲霉Aspergillus niger组合。
  7. 根据权利要求1所述的一种调理肠胃功能酵素的制备方法，其特征在于，步骤(4)所述的壳聚糖脱乙酰度为50～60％。
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