WO2023006105A1 - 一种复合果蔬发酵组合物及其制备方法 - Google Patents

一种复合果蔬发酵组合物及其制备方法 Download PDF

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WO2023006105A1
WO2023006105A1 PCT/CN2022/109221 CN2022109221W WO2023006105A1 WO 2023006105 A1 WO2023006105 A1 WO 2023006105A1 CN 2022109221 W CN2022109221 W CN 2022109221W WO 2023006105 A1 WO2023006105 A1 WO 2023006105A1
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fermentation
parts
acid bacteria
vegetable
preparation
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French (fr)
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彭路
吴冬
代陈胜
侯丽丽
成丽丽
杨智明
凌远建
张大桥
何小明
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深圳市全酵源生物技术有限公司
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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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
    • 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
    • A61K36/02—Algae
    • A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus
    • 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)
    • 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)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • 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)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • 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)
    • A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
    • 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)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • A61K36/605—Morus (mulberry)
    • 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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • 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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/734—Crataegus (hawthorn)
    • 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)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • 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)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • 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)
    • A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a health care product, in particular to a compound fruit and vegetable fermented composition and a preparation method thereof.
  • HepG2 cells have the physiological functions of normal hepatocytes and the characteristics of rapid proliferation of cancer cells, so they are widely used in the study of hepatotoxicity, hepatic lipid metabolism and in vitro metabolism, and are a good model for studying hepatocyte injury from the cellular level.
  • the inert gas such as nitrogen or carbon dioxide
  • the inert gas can be filled multiple times to achieve sufficient deoxygenation, thereby changing the microbial metabolism, suspending the aerobic fermentation reaction, and facilitating better preservation of activity.
  • Nitrogen is a colorless, odorless inert gas. Nitrogen is generally used to exclude oxygen, slow down the oxidation and respiration of microorganisms, change the growth balance and metabolism of microorganisms, and suspend the aerobic fermentation reaction. Conducive to better preservation of activity.
  • Carbon dioxide (CO 2 ) is the highest oxidation state of carbon atoms, exists as a colorless and odorless gas at room temperature, and is slightly soluble in water. Carbon dioxide is a natural antimicrobial agent that inhibits the growth of microorganisms. Because the carbon dioxide molecule is a non-polar molecule, it is easy to dissolve in the lipid bilayer of the microbial cell membrane and can increase the fluidity of the membrane, thereby exposing the cytoplasm to a toxic environment. The cytoplasm of microorganisms contains water, and the hydration reaction between carbon dioxide and water produces carbonic acid, which leads to a decrease in the pH value of the cytoplasm and increases the pressure on the cells. When carbon dioxide accumulates in the cells, microorganisms regard it as metabolic waste and need to consume more metabolic energy, and at the same time replace oxygen to a minimum, and degradation reactions occur, disturbing the normal physiological balance of microorganisms.
  • Small berries refer to the general term for a class of fruit trees whose fruits are relatively small and tender and juicy. It mainly includes raspberries, blueberries, grapes, mulberries, etc. It is rich in nutritional value, rich in polyphenols, flavonoids, anthocyanins and other natural substances, has a very good antioxidant effect, and can help the liver protect human cells from free radicals and oxidative stress. Selected small berries are used as a combination of fermented products, which has the effect of relieving alcohol and protecting the liver.
  • the present invention provides a nutrient-rich compound fruit and vegetable fermentation composition, also known as phytomycin, which undergoes step-by-step submerged fermentation, and at the same time passes inert gas several times to change the fermentation mode to achieve sufficient Deoxygenation, thereby changing the microbial metabolism, suspending the aerobic fermentation reaction, which is conducive to better preservation of activity, and finally pasteurized to release more small molecular substances that are easy to absorb, and obtain the compound fruit and vegetable fermentation liquid-phytophyte.
  • the invention also provides a preparation method of the compound fruit and vegetable fermentation composition.
  • inert gas is introduced several times at the same time, the fermentation mode is changed, and sufficient deoxygenation is achieved, thereby changing the microbial metabolism, making the aerobic fermentation reaction suspended, which is conducive to better preservation of activity, and finally pasteurized
  • more small molecular substances that are conducive to absorption are released, and the compound fruit and vegetable fermentation broth-phytomycin is obtained.
  • the invention provides a compound fruit and vegetable fermentation composition, which is characterized in that it is made by fermentation of the following raw materials in parts by weight:
  • Fruits Contains pomegranate: 2 to 11 parts, hawthorn: 1 to 9 parts, grapefruit: 10 to 20 parts, grapefruit: 10 to 20 parts, tomato: 10 to 20 parts, blackberry: 1 to 10 parts, mulberry: 1 to 10 parts, red raspberry: 1 to 10 parts, black currant: 1 to 10 parts, blueberry: 1 to 10 parts;
  • Vegetables including celery: 6 to 16 servings, and at least one cruciferous vegetable: 4 to 14 servings;
  • Algae including kelp: 1 to 10 parts, spirulina: 1 to 10 parts;
  • the ratio of the raw materials (1):(2):(3) is 50 to 90 parts: 2 to 40 parts: 1 to 20 parts;
  • the fermented composition further comprises live lactic acid bacteria, acetic acid bacteria and yeast each accounting for 0.1% to 5% of the total weight of the composition.
  • the cruciferous vegetable is broccoli or/and mustard greens.
  • the compound fruit and vegetable fermentation composition is made by fermentation of the following raw materials in parts by weight:
  • Fruits including pomegranate, hawthorn, grape, grapefruit, tomato, blackberry, mulberry, red raspberry, black currant and blueberry, the ratio is 7:5:14:12:12:4:4:4:4 ;
  • Vegetables Contains celery and at least one cruciferous vegetable in a ratio of 11:9;
  • Algae including kelp and spirulina, the ratio is 5:5;
  • the ratio of the raw materials (1):(2):(3) is 70:20:10;
  • the fermented composition also comprises 3% live lactic acid bacteria, 3% acetic acid bacteria and 3% yeast.
  • the present invention also provides a method for preparing the above-mentioned composite fruit and vegetable fermentation composition, which specifically includes the following steps:
  • feed liquid the raw material fruits, vegetables, and algae are cleaned, pulverized, stirred, and brown sugar is optionally added;
  • Acetic acid bacteria fermentation add 0.1% to 5% acetic acid bacteria to the feed liquid for fermentation, after the fermentation is completed, feed sterile inert gas, seal and wait for the next step of fermentation;
  • Lactic acid bacteria fermentation Add 0.1% to 5% live lactic acid bacteria to the feed liquid for fermentation, after the fermentation is completed, feed sterile inert gas, seal and other next steps of fermentation;
  • the fermentin added is respectively 3% live lactic acid bacteria, 3% acetic acid bacteria and 3% yeast.
  • an inert gas including 99% pure sterile carbon dioxide or nitrogen is introduced, and the aeration time is determined according to the time fermentation capacity, generally From 30 minutes to 2 hours, the function of introducing inert gas is to change the fermentation mode and achieve sufficient deoxygenation, thereby changing the microbial metabolism and suspending the aerobic fermentation reaction, which is conducive to better preservation of activity.
  • each fermentation period is 1-4 weeks, preferably 2 weeks.
  • the fermented composition can be prepared in the form of solution, suspension, syrup, honey paste, dew, soft capsule, powder, hard capsule, tablet or granule.
  • the present invention also provides the use of the compound fruit and vegetable fermented composition, which contains one or more medicinal active ingredients for hangover protection and liver protection, drugs or health products for hangover protection and liver protection.
  • Fig. 1 is the SOD detection result figure of obtained product, grape freshly squeezed juice, blueberry freshly squeezed juice, red raspberry freshly squeezed juice and commercially available enzyme in embodiment case 1;
  • Fig. 2 is a graph showing the results of cell viability measured by the CCK8 method after incubation with various reagents.
  • Yeast fermentation add 3% yeast, stir well, cover with air-permeable film, and ferment at room temperature for 2 weeks, observe the fermentation every day during this period, stir once every 3 days, 30 minutes each time. After the fermentation is completed, sterile carbon dioxide with a purity of 99% is introduced for 45 minutes and sealed.
  • Acetic acid bacteria fermentation After 2 weeks of yeast fermentation, add 3% acetic acid bacteria, stir well, and ferment at room temperature for 2 weeks. During this period, observe the fermentation situation every day, and stir once every 3 days, 30 minutes each time. After the fermentation is completed, sterile carbon dioxide with a purity of 99% is introduced for 45 minutes and sealed.
  • Lactic acid bacteria fermentation After 2 weeks of acetic acid bacteria fermentation, add 3% lactic acid bacteria, stir well, feed sterile nitrogen at a flow rate of 50L/min for 30 minutes, seal, and ferment at room temperature for 2 weeks, during which the fermentation situation is observed every day. After the fermentation is completed, sterile carbon dioxide with a purity of 99% is introduced for 45 minutes and sealed.
  • Yeast fermentation add 3% yeast, stir well, cover with air-permeable film, and ferment at room temperature for 2 weeks, observe the fermentation every day during this period, stir once every 3 days, 30 minutes each time. After the fermentation is completed, sterile carbon dioxide with a purity of 99% is introduced for 45 minutes and sealed.
  • Acetic acid bacteria fermentation After 2 weeks of yeast fermentation, add 3% acetic acid bacteria, stir well, and ferment at room temperature for 2 weeks. During this period, observe the fermentation situation every day, and stir once every 3 days, 30 minutes each time. After the fermentation is completed, sterile carbon dioxide with a purity of 99% is introduced for 45 minutes and sealed.
  • Lactic acid bacteria fermentation After 2 weeks of acetic acid bacteria fermentation, add 3% lactic acid bacteria, stir well, pass in sterile nitrogen, flow rate 50L/min, time 30 minutes, seal, ferment at room temperature for 2 weeks, observe the fermentation situation every day during this period. After the fermentation is completed, sterile carbon dioxide with a purity of 99% is introduced for 45 minutes and sealed.
  • phytophytes that is, SOD in the compound fruit and vegetable fermentation composition, had high activity and antioxidative properties.
  • mice SPF grade KM mice, 60, 50 were selected for the test, and the other 10 were blank controls.
  • the mice were all male, 4-5 weeks old, purchased from the Guangdong Medical Experimental Animal Center, and the average weight of the animals was 21.3 ⁇ 1.4g at the beginning of the experiment.
  • Test grouping and design The test was divided into model group, Neptune golden bottle group, acetic acid bacteria group, Hydrodol group, and phytomycin group (the product obtained in Example 1 was 30 times the recommended dose for human use).
  • the proposed dosage of Phytomycin is 60ml/d
  • the clinical dosage of Haiwang Jinzun Tablets is 9g/d
  • the clinical dosage of Acetobacter is 60ml/d
  • the clinical dosage of Hydrodol is 4 capsules/d.
  • People are grouped according to 60kg body weight by random grouping method, and the dose design is shown in Table 1 below.
  • the incidence of drunkenness the number of drunken animals in each group/10*100%; the time from gavage with 50% ethanol to the disappearance of the righting reflex is the drunken latency; the time from the disappearance of the righting reflex to awakening is the sobering time. 6 hours are counted as 6 hours.
  • ALT and AST alanine aminotransferase (ALT) and aspartate aminotransferase (AST): 6 hours after intragastric administration of 50% ethanol, all mice were removed from the eyeballs to collect blood, centrifuged at 3000r/min for 10min to separate the serum, and the Blood biochemical analysis was used to detect the contents of serum ALT and AST. The blood of the remaining mice after the test grouping was collected, and the content of serum ALT and AST was detected, which was used as a blank control.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • phytophytin that is, the compound fruit and vegetable fermented composition, had obvious sobering effect.
  • HepG2 cells The number of HepG2 cells was adjusted to 5 ⁇ 10 4 cells/mL, inoculated in 96-well culture plates, and cultured for 24 hours, with 6 replicate wells in each group. Set different groups, as shown in the following table:
  • phytomycin has the function of protecting liver cells from alcohol damage.
  • the raw materials used are all from food materials, and the bacteria participating in the fermentation are components of the micro-ecological system of the human body. Therefore, its side effects are almost zero.
  • the inert gas is fed multiple times to change the fermentation mode to achieve sufficient deoxygenation, thereby changing the microbial metabolism, suspending the aerobic fermentation reaction, and facilitating better preservation of activity.
  • the composite fruit and vegetable composition of the present invention through submerged fermentation, after the final product is sealed and then pasteurized, can not only be stored for a long time, but also help to release a large amount of active small molecules that are good for absorption. Therefore, it has a good anti-alcoholic and liver-protecting effect.
  • the present invention provides a new compound fruit and vegetable fermentation technology.
  • the quality of the obtained product is controllable, rich in active small molecules, free of methanol, formaldehyde and butyric acid, which can relieve alcohol and protect the liver, and protect liver cells from Infringed by alcohol, industrialized production can be realized.

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Abstract

一种复合果蔬发酵组合物及其制备方法，所述组合物由包括选自多种不同的小浆果水果、芹菜和十字花科蔬菜和藻类等原料经过添加乳酸菌、醋酸菌和酵母菌深层发酵，多次通入氮气的发酵模式制成。复合果蔬发酵组合物具有解酒护肝效果。

Description

一种复合果蔬发酵组合物及其制备方法 技术领域
本发明涉及一种保健品，尤其涉及一种复合果蔬发酵组合物及其制备方法。
背景技术
中国的饮酒文化源远流长。随着物质生活水平的提高，含酒精饮料的消耗量也在增加，近年来，成人酒精中毒的发生率也相应升高。众所周知，长期饮酒可造成全身多***的损伤，尤其容易形成酒精性脂肪肝，因此解酒护肝是临床医学中一项十分重要的研究课题。目前已有很多解酒护肝类中药和保健品，证明对减少酒精损伤肝原纤维，加快乙醇分解，减少引发酒精肝等具有疗效，但仍存在效果不佳，口感单一、含有防腐剂、配方不科学、疗效不确切、工艺不合理、剂型落后等缺陷。其中，市场上一些具有解酒护肝功能的发酵类产品，质量层次不齐，存在发酵工艺简单，效果不佳，无法真正达到护肝效果。
HepG2细胞具有正常肝细胞的生理功能，又具有癌细胞增殖速度快的特点，因此被广泛应用于肝细胞毒性、肝脂质代谢和体外代谢研究，是从细胞层面研究肝细胞损伤的良好模型。
一般微生物的正常生长需要气体交换。在发酵中通过多次充入惰性气体(如氮气或者二氧化碳)，可以实现充分的脱氧，从而改变微生物代谢，使得耗氧发酵反应暂停，利于活性更好的保存。
氮气(N 2)是一种无色、无臭的惰性气体，使用氮气一般是利用它来排除氧气，减缓微生物的氧化作用和呼吸作用，改变微生物生长平衡以及代谢，使得耗氧发酵反应暂停，利于活性更好的保存。
二氧化碳(CO 2)是碳原子的最高氧化状态，常温下以无色无味的气体状态存在，微溶于水。二氧化碳是一种天然抗微生物剂，具有抑制微生物生长的作用。因为二氧化碳分子是非极性分子，易溶解在微生物细胞膜的脂质双分子层中，且能增加膜的流动性，从而使细胞质暴露在有毒的环境中。微生物的细胞质含有水分，而二氧化碳与水发生水合反应生成碳酸，导致细胞质pH值降低，使细胞所受压强增大。当二氧化碳在细胞内积累时，微生物视其为代谢废物而需消耗更多 的代谢能量，同时取代氧气最小限度，发生降解反应，扰乱微生物正常的生理平衡。
小浆果是指的果实相对较小而柔嫩多汁的一类果树总称。主要包括树莓、蓝莓、葡萄、桑葚等。其营养价值丰富，富含多酚，类黄酮，花青素等天然物质，具有非常好的抗氧化作用，能帮助肝脏保护人体细胞免受自由基和氧化应激反应的攻击。精选小浆果作为发酵物组合，具有解酒护肝效果。
发明内容
为了解决现有技术中存在的问题，本发明提供一种营养丰富的复合果蔬发酵组合物，也称植菌素，经过分步深层发酵，同时多次通入惰性气体改变发酵模式，实现充分的脱氧，从而改变微生物代谢，使得耗氧发酵反应暂停，利于活性更好的保存，最后进行巴氏灭菌处理，释放出更多利于吸收的小分子物质，得到复合果蔬发酵液-植菌素。通过体内和体外实验验证，能够达到解酒护肝的显著功效，保护肝脏细胞免受酒精侵害。本发明还提供一种复合果蔬发酵组合物制备方法。该方法是经过深层发酵后，同时多次通入惰性气体，改变发酵模式，实现充分的脱氧，从而改变微生物代谢，使得耗氧发酵反应暂停，利于活性更好的保存，最后进行巴氏灭菌处理，释放出更多利于吸收的小分子物质，得到复合果蔬发酵液-植菌素。通过体内和体外实验验证，能够达到解酒护肝的功效，保护肝脏细胞免受酒精侵害。
为了实现上述目的，本发明的技术方案为：
本发明提供一种复合果蔬发酵组合物，其特征在于，由以下重量份的原料经发酵制成：
(1)水果：包含石榴：2至11份，山楂：1至9份，葡萄：10至20份，柚子：10至20份，西红柿：10至20份，黑莓：1至10份，桑葚：1至10份，红树莓：1至10份，黑加仑：1至10份，蓝莓：1至10份；
(2)蔬菜：包含芹菜:6至16份,和至少一种十字花科蔬菜：4至14份；
(3)藻类：包含海带：1至10份，螺旋藻:1至10份；
所述原料(1)：(2)：(3)的比例为50至90份：2至40份：1至20份；
所述发酵组合物还包含各自占组合物总重量0.1％至5％的活的乳酸菌、醋酸菌以及酵母。
进一步的，所述十字花科蔬菜为西兰花或/和芥菜。
优选的，所述的复合果蔬发酵组合物，由以下重量份的原料经发酵制成：
(1)水果：包含石榴、山楂、葡萄、柚子、西红柿、黑莓、桑葚、红树莓、黑加仑和蓝莓，比例为7：5：14：12：12：4：4：4：4：4；
(2)蔬菜：包含芹菜和至少一种十字花科蔬菜，其比例为11：9；
(3)藻类：包含海带和螺旋藻，其比例为5：5；
所述原料(1)：(2)：(3)的比例为70：20：10；
所述发酵组合物还包含3％的活的乳酸菌、3％的醋酸菌以及3％的酵母。
进一步的，所述复合果蔬发酵组合物，其由选自以下的原料发酵制成：石榴、山楂、葡萄、柚子、西红柿、黑莓、桑葚、红树莓、黑加仑、蓝莓、芹菜、西兰花、海带、螺旋藻，并且所述发酵组合物包含活的乳酸菌、醋酸菌以及酵母，并且其中所述原料的重量百分比为：石榴：山楂：葡萄：柚子：西红柿：黑莓：桑葚：红树莓：黑加仑：蓝莓：芹菜：西兰花：海带：螺旋藻＝7％：5％：14％：12％：12％：4％：4％：4％：4％：4％：11％：9％：5％：5％。
本发明还提供一种制备上述复合果蔬发酵组合物的方法，具体包括以下步骤：
(1)制备料液：将所述原料水果、蔬菜、藻类清洗、粉碎，搅拌，任选地添加红糖；
(2)酵母发酵：向料液中加入0.1％至5％的酵母菌进行发酵，发酵完成后通入无菌的惰性气体，密封等下一步发酵；
(3)醋酸菌发酵：向料液中加入0.1％至5％的醋酸菌进行发酵，发酵完成后通入无菌的惰性气体，密封等下一步发酵；
(4)乳酸菌发酵：向料液中加入0.1％至5％的活的乳酸菌进行发酵，发酵完成后通入无菌的惰性气体，密封等下一步发酵；
(5)过滤，通入无菌的惰性气体，密封静置；
(6)灌装，巴氏灭菌即为成品。
优选的，所述的复合果蔬发酵组合物的制备方法，添加的发酵菌素分别为3％的活的乳酸菌、3％的醋酸菌以及3％的酵母菌。
进一步的，所述的复合果蔬发酵组合物的制备方法，制备步骤(2)-(6)，通入惰性气体包括99％纯度无菌的二氧化碳或者氮气，通气时间根据时间发酵容 量来确定，一般30min到2h，通入惰性气体的作用是改变发酵模式，实现充分的脱氧，从而改变微生物代谢，使得耗氧发酵反应暂停，利于活性更好的保存。
进一步的，所述的复合果蔬发酵组合物的制备方法，各发酵周期为1-4周，优选2周。
进一步的，依据上述方法，可以将所述发酵组合物制备成溶液剂、混悬剂、糖浆剂、蜜膏、露剂、软胶囊、散剂、硬胶囊、片剂或颗粒的形式。
本发明还提供所述复合果蔬发酵组合物的用途，其包含有一种或多种用于解酒护肝的药物活性成分，用于解酒护肝的药物或保健品。
附图说明
图1为实施案例1中所得产品、葡萄鲜榨汁、蓝莓鲜榨汁、红树莓鲜榨汁以及市售酵素的SOD检测结果图；
图2为各试剂孵育后的细胞经CCK8法测定细胞活性的结果图。
具体实施方式
制备实施例1：
原料：
石榴：山楂：葡萄：柚子：西红柿：黑莓：桑葚：红树莓：黑加仑：蓝莓：芹菜：西兰花：海带：螺旋藻＝7：5：14：12：12：4：4：4：4：4：11：9：5:5。
制法：
原料分拣，去除坏果，按上述比例洗净，用臭氧水消毒处理后，用无菌水漂洗，以去除材料上残留的臭氧，在洁净的条件下，消毒的粉碎机打碎，转入发酵桶，搅拌器60转/分钟处理2小时，按照原料总重加15％的红糖，搅拌均匀。
酵母发酵：加入3％的酵母菌，搅匀后，盖好透气膜，在常温下发酵2周，期间每天观察发酵情况，3天搅拌一次，每次30分钟。发酵完成后，通入无菌的99％纯度二氧化碳45min，密封。
醋酸菌发酵：酵母菌发酵2周后，加入3％的醋酸菌，搅匀，常温发酵2周，期间每天观察发酵情况，3天搅拌一次，每次30分钟。发酵完成后，通入无菌的99％纯度二氧化碳45min，密封。
乳酸菌发酵：醋酸菌发酵2周后，加入3％的乳酸菌，搅匀，通入无菌氮气，流 速50L/分钟，时间30分钟，密封，常温发酵2周，期间每天观察发酵情况。发酵完成后，通入无菌的99％纯度二氧化碳45min，密封。
乳酸菌发酵两周后，用400目滤网，过滤得到发酵液，发酵液中加入15％蜂蜜，通入无菌的99％纯度二氧化碳45min，密封静置；灌装，巴氏灭菌即为成品。
制备实施例2：
原料：
石榴：山楂：葡萄：柚子：西红柿：黑莓：桑葚：红树莓：黑加仑：蓝莓：芹菜：芥菜：海带：螺旋藻＝7：5：14：12：12：4：4：4：4：4：11：9：5:5。
制法：
原料分拣，去除坏果，按上述比例洗净，用臭氧水消毒处理后，用无菌水漂洗，以去除材料上残留的臭氧，在洁净的条件下，消毒的粉碎机打碎，转入发酵桶，搅拌器60转/分钟处理2小时，按照原料总重加15％的红糖，搅拌均匀。
酵母发酵：加入3％的酵母菌，搅匀后，盖好透气膜，在常温下发酵2周，期间每天观察发酵情况，3天搅拌一次，每次30分钟。发酵完成后，通入无菌的99％纯度二氧化碳45min，密封。
醋酸菌发酵：酵母菌发酵2周后，加入3％的醋酸菌，搅匀，常温发酵2周，期间每天观察发酵情况，3天搅拌一次，每次30分钟。发酵完成后，通入无菌的99％纯度二氧化碳45min，密封。
乳酸菌发酵：醋酸菌发酵2周后，加入3％的乳酸菌，搅匀，通入无菌氮气，流速50L/分钟，时间30分钟，密封，常温发酵2周，期间每天观察发酵情况。发酵完成后，通入无菌的99％纯度二氧化碳45min，密封。
乳酸菌发酵两周后，用400亩滤网，过滤得到发酵液，发酵液中加入15％蜂蜜，通入无菌的99％纯度二氧化碳45min，密封静置；灌装，巴氏灭菌即为成品。
几组实验数据：
1、抗氧化研究实验：
按照SOD检测试剂盒(南京建成)的操作规程对实施案例1中所得产品、葡萄鲜榨汁、蓝莓鲜榨汁、红树莓鲜榨汁以及市售酵素进行测定。
SOD结果如图1所示，
结果表明：植菌素，即复合果蔬发酵组合物中的SOD活性高，具有抗氧化性。
2、解酒护肝功效研究试验：
试验动物：SPF级KM小鼠，60只，试验选用50只，其他10只为空白对照。小鼠均为雄性，4-5周龄，购自广东省医学实验动物中心，实验开始时动物体重平均值为21.3±1.4g。
试验分组和设计：试验分为试验设模型组、海王金樽组、醋酸菌组、Hydrodol组、植菌素组(实施案例1中所得产品，按人用推荐剂量的30倍)。植菌素人拟用剂量为60ml/d，海王牌金樽片临床用量为9g/d，醋酸菌临床用量为60ml/d，Hydrodol临床用量为4粒/d。人按照60kg体重计，利用随机分组方法进行分组，剂量设计如下表1所示。
表1、单次给药对醉酒小鼠解酒试验动物组别及剂量设置
Figure PCTCN2022109221-appb-000001
注：给药0.5h后灌胃给予50％乙醇造模。
给药前禁食不禁水12h-16h，给药顺序先模型组、植菌素组、海王金樽组、醋酸菌组、Hydrodol组。给药30min后，各组分别以0.2ml/10g体重灌胃给予50％乙醇。灌胃给予50％乙醇后，立即观察各动物的醉酒发生率、醉酒潜伏期以及醉酒时间。小鼠灌酒后将其背向下轻轻放入鼠笼，若小鼠背向下的姿势保持30秒以上，则认为翻正反射消失，即为醉酒。醉酒发生率＝每组发生醉酒的动物数/10*100％；从灌胃给予50％乙醇至翻正反射消失的时间为醉酒潜伏时间；从翻正反射消失到苏醒的时间为醒酒时间，超过6小时的以6小时计。
丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)的含量：50％乙醇灌胃6h后，所有小鼠摘眼球取血，3000r/min离心10min分离血清，用全自动血液生 化分析检测血清ALT、AST的含量。采集试验分组后剩余小鼠血液，进行血清ALT、AST的含量检测，作为空白对照。
结果与分析如下：
1、解酒作用
小鼠灌胃给予50％乙醇后，模型组、海王金樽组、醋酸菌组、Hydrodol组、植菌素组醉酒发生率分别为100％、89％、80％、90％、56％。与模型组相比较，植菌素组潜伏期显著延长，差异具有统计学意义(p<0.01)；醋酸菌组合Hydrodol组醉酒潜伏期有所延长，但是差异不具有统计学意义。与模型组相比较，植菌素组醉酒时间明显缩短，差异具有统计学意义(p<0.01)。见表2所示。
表2、单次给药对醉酒小鼠醒酒作用
Figure PCTCN2022109221-appb-000002
组别 醉酒发生率/％ 醉酒潜伏期/min 醉酒时间/min
模型组(50％乙醇) 100(n＝9) 25.0±18.1(n＝9) 360.0±0.00(n＝9)
海王金樽组 89(n＝9) 25.3±18.0(n＝8) 360.0±0.00(n＝8)
醋酸菌组 80(n＝10) 31.3±22.9(n＝8) 360.0±0.00(n＝8)
Hydrodol组 90(n＝10) 29.9±15.3(n＝9) 360.0±0.00(n＝9)
植菌素组 56(n＝9) 99.4±60.0 **(n＝5) 239.2±104.4 **(n＝5)
注：与模型组比较，*：p<0.05，**：p<0.01。
结果表明：植菌素，即复合果蔬发酵组合物具有明显的醒酒作用。
2、对动物血清ALT、AST含量影响
与空白组相比，模型组AST含量显著升高(p<0.01)，ALT含量未见明显差异。与模型组相比，醋酸菌组合植菌素组AST含量显著下降，差异具有统计学意义(p<0.05和p<0.05)。见表3所示。
表3、单次给药对醉酒小鼠血清ALT、AST含量的影响
Figure PCTCN2022109221-appb-000003
组别 ALT(U/L) AST(U/L)
空白组 32±10(n＝9) 99±24(n＝9)
模型组(50％乙醇) 28±11(n＝9) 262±119 **(n＝9)
海王金樽组 25±7(n＝8) 178±51(n＝8)
醋酸菌组 33±16(n＝10) 140±16 *(n＝10)
Hydrodol组 29±16(n＝6) 174±45(n＝6)
植菌素组 42±10 *(n＝9) 164±29 *(n＝9)
注：与空白组比较，*：p<0.05，**：p<0.01。与模型组比较，*：p<0.05，**：p<0.01。
结果表明：植菌素具有护肝的功效。
3、对HepG2细胞酒精性损伤的保护作用。
将HepG2细胞数调整到5×10 4个/mL,接种于96孔培养板,培养24h,每组6个复孔。设置不同分组，如下表：
  乙醇 植菌素
空白 / /
模型组(乙醇组) 6％ /
低剂量对照组 / 2％
中剂量对照组 / 3％
高剂量对照组 / 4％
低剂量试验组 6％ 2％
中剂量试验组 6％ 3％
高剂量试验组 6％ 4％
剩余用无菌纯水补充。
将各试剂按照上表进行配制后，混匀反应30min，过0.22滤膜，向各孔加入10ul，孵育24h，CCK8法测定细胞活性。
结果如图2所示，注：p<0.01，
结果表明：植菌素具有保护肝脏细胞免受酒精侵害的作用。
本发明的复合果蔬组合物，所用的原料都是来自于食材，且发酵参与菌更是人体微生态***的组成部分。因此，其副作用几乎为零。
本发明的复合果蔬组合物所用制备方式中多次通入惰性气体，改变发酵模式，实现充分的脱氧，从而改变微生物代谢，使得耗氧发酵反应暂停，利于活性更好的保存。
本发明的复合果蔬组合物，经过深层发酵，最终成品密封后，经过巴氏灭菌，不仅能长期保存，而且有利于释放大量利于吸收的活性小分子。因此，其解酒护肝效果好
因此，本发明提供了一种新的复合果蔬发酵技术，得到的产品质量可控，富含活性小分子，不含甲醇、甲醛和丁酸，能够起到解酒护肝功效，保护肝脏细胞免受酒精侵害，可以实现工业化生产。

Claims (10)

  1. 一种复合果蔬发酵组合物，其特征在于，由以下重量份的原料经发酵制成：
    (1)水果：包含石榴：2至11份，山楂：1至9份，葡萄：10至20份，柚子：10至20份，西红柿：10至20份，黑莓：1至10份，桑葚：1至10份，红树莓：1至10份，黑加仑：1至10份，蓝莓：1至10份；
    (2)蔬菜：包含芹菜:6至16份,和至少一种十字花科蔬菜：4至14份；
    (3)藻类：包含海带：1至10份，螺旋藻:1至10份；
    所述原料(1)：(2)：(3)的比例为50至90份：2至40份：1至20份；
    所述发酵组合物还包含各自占组合物总重量0.1％至5％的活的乳酸菌、醋酸菌以及酵母。
  2. 根据权利要求1所述的复合果蔬发酵组合物，其特征在于，所述十字花科蔬菜为西兰花或/和芥菜。
  3. 根据权利要求1所述的复合果蔬发酵组合物，其特征在于，由以下重量份的原料经发酵制成：
    (1)水果：包含石榴、山楂、葡萄、柚子、西红柿、黑莓、桑葚、红树莓、黑加仑和蓝莓，比例为7：5：14：12：12：4：4：4：4：4；
    (2)蔬菜：包含芹菜和至少一种十字花科蔬菜，其比例为11：9；
    (3)藻类：包含海带和螺旋藻，其比例为5：5；
    所述原料(1)：(2)：(3)的比例为70：20：10；
    所述发酵组合物还包含3％的活的乳酸菌、3％的醋酸菌以及3％的酵母。
  4. 根据权利要求1所述的复合果蔬发酵组合物，其特征在于，其由选自以下的原料发酵制成：石榴、山楂、葡萄、柚子、西红柿、黑莓、桑葚、红树莓、黑加仑、蓝莓、芹菜、西兰花、海带、螺旋藻，并且所述发酵组合物包含活的乳酸菌、醋酸菌以及酵母，并且其中所述原料的重量百分比为：石榴：山楂：葡萄：柚子：西红柿：黑莓：桑葚：红树莓：黑加仑：蓝莓：芹菜：西兰花：海带：螺旋藻＝7％：5％：14％：12％：12％：4％：4％：4％：4％：4％：11％：9％：5％：5％。
  5. 一种制备权利要求1复合果蔬发酵组合物的方法，其特征在于，包括以下步骤：
    (1)制备料液：将所述原料水果、蔬菜、藻类清洗、粉碎，搅拌，任选地添加红糖；
    (2)酵母发酵：向料液中加入0.1％至5％的酵母菌进行发酵，发酵完成后通入无菌的惰性气体，密封等下一步发酵；
    (3)醋酸菌发酵：向料液中加入0.1％至5％的醋酸菌进行发酵，发酵完成后通入无菌的惰性气体，密封等下一步发酵；
    (4)乳酸菌发酵：向料液中加入0.1％至5％的活的乳酸菌进行发酵，发酵完成后通入无菌的惰性气体，密封等下一步发酵；
    (5)过滤，通入无菌的惰性气体，密封静置；
    (6)灌装，巴氏灭菌即为成品。
  6. 根据权利要求5所述的复合果蔬发酵组合物的制备方法，其特征在于，添加的发酵菌素分别为3％的活的乳酸菌、3％的醋酸菌以及3％的酵母菌。
  7. 根据权利要求6所述的复合果蔬发酵组合物的制备方法，其特征在于，制备步骤(2)-(6)，通入惰性气体包括99％纯度无菌的二氧化碳或者氮气，通气时间根据时间发酵容量来确定，一般30min到2h，通入惰性气体的作用是改变发酵模式，实现充分的脱氧，从而改变微生物代谢，使得耗氧发酵反应暂停，利于活性更好的保存。
  8. 根据权利要求6或7所述的复合果蔬发酵组合物的制备方法，其特征在于，各发酵周期为1-4周。
  9. 根据权利要求1-8所述的解酒护肝植菌素及其制备方法，其特征在于，所述发酵组合物可以制备成溶液剂、混悬剂、糖浆剂、蜜膏、露剂、软胶囊、散剂、硬胶囊、片剂或颗粒的形式。
  10. 根据权力要求1的复合果蔬发酵组合物的用途，其包含有一种或多种用于解酒护肝的药物活性成分，用于解酒护肝的药物或保健品。
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