WO2017186144A1 - 植物药的微生物发酵 - Google Patents

植物药的微生物发酵 Download PDF

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WO2017186144A1
WO2017186144A1 PCT/CN2017/082207 CN2017082207W WO2017186144A1 WO 2017186144 A1 WO2017186144 A1 WO 2017186144A1 CN 2017082207 W CN2017082207 W CN 2017082207W WO 2017186144 A1 WO2017186144 A1 WO 2017186144A1
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Prior art keywords
lactobacillus
fermentation
fungus
acetobacter
yeast
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PCT/CN2017/082207
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English (en)
French (fr)
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刘东波
王蕾
康信聪
张思
赖锡湖
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刘东波
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Priority to JP2019507981A priority Critical patent/JP2019514424A/ja
Priority to EP17788796.5A priority patent/EP3444353B1/en
Priority to US16/096,059 priority patent/US11414682B2/en
Priority to KR1020187031678A priority patent/KR102541968B1/ko
Publication of WO2017186144A1 publication Critical patent/WO2017186144A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • 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
    • 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
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • A61K36/06—Fungi, e.g. yeasts
    • 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
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • 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
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/066—Clavicipitaceae
    • A61K36/068—Cordyceps
    • 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
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/074—Ganoderma
    • 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)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/488—Pueraria (kudzu)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously

Definitions

  • the present invention relates to the field of botanical drug fermentation, and more particularly to a method for directional fermentation of botanicals by microorganisms, promotion of release of botanical active ingredients, and/or increase of botanical efficacy.
  • the present application provides a safe and efficient method of promoting the release of active ingredients of botanicals.
  • the method first ferments the botanical drug by the broken wall fungus, and then ferments the botanical drug one or more times by the probiotic bacteria.
  • the method of the present application can effectively destroy the cell wall of the botanical drug, promote the release of the active ingredient of the botanical drug, and improve the utilization rate of the botanical drug.
  • the term "botanical medicine” as used herein mainly refers to a material of a stem, a leaf, a root, and/or a fruit of a plant, which is a medicinal material of a Chinese medicine.
  • the botanical drug herein is a botanical drug listed in the Chinese Pharmacopoeia or other national pharmacopoeia.
  • the botanical drug herein is ginseng or puerarin. It is known that ginseng belongs to the class of dicotyledon, Umbelliferae, Araliaceae, and Panax ginseng; Pueraria lobata belongs to Magnolia, Rosaceae, Leguminosae, and Genus.
  • ginseng and puerarin are very different species.
  • the application of the method of the present application on ginseng and puerarin indicates that the method of the present application has a wide application range and can be applied to a plurality of different botanical drugs.
  • broken wall fungus refers to an edible fungus that has a wall-breaking effect on a hard surface structure such as a plant cell wall or an insect body wall.
  • the wall breaking effect mentioned here does not require that the wall be completely broken. As long as a hole is formed in the wall, the contents are more easily released outward.
  • broken wall fungus include medicinal fungi, such as the multicellular fungi listed in the National Pharmacopoeia.
  • the disrupted fungus is a large fungus capable of producing a fruiting body visible to the naked eye.
  • the main components of plant cell walls are known to be cellulose, hemicellulose and pectin. Accordingly, edible fungi capable of producing cellulase, hemicellulase, and/or pectinase are capable of causing the wall-breaking effect described herein on plant cell walls and are therefore all of the wall-breaking fungi described herein.
  • wood fungi are the wall-breaking fungi described herein, and specific examples are: Ganoderma, Poria cocos, Grifola umbellata, and the like.
  • insects and their larvae In addition to the hard surface of plant cells such as trees, insects and their larvae also have hard cellulose and chitin in the body wall. Some fungi can infect insects (such as Lepidoptera) larvae or pupa, causing insects that are going through the larval ⁇ ⁇ ⁇ adult growth stage to terminate at the stage of larvae or pupa, which uses insect larvae or sputum bodies for nutrition. Carry out growth. Cordyceps is such a fungus. In this context, "C. cordyceps" refers to Cordyceps fungi in a broad sense, which parasitize insects, turn the worm into a hyphae filled with hyphae, and produce a stem-like or stick-like child from the front end of the worm.
  • Cordyceps fungi have been reported in more than 500 species, the most widely known of which include Cordyceps sinensis, Cordyceps militaris and the like. Cordyceps can break through the body wall of insects, indicating that it can produce corresponding wall-breaking substances, such as cellulase, chitinase, etc., and therefore belong to the broken-wall fungus described herein.
  • the wall-breaking fungi described herein include woody fungi and cordyceps.
  • the disrupted fungus described herein is selected from the group consisting of Ganoderma lucidum, Lycium barbarum, Poria sinensis, Cordyceps sinensis, and/or Cordyceps militaris.
  • “fermentation” refers to a process in which a microorganism is inoculated in a plant or a fruit thereof, a stem or the like, and the like. Due to the life activities of microorganisms, carbohydrates can be converted into substances such as alcohols and organic acids, and components (such as nutrients) contained in plants can be changed, for example, they are more easily released, more active, or become more beneficial to the human body. The form of absorption.
  • fertilizers refers to a mixture of various biologically active substances obtained by fermenting a probiotic by a plant, which comprises: a fermentation-involving bacteria, an ingredient for fermentation, an active ingredient of a fermentation-involved bacteria or a foodstuff,
  • a variety of enzymes also include ingredients that are modified by fermentation, such as ingredients that are more active or more soluble, and the like.
  • probiotics refers to microorganisms that are beneficial to health in the human body. Probiotics are commonly used to make foods such as yogurt and enzymes. Commonly used probiotics include yeast, yeast, Lactobacillus, Bifidobacterium, Leuconostoc, Streptococcus, Lactococcus, Acetobacterium, etc. Wait.
  • the yeast such as Saccharomyces cerevisiae and Saccharomyes eerevisiae, Saccharomyces, Pichia pastoris, Pichia ohmeri, membrane Yeast of the genus Hansenula, such as the yeast of Pichia, such as Pichia membranaefaciens, and Hansenula anomala.
  • the lactobacillus for example, Lactobacillus buchneri, Lactobacillus panis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermenti, saliva milk Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus veridescens, Lactobacillus delbrueckii bulgaricus, Lactobacillus rhamnosus, cellobiose Lactobacillus cellobiosus, Lactobacillus casei subsp casei, Lactobacillus helveticus subsp. jugurti, Lacticum Jansen, Lactobacillus Pentosus), and so on.
  • the bifidobacteria are, for example, Bifidobacterium adolescents, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, and the like.
  • the Acetobacter such as Acetobacter pasteuranus, Acetobacter rancens, and the like.
  • Other probiotics include: Leuconostoc mesenteroides aureus, Streptococcus salivarius subsp. thermophilus, Lactococcus lactis subsp. Cremoris, and the like.
  • the Applicant has found that different botanical drugs are fermented by broken-wall fungi and fermented by probiotics, which can greatly improve the content of botanical active ingredients in the liquid medicine compared with traditional decoction soups, thereby improving the traditional Chinese medicine administration method. And the low efficacy of existing Chinese medicine cultivation methods.
  • the botanical fermented product obtained by the method of the present application comprises a botanical active ingredient and various active enzymes or active substances derived from a broken-wall fungus and a probiotic, and is a nutrient-rich preparation. Such a preparation can be used to condition human health.
  • the present application also relates to a fermentation method which combines the fermentation of a broken-wall fungus with the fermentation of a probiotic.
  • the fermentation method may be a fermentation method of a botanical drug.
  • the present application involves the first fermentation of a broken-wall fungus followed by the fermentation of a probiotic.
  • the probiotic fermentation of the present application is carried out using yeast, lactobacilli, and Acetobacter.
  • the probiotic fermentation of the present application is carried out in a stepwise manner, for example, by performing a mixed fermentation of yeast and lactobacillus, followed by fermentation of Acetobacter. Both the fermentation of the broken-wall fungus and the fermentation of the probiotic bacteria are carried out by using culture conditions such as the applicable temperature and pH of the respective strains, and the specific conditions can be confirmed by those skilled in the art according to the routine.
  • the culture medium of the broken wall fungus such as Ganoderma lucidum, Cordyceps, etc. is also common in the art.
  • the composition of one medium is as follows (by mass ratio): glucose 1 to 6%, yeast extract 0.1% to 0.4%, peptone 0.1 % to 0.8%, potassium dihydrogen phosphate 0.05% to 0.3%, magnesium sulfate 0.05% to 0.3%, supplemented with water.
  • Glucose as a carbon source can be replaced with potato juice, and other ingredients can be adjusted, and these adjustments are easily made by those skilled in the art.
  • the disrupted fungus that can share the same or similar culture conditions can also be cultured together in the same medium, optionally further probiotic fermentation, such that the final product contains more than one type of broken fungus And / or its active ingredients.
  • the fermentation of the broken-wall fungus may be: 50 to 250 rpm, 7 to 30 ° C, and stirred for 3 to 45 days. These conditions are readily determined by those skilled in the art based on the growth requirements of specific species of the broken wall fungus.
  • each of the probiotics of the present application is selected from a bacterial liquid having a million viable counts and is inoculated in an amount of 1 to 3 Torr of the total raw material.
  • the fermentation process of the present application does not involve the step of extracting the active ingredient of the broken-wall fungus, that is, the method does not require extraction of the large fungal active ingredient before or after the fermentation of the broken-wall fungus, but the fermentation of the broken-wall fungus and the probiotic
  • the fermentation can be carried out sequentially in the same culture system or fermentation unit.
  • the method of the present application includes the following steps:
  • the probiotic bacteria are connected, sealed, and cultured in the environment of 10-40 ° C for more than 48 hours to complete the second fermentation;
  • the invention has the advantages that the preliminary fermentation of the botanical drug by the broken-wall fungus, and the subsequent propagation of the probiotic bacteria to the botanical drug again (or even more) can effectively destroy the cell wall of the botanical drug and promote the effective botanical drug.
  • the release of ingredients improves the utilization of botanicals.
  • the botanical drug solution also contains the active ingredient of the broken-wall fungus, thereby increasing the efficacy of the botanical drug.
  • a method for microbial fermentation of a botanical drug comprising first fermenting a broken-wall fungus, and then fermenting the probiotic.
  • any one of items 1 to 2 wherein the wall-breaking fungus is selected from the group consisting of ganoderma lucidum, pig mites, cockroaches, cordyceps sinensis, and/or cordyceps militaris.
  • probiotic selected from one or more of the group consisting of: sugar yeast, Pichia pastoris, Hansenula, lactobacillus, bifidobacteria, Leuconostoc, streptococcus, Lactococcus, Acetobacter.
  • probiotic selected from one or more of the following: Saccharomyces cerevisiae, elliptic yeast, Pichia pastoris, Pichia pipiens, Pichia pastoris, Hansenula Lactobacillus brevis, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus glabrata, Lactobacillus delbrueckii subsp.
  • Lactobacillus rhamnosus fiber Lactobacillus disaccharide, Lactobacillus casei subsp., Lactobacillus helveticus, G. johnsonii, Lactobacillus pentosus), Acetobacter baumannii, Acetobacter odorum, Bifidobacterium adolescentis, Bifurcation bifurcation Bacillus, Bifidobacterium infantis, Bifidobacterium longum, Streptococcus faecalis, S. salivarius subsp. thermophilus, and Lactococcus lactis subsp.
  • Acetobacter (Acetobacter odoratum AS.1.41, purchased From the brewing plant in Shanghai, the number of million live bacteria), static culture at 30 ° C for 7 days, filtration, collection of liquid.
  • ginseng application processing liquid wherein the total ginsenoside content of 1.06mg / mL (see the measurement method, the measurement of total ginsenoside content, high Li Ping et al., Journal of Zhejiang College of Engineering, 2012,31 (3): 382-388), which is 7.01 times higher than the traditional decoction (ginseng: water 1:8 weight ratio); the ganoderma lucidum triterpene content is also detected in the liquid solution is 0.75 mg/mL (for the measurement method, see the People's Republic of China) Pharmacopoeia 2015 edition). All data are the average of triplicate samples.
  • the ginseng solution treated by the present invention was determined, wherein the total ginsenoside content reached 1.03 mg/mL (the measurement method was the same as in Example 1), which was 6.81 higher than the traditional decoction (ginseng: water: 1:8 weight ratio).
  • the ratio of Ganoderma lucidum triterpene was also determined to be 0.64 mg/mL in the drug solution (the measurement method is the same as in Example 1). All data are the average of triplicate samples.
  • the puerarin solution treated by the invention is determined, wherein the puerarin content reaches 0.119 mg/mL (for the determination method, the determination of puerarin in the health food of Chinese national standard GB/T 22251-2008), compared with the traditional decoction (puera) : Water is 1:10 by weight) increased by 1.25 times.
  • the cordycepin content was also detected in the drug solution at 11.3 ⁇ g/mL (for the method of assay, the screening of high-yield strains of cordycepin and the effect of different additives on the yield of cordycepin, Wang Lei et al., Journal of Fungal Sciences , 2012, 31 ( 3): 382-388). All data are the average of triplicate samples.
  • the puerarin solution treated by the present invention was determined to have a puerarin content of 0.113 mg/mL, which was 1.19 times higher than that of the conventional decoction (pusen: water: 1:10 weight ratio) (the measurement method is the same as in the third embodiment). .
  • the cordycepin content was also detected in the drug solution to be 13.7 ⁇ g/mL (the measurement method was the same as in Example 3). All data are the average of triplicate samples.

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Abstract

一种植物药的微生物发酵方法，包括先进行破壁真菌的发酵再进行益生菌发酵。

Description

植物药的微生物发酵 技术领域
本发明涉及植物药发酵领域，更具体地说，是涉及利用微生物定向发酵植物药，促进植物药有效成分释放和/或增加植物药药效的方法。
背景技术
天然植物药，尤其是中药，在保健和治疗领域的很多方面有化学方法制备的药物不具备的优势，应用越来越广泛，但其有效成分的利用率一直存在问题。不仅传统的煎煮服用方式受制于植物药有效成分难以突破坚硬的植物细胞壁而释放的障碍，而且现有的中药种植技术也面临植物药有效成分含量普遍较低、重金属污染、和农药残留等问题，其药效与野生植物药也相差甚远。因此，植物药领域亟需安全、高效的促进植物药有效成分释放的工艺。
发明内容
本申请提供了安全、高效的促进植物药有效成分释放的方法。该方法先通过破壁真菌对植物药进行发酵，后通过益生菌对植物药进行一次或多次发酵。本申请的方法能有效破坏植物药细胞壁，促进植物药有效成分的释放，提高植物药利用率的目的。
本文中“植物药”主要是指植物的茎、叶、根、和/或果等部位、作为中医的药材的材料。优选地，本文的植物药是列入中国药典或其它国家药典的植物药。在更具体的实施方案中，本文的植物药是人参或葛根。已知人参属于双子叶植物纲，伞形目，五加科，人参属；葛根属于木兰纲，蔷薇目，豆科，葛属。可见，人参和葛根是非常不相同的两个物种。本申请的方法在人参和葛根上的适用，说明本申请的方法的适用范围较广，可以适用于多种不同的植物药。
本文中“破壁真菌”是指，能对植物细胞壁或昆虫体壁等坚硬的表面结构产生破壁效果的食用真菌。这里提到的破壁效果，并不要求使壁完全破裂，只要使壁上出现漏洞，内含物更容易向外释放即可。所述破壁真菌的范畴内 包括药用真菌，例如各国药典中所列的多细胞真菌。在具体实施方案中，所述破壁真菌是能生成肉眼可见的子实体的大型真菌。
已知植物细胞壁的主要成分有纤维素、半纤维素和果胶。相应地，能产生纤维素酶、半纤维素酶、和/或果胶酶的食用真菌，都是能对植物细胞壁造成本文所述破壁效果的，因此都属于本文所述的破壁真菌。
事实上，有不少食用真菌能在木质如树木上生长，它们能突破坚硬的木质表面，固着于其中，说明它们都具有本文所述的破壁效果，因此，这些能在木质上生长的多细胞真菌(简称“木生真菌”)属于本文所述的破壁真菌，具体例子有：灵芝(Ganoderma)、茯苓(Poria cocos)、猪苓(Grifola umbellata)，等等。
除了树木等植物细胞有坚硬表面以外，昆虫及其幼虫在体壁中也有坚硬的纤维素、几丁质成分。有些真菌能感染昆虫(如鳞翅目)的幼虫或蛹，使得本来要经历幼虫→蛹→成虫生长阶段的昆虫终止于幼虫或蛹的阶段，该类真菌则利用昆虫幼虫或蛹的身体为营养进行生长。虫草菌就是这样的真菌。在本文中，“虫草菌”是指广义上的虫草属(Cordyceps)真菌，它们寄生于昆虫，把虫体变成充满菌丝的僵虫，从僵虫前端生出柄头状或棍棒状的子座。虫草属真菌目前已报道的超过500多种，最广为人知的包括冬虫夏草(Cordyceps sinensis)、蛹虫草(Cordyceps militaris)等。虫草菌能突破昆虫体壁，说明能产生相应的破壁物质，例如纤维素酶、几丁质酶等等，因此也属于本文所述的破壁真菌。
相应地，在具体实施方案中，本文所述破壁真菌包括木生真菌和虫草菌。在更优选的实施方案中，本文所述破壁真菌选自灵芝、茯苓、猪苓、冬虫夏草、和/或蛹虫草。
本文中“发酵(fermentation)”是指在植物或其果实、茎叶等材料中接种微生物进行培养的过程。由于微生物的生命活动，可以将碳水化合物转化为醇和有机酸等物质，也可以使植物所含的成分(例如营养成分)发生变化，例如更容易释放，活性更强，或成为更有利于被人体吸收的形式。
本文中“酵素(ferment nutrients)”是指植物经益生菌发酵而得到的多种生物活性物质的混合体，其包括：发酵参与菌、用于发酵的食材、发酵参与菌或食材的活性成分，例如多种酶(enzyme)，还包括因发酵而被改良的成分，例如活性更强或更有利于吸收的成分，等等。
本文中“益生菌(probiotics)”是指在人体内对健康有益的微生物。益生菌常用于制作酸奶、酵素等食用物。常用的益生菌有，酵母菌(yeast)、乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)、明串珠菌(Leuconostoc)、链球菌(Streptococcus)、乳球菌(Lactococcus)，醋酸杆菌(Acetobacterium)等等。所述酵母菌例如酿酒酵母(Saccharomyces cerevisiae)和椭圆酵母(Saccharomyes eerevisiae)等糖酵母属(Saccharomyces)的酵母、巴斯德毕赤酵母(Pichia pastoris)、奥默毕赤酵母(Pichia ohmeri)、膜璞毕赤酵母(Pichia membranaefaciens)等毕赤酵母属(Pichia)的酵母、异常汉逊酵母(Hansenula anomala)等汉逊酵母属(Hansenula)的酵母。所述乳酸杆菌例如，布氏乳杆菌(Lactobacillus buchneri)、面包乳杆菌(Lactobacillus panis)、嗜酸乳杆菌(Lactobacillus acidophilus)、植物乳杆菌(Lactobacillus plantarum)、发酵乳杆菌(Lactobacillus fermenti)、唾液乳杆菌(Lactobacillus salivarius)、短乳杆菌(Lactobacillus brevis)、淡绿色乳杆菌(Lactobacillus veridescens)、德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii bulgaricus)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、纤维二糖乳杆菌(Lactobacillus cellobiosus)、干酪乳杆菌干酪亚种(Lactobacillus casei subsp casei)、瑞士乳杆菌约古特亚种(Lactobacillus helveticus subsp.jugurti)、詹森乳杆菌(Lacticum Jansen)、戊糖乳杆菌(Lactobacillus pentosus)，等等。所述双歧杆菌例如，青春双歧杆菌(Bifidobacterium adolescents)、分叉双歧杆菌(Bifidobacterium bifidum)、婴儿双歧杆菌(Bifidobacterium infantis)、长双歧杆菌(Bifidobacterium longum)，等等。所述醋酸杆菌例如巴氏醋酸杆菌(Acetobacter pasteuranus)、恶臭醋酸杆菌(Acetobacter rancens)等。其他益生菌还有：肠膜明串珠球菌(Leuconostoc mesenteroides aureus)、唾液链球菌嗜热亚种(Streptococcus salivarius subsp.thermophilus)、乳球菌乳脂亚种(Lactococcus lactis subsp.Cremoris)，等等。
本申请人发现，不同的植物药经过破壁真菌发酵和益生菌发酵，相比于传统煎服的汤药，可以大大提高药液中的植物药有效成分的含量，由此可以改进传统中药服用方式和现有中药种植方式所面临的药效低下问题。经过本申请的方法制得的植物药发酵制品，包含植物药有效成分以及来自破壁真菌和益生菌的各种活性酶类或活性物质，是营养丰富的制剂。这样的制剂，可用于调理人体健康。
相应地，本申请还涉及一种发酵方法，其是将破壁真菌的发酵和益生菌的发酵组合。所述发酵方法可以是植物药的发酵方法。在更具体的实施方案中，本申请涉及先进行破壁真菌的发酵，再进行益生菌的发酵。在更具体的实施方案中，本申请的益生菌发酵用酵母菌、乳酸杆菌和醋酸杆菌进行。在更具体的实施方案中，本申请的益生菌发酵是分步骤进行，例如先进行酵母菌和乳酸杆菌的混合发酵，再进行醋酸杆菌的发酵。无论是破壁真菌的发酵，还是益生菌的发酵，都采用各自菌种的适用温度和pH等培养条件进行培养，具体条件是本领域技术人员根据常规可以确认的。
破壁真菌如灵芝、虫草等的培养基也是本领域常见的，作为举例，其中一种培养基的组成如下(按质量比)：葡萄糖1～6％，酵母膏0.1％～0.4％，蛋白胨0.1％～0.8％，磷酸二氢钾0.05％～0.3％，硫酸镁0.05％～0.3％，以水补足。作为碳源的葡萄糖可以用马铃薯汁替换，其它成分也可以调整，这些调整是本领域技术人员容易做到的。在本申请的实施例中，为了统一实验体系，采用了以下的组成(按质量比)：葡萄糖2.4％，酵母膏0.2％，蛋白胨0.4％，磷酸二氢钾0.1％，硫酸镁0.1％，蒸馏水96.8％。
在优选实施方案中，能共享相同或相似培养条件的破壁真菌还可以一起在同一培养基中培养，可选地，再进一步进行益生菌发酵，使得最终的产品中包含不止一种破壁真菌和/或其活性成分。
破壁真菌的发酵可以是：50～250rpm，7～30℃，搅拌发酵3～45天。这些条件是本领域技术人员基于破壁真菌具体物种的生长需求很容易确定的。
益生菌的发酵可以是：密封，10～40℃静置发酵48h以上。在具体实施方案中，本申请的益生菌每一种都选用百万级活菌数的菌液、并按总原料质量分数为1～3‰的量接种。这些条件也是本领域技术人员基于益生菌具体物种的生长需求很容易确定的。
优选地，本申请的发酵方法不涉及提取破壁真菌活性成分的步骤，即，所述方法无需在破壁真菌发酵之前或之后提取大型真菌活性成分，而是破壁真菌的发酵和益生菌的发酵可以在同一个培养系统或发酵装置中依次进行。
在一个具体举例的实施方案中，本申请的方法包括如下步骤：
1、选用符合规定的植物药鲜药材及植物药饮片，粉碎至10～30目，倒入发酵罐中，加入5～20倍量(质量)的培养液，搅拌混合均匀；
2、用食用醋或者食用氢氧化钠溶液调整pH值至5.0～8.0，后通入蒸汽 灭菌30～60min；
3、冷却至室温后，接入药用菌菌种，控制发酵罐搅拌速度为50～250rpm，控制温度为7～30℃，发酵3～45天，完成初次发酵；
4、完成初次发酵后，接入益生菌菌种，密封，于10～40℃环境下静置培养48h以上，完成第二次发酵；
5、滤去药渣，收集发酵液。
本发明的优点在于，通过破壁真菌对植物药进行初步发酵，后接入益生菌菌种对植物药进行再次(甚至更多次)发酵，可以有效的破坏植物药的细胞壁，促进植物药有效成分的释放，提高植物药的利用率。另外，由于使用了破壁真菌发酵，植物药药液中还包含有破壁真菌的有效成分，从而起到了增加植物药药效的目的。
更具体举例地，本申请涉及
1.一种植物药的微生物发酵方法，包括先进行破壁真菌的发酵，再进行益生菌发酵。
2.项1的方法，其中所述破壁真菌是木生真菌和/或虫草菌。
3.项1～2之一的方法，其中所述破壁真菌选自灵芝、猪苓、茯苓、冬虫夏草、和/或蛹虫草。
4.项1～3之一的方法，其中所述益生菌选自以下的一或多种：糖酵母、毕赤酵母、汉逊酵母、乳酸杆菌、双歧杆菌、明串珠菌、链球菌、乳球菌，醋酸杆菌。
5.项4的方法，其中的益生菌选自以下的一或多种：酿酒酵母、椭圆酵母、巴斯德毕赤酵母、奥默毕赤酵母、膜璞毕赤酵母、异常汉逊酵母、布氏乳杆菌、面包乳杆菌、嗜酸乳杆菌、植物乳杆菌、发酵乳杆菌、唾液乳杆菌、短乳杆菌、淡绿色乳杆菌、德氏乳杆菌保加利亚亚种、鼠李糖乳杆菌、纤维二糖乳杆菌、干酪乳杆菌干酪亚种、瑞士乳杆菌约古特亚种、詹森乳杆菌、戊糖乳杆菌)、巴氏醋酸杆菌、恶臭醋酸杆菌、青春双歧杆菌、分叉双歧杆菌、婴儿双歧杆菌、长双歧杆菌、肠膜明串珠球菌、唾液链球菌嗜热亚种、和乳球菌乳脂亚种。
6.项5的方法，其中所述益生菌是酵母菌、乳酸杆菌和醋酸杆菌。
7.向6的方法，其中先进行酵母菌和乳酸杆菌的混合发酵，再进行醋酸杆菌的发酵。
8.项1～7之一的方法，其中所述植物药是人参。
9.项1～7之一的方法，其中所述植物药是葛根。
10.项1～10之一的发酵方法得到的产物。
具体实施例
下面的实施例是对本申请的具体描述，这些实施例旨在对本申请进行进一步说明，不能理解为对发明的保护范围有任何限制，本领域的技术人员基于本申请作出的改进和调整也属于受保护的范围。
实施例1
称取人参10Kg，粉碎至30目，倒入发酵罐中，加入80Kg的培养液(按质量比：葡萄糖2.4％，酵母膏0.2％，蛋白胨0.4％，磷酸二氢钾0.1％，硫酸镁0.1％，蒸馏水96.8％)，充分搅拌混合，用氢氧化钠溶液调整pH值为6.8，通入蒸汽灭菌30min。冷却至室温后，接入灵芝菌(湘赤芝1号，湖南省非主要农作物品种登记证书编号XPD010-2013，获自国家中医药管理局亚健康干预技术实验室)，控制发酵罐搅拌速度为120rpm，控制温度为20℃，发酵15天；后加入1‰(总原料质量分数)酵母菌(奥默毕赤酵母，CGMCC编号2.1803，百万级活菌数)和1‰(总原料质量分数)乳酸菌(植物乳杆菌，CGMCC编号1.6971，百万级活菌数)，于28℃环境下静置发酵7天；后加入1‰(总原料质量分数)醋酸杆菌(恶臭醋酸杆菌AS.1.41，购自上海酿造一厂，百万级活菌数)，30℃静置培养7天，过滤，收集药液。
经测定，应用本发明处理的人参药液，其中总人参皂苷含量达到了1.06mg/mL(测定方法参见，人参总皂苷的含量测定，高丽萍等，浙江工程学院 学报,2012,31(3):382-388)，较传统汤剂(人参:水为1:8重量比)提高了7.01倍；还在该药液中检测到灵芝三萜含量为0.75mg/mL(测定方法参见，中华人民共和国药典2015版)。所有数据都是一式三份试样的平均结果。
实施例2
称取人参10Kg，粉碎至30目，倒入发酵罐中，加入80Kg的实施例1所述培养液，充分搅拌混合，用氢氧化钠溶液调整pH值为6.8，通入蒸汽灭菌30min。冷却至室温后，接入灵芝菌(紫光灵芝，CGMCC 5.772，属于紫 芝)，控制发酵罐搅拌速度为100rpm，控制温度为23℃，发酵15天；后加入1‰(总原料质量分数)酵母菌(酿酒酵母，CGMCC编号2.3888，百万级活菌数)和1‰(总原料质量分数)乳酸菌(面包乳杆菌，CGMCC编号1.3925，百万级活菌数)，于28℃静置发酵7天；后加入1‰(总原料质量分数)醋酸杆菌(恶臭醋酸杆菌AS.1.41，百万级活菌数)，30℃静置培养7天，过滤，收集药液。
经测定，应用本发明处理的人参药液，其中总人参皂苷含量达到了1.03mg/mL(测定方法同实施例1)，较传统汤剂(人参:水为1:8重量比)提高了6.81倍；还在该药液中检测到灵芝三萜含量为0.64mg/mL(测定方法同实施例1)。所有数据都是一式三份试样的平均结果。
实施例3
称取葛根10Kg，粉碎至20目，倒入发酵罐中，加入100Kg的实施例1所述培养液，充分搅拌混合，用氢氧化钠溶液调整pH值为6.5，121℃高温灭菌30min。冷却至室温后，接入虫草菌(湘北虫草1号，湖南省非主要农作物品种登记证书编号XPD009-2013，得自国家中医药管理局亚健康干预技术实验室)，控制发酵罐搅拌速度为150rpm，控制温度为25℃，发酵10天；后加入3‰(总原料质量分数)酵母菌(酿酒酵母，CGMCC编号2.3973，百万级活菌数)和1‰(总原料质量分数)乳酸菌(布氏乳杆菌，CGMCC编号1.3114，百万级活菌数)，于28℃静置发酵8天；后加入1‰(总原料质量分数)醋酸杆菌(巴氏醋酸杆菌亚种1.01，百万级活菌数)，30℃静置培养7天，过滤，收集药液。
经测定，应用本发明处理的葛根药液，其中葛根素含量达到了0.119mg/mL(测定方法参见中国国家标准GB/T 22251-2008保健食品中葛根素的测定)，较传统汤剂(葛根:水为1:10重量比)提高了1.25倍。还在该药液中检测到虫草素含量为11.3μg/mL(测定法参见，虫草素高产菌株的筛选及不同添加物对虫草素产量的影响研究，王蕾等，菌物学报,2012,31(3):382-388)。所有数据都是一式三份试样的平均结果。
实施例4
称取葛根10Kg，粉碎至20目，倒入发酵罐中，加入100Kg的实施例 1所述培养液，充分搅拌混合，用氢氧化钠溶液调整pH值为6.5，121℃高温灭菌30min。冷却至室温后，接入虫草菌(蛹虫草，CGMCC 5.856)，控制发酵罐搅拌速度为130rpm，控制温度为28℃，发酵10天；后加入3‰(总原料质量分数)酵母菌(膜醭毕赤酵母，CGMCC 2.661，百万级活菌数)和1‰(总原料质量分数)乳酸菌(面包乳杆菌，CGMCC 1.3925，百万级活菌数)，28℃静置发酵8天；后加入1‰(总原料质量分数)醋酸杆菌(醋酸杆菌沪酿1.01，百万级活菌数)，30℃静置培养7天，过滤，收集药液。
经测定，应用本发明处理的葛根药液，其中葛根素含量达到了0.113mg/mL，较传统汤剂(葛根:水为1:10重量比)提高了1.19倍(测定方法同实施例3)。还在该药液中检测到虫草素含量为13.7μg/mL(测定方法同实施例3)。所有数据都是一式三份试样的平均结果。

Claims (10)

  1. 一种植物药的微生物发酵方法，包括先进行破壁真菌的发酵，再进行益生菌发酵。
  2. 权利要求1的方法，其中所述破壁真菌是木生真菌和/或虫草菌。
  3. 权利要求1～2之一的方法，其中所述破壁真菌选自灵芝、猪苓、茯苓、冬虫夏草、和/或蛹虫草。
  4. 权利要求1～3之一的方法，其中所述益生菌选自以下的一或多种：糖酵母、毕赤酵母、汉逊酵母、乳酸杆菌、双歧杆菌、明串珠菌、链球菌、乳球菌，醋酸杆菌。
  5. 权利要求4的方法，其中的益生菌选自以下的一或多种：酿酒酵母、椭圆酵母、巴斯德毕赤酵母、奥默毕赤酵母、膜璞毕赤酵母、异常汉逊酵母、布氏乳杆菌、面包乳杆菌、嗜酸乳杆菌、植物乳杆菌、发酵乳杆菌、唾液乳杆菌、短乳杆菌、淡绿色乳杆菌、德氏乳杆菌保加利亚亚种、鼠李糖乳杆菌、纤维二糖乳杆菌、干酪乳杆菌干酪亚种、瑞士乳杆菌约古特亚种、詹森乳杆菌、戊糖乳杆菌)、巴氏醋酸杆菌、恶臭醋酸杆菌、青春双歧杆菌、分叉双歧杆菌、婴儿双歧杆菌、长双歧杆菌、肠膜明串珠球菌、唾液链球菌嗜热亚种、和乳球菌乳脂亚种。
  6. 权利要求5的方法，其中所述益生菌是酵母菌、乳酸杆菌和醋酸杆菌。
  7. 权利要求6的方法，其中先进行酵母菌和乳酸杆菌的混合发酵，再进行醋酸杆菌的发酵。
  8. 权利要求1～7之一的方法，其中所述植物药是人参。
  9. 权利要求1～7之一的方法，其中所述植物药是葛根。
  10. 权利要求1～9之一的发酵方法得到的产物。
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