CN113966832A

CN113966832A - Dendrobium nobile fermentation product and preparation method and application thereof

Info

Publication number: CN113966832A
Application number: CN202010713499.6A
Authority: CN
Inventors: 彭宁; 熊涛; 李知洪; 覃敬羽; 张彦; 张海波; 王欢; 杨树金; 冯玖
Original assignee: Angel Nutt Co ltd
Current assignee: Angel Nutt Co ltd
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2022-01-25

Abstract

The invention relates to the field of additives, and particularly relates to a dendrobe fermentation product and a preparation method and application thereof. The invention provides a dendrobium nobile fermentation product with oxidability and capable of improving immunity, which comprises the following raw materials: dendrobe, an enzymolysis agent and fermentation thalli; on the other hand, the invention also provides a preparation method of the dendrobium nobile fermentation product, which comprises the following steps: activating fermentation thalli, processing dendrobe raw materials, performing enzymolysis, fermenting and post-processing to prepare an oral and external dendrobe fermentation product, and realizing the release of polyphenol, polysaccharide and flavonoid substances in dendrobe and the enrichment of microbial metabolites such as short chain fatty acid, polypeptide, amino acid and the like. The fermented dendrobium has special fermented fragrance and good taste, and can realize the synergistic effect of oral administration and external use.

Description

Dendrobium nobile fermentation product and preparation method and application thereof

Technical Field

The invention relates to the field of additives, and particularly relates to a dendrobe fermentation product and a preparation method and application thereof.

Background

Dendrobe (Dendrobium), the second major genus of Orchidaceae (Orchidaceae) plants, traditional Chinese medicine dendrobe, is perennial herb, about 500 plants are more than 1500 in the world, about 76 plants are in China, and 33 varieties of original plants serving as commodity dendrobe are purchased and circulated. In 2005, "pharmacopoeia of the people's republic of China" recorded three types of dendrobe which can be used as a traditional Chinese medicine, including: herba Dendrobii, and herba Verbenae. Three dendrobium stems recorded in pharmacopoeia of people's republic of China in 2010 and 2015 are stems of dendrobium nobile, dendrobium chrysotoxum and dendrobium fimbriatum, and dendrobium officinale is listed separately. The most common varieties are dendrobium officinale and dendrobium nobile. The dendrobium officinale in the effective substances is mainly polysaccharide, and the dendrobium nobile is mainly alkaloid. Compendium of materia Medica: the dendrobium stem can remove arthralgia to expel qi, tonify internal organs, lose fatigue and thin, strengthen yin and replenish vital essence, is taken for a long time, thicken intestines and stomach, tonify internal organs without enough, calm stomach qi, grow muscles, dispel skin evil and heat prickly heat qi, relieve pain and cold arthralgia of feet and knees, fix will and remove fright, lighten body and prolong life, tonify qi and remove heat, treat weak waist and knees of men, strengthen yang, dispel wind arthralgia, cool bones for a long time, tonify kidney and improve strength, strengthen bones and muscles, warm water and stop, promote intelligence and clear qi, treat fever and spontaneous perspiration, carbuncle and cellulitis to discharge pus and internal embolism.

Modern scientific research on active ingredients contained in dendrobium nobile are mainly alkaloids, flavonoids, polysaccharides, terpenoids, phenolic compounds and the like. The product has effects of enhancing immunity, resisting tumor, resisting oxidation, reducing blood lipid and blood pressure, and regulating intestinal flora balance. The method has the advantages that the microorganism is used for fermentation on the basis of the dendrobium raw material, the fermentation capacity and rich metabolites of the microorganism are used, the mild release and conversion of the functional substances in the dendrobium in the fermentation process can be realized, and the loss of the functional substances caused by high-temperature extraction is avoided. Different microorganism individuals have different fermentation characteristics, and under the condition that polysaccharide is used as prebiotics, the microbial fermentation agent can promote the metabolic growth of various beneficial microorganisms, secrete short-chain fatty acids, polypeptides and amino acids and metabolize to generate various enzymes. The fermented dendrobium product has more components beneficial to human bodies, has better function embodiment, has important value in the development of medicines, health-care foods, common foods and skin care products, and solves the body problems of various modern people by oral administration and external use.

The technical solutions disclosed in the prior art include:

CN 104585762A: a herba Dendrobii fermented product is prepared by fermenting fruit and vegetable juice with mixture of Pleurotus Citrinopileatus Sing, lactobacillus and yeast

CN 104940644A: a lactobacillus fermentation method of herba Dendrobii, and its fermented product and application are provided; fermenting herba Dendrobii with lactobacillus to obtain lyophilized powder.

CN 105534876A: a herba Dendrobii fermented raw stock and its preparation method and application are provided; dendrobe powder and lactobacillus fermented raw stock are a patent research aiming at skin care products.

CN 105962368A: a preparation method of dendrobium officinale fermentation liquor uses a product which is prepared by fermenting raw materials which mainly contain a plurality of auxiliary materials such as purple rice, millet, coix seed, tuckahoe, red date, dark plum, dried orange peel, scorched hawthorn fruit and the like. The zymocyte is lactobacillus.

CN 107629965A: a method for preparing herba Dendrobii fungus fermented product, its fermented product and application, the patent adopts fungus to ferment, the fungus species include Grifola frondosa, round Ganoderma lucidum, Poria cocos, Coriolus versicolor, Ganoderma lucidum, and Hericium erinaceus.

CN 108042456A: a preparation method and application of a dendrobium candidum fermentation product are provided, wherein the dendrobium candidum fermentation product is prepared by fermenting dendrobium candidum with bacillus subtilis and bacillus coagulans, and is mainly used for improving skin.

Disclosure of Invention

According to the prior art, firstly, fermentation strains and fermentation processes are incomplete, and fermentation is mostly carried out by lactic acid bacteria, and individual fungi, bacillus subtilis, bacillus coagulans and the like are adopted; the fermentation mode is mainly single fermentation, the advantage of flora competitive fermentation cannot be embodied by single fermentation, and part of patents relate to multi-strain step-by-step fermentation, but the fermentation mode is relatively single for the strains for fermentation; meanwhile, a large amount of auxiliary materials are used as fermentation substrates in a plurality of fermentation processes, the auxiliary materials are high in content and complex in components, and the final fermentation liquid has efficacy, but cannot represent the real characteristics of the dendrobium and is not the expression of the fermented dendrobium.

The technical problems in the prior art are as follows: in the prior art, the fermentation strains and the fermentation process are not comprehensive, the fermentation strains and the fermentation mode are relatively single, a large amount of auxiliary materials are used as fermentation substrates in a plurality of fermentation processes, the auxiliary materials are high in content and complex in components, and the final fermentation liquid has effect, but cannot represent the real characteristics of the dendrobium and is not the expression of the dendrobium after fermentation.

The invention aims to solve the technical problems and provides a process for preparing a dendrobium fermentation product by using dendrobium as a main raw material through microbial fermentation and a product of the dendrobium fermentation product; meanwhile, a preparation process of the dendrobium nobile fermentation product is provided, which comprises the following steps: (1) activating and culturing strains; (2) processing the dendrobium raw material; (3) enzymolysis treatment (4) inoculation fermentation; and (5) post-treatment. The obtained herba Dendrobii fermented product has good effects of resisting oxidation and inhibiting tyrosinase activity, and can improve intestinal tract and improve immunity by oral administration.

Specifically, the invention provides the following technical scheme:

a herba Dendrobii fermented product with antioxidant and immunity enhancing effects is prepared by fermenting herba Dendrobii, enzymolysis agent and fermentation thallus;

wherein the enzymolysis agent is selected from one or more of protease, amylolytic enzyme, pectolytic enzyme, saccharifying enzyme and cellulolytic enzyme;

the fermentation thallus is selected from one or more of yeast, lactobacillus, Aspergillus niger and acetic acid bacteria.

Optionally, for the dendrobium fermentation product, the dendrobium in the raw materials is one or more than two selected from dendrobium officinale, dendrobium nobile, dendrobium chrysotoxum and dendrobium fimbriatum, preferably dendrobium officinale;

preferably, the using part of the dendrobium comprises at least one of dendrobium stem, dendrobium leaf and dendrobium flower; further preferably dendrobium stem;

preferably, the dendrobe is used in a form including at least one of a fresh survival state, a semi-dried state, a dried state and an extract form.

Optionally, in the dendrobe fermented product, the proteolytic enzyme in the enzymolysis agent is selected from one or more of papain, bromelain, trypsin, pepsin and peptidase, preferably bromelain and/or pepsin;

wherein, the amylolytic enzyme in the enzymolysis agent is selected from one or more than two of alpha-amylase, beta-amylase, high-temperature amylase and saccharifying enzyme, preferably high-temperature amylase and/or saccharifying enzyme;

wherein the pectic decomposition enzyme in the enzymolysis agent is one or more selected from pectin depolymerase, pectin lyase or pectin demethoxylase, preferably pectin lyase;

wherein, the cellulolytic enzyme in the enzymolysis agent is selected from one or more than two of cellulase, hemicellulase, mannanase, xylanase, ligninase and glucanase, preferably cellulase and/or ligninase, and more preferably ligninase.

Optionally, for the dendrobe fermentation product, the yeast in the fermentation thallus is selected from one or more of saccharomyces cerevisiae, saccharomyces boulardii BLD, pichia pastoris, candida albicans or saccharomyces cerevisiae; preferably selected from the group consisting of Bld, Candida or Babylonia, more preferably Candida or Babylonia;

wherein the lactobacillus in the fermented thallus is selected from one or more of Lactobacillus pentosus, Lactobacillus casei, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, LGG or BB 12; preferably one or more selected from Lactobacillus pentosus, Lactobacillus plantarum or Lactobacillus acidophilus; more preferably lactobacillus pentosus.

A preparation method of the dendrobium nobile fermentation product comprises the following steps:

(1) activating and culturing the fermentation thalli;

(2) processing a dendrobe raw material;

(3) performing enzymolysis on the dendrobium processed in the step (2);

(4) inoculating the activated zymophyte in the step (1) into the enzymolysis product in the step (3) for fermentation;

(5) and (4) carrying out subsequent treatment on the product obtained in the step (4) to obtain the dendrobium fermentation product.

Optionally, for the preparation method, the inoculating the activated zymophyte of step (1) into the enzymolysis product of step (3) in step (4) for fermentation includes at least one of the following four fermentation methods:

A. each microorganism fermented separately: respectively inoculating each activated fermentation thallus, and fermenting each fermentation thallus independently;

B. mixing and synchronously fermenting: inoculating at least two activated fermentation thalli together, mixing and fermenting synchronously;

C. separately fermenting, and mixing, wherein at least two activated fermentation thalli are adopted, each of which is inoculated separately, and after separate fermentation, fermentation products of different fermentation strains are mixed;

D. step-by-step gradient fermentation: at least two activated fermentation thalli are adopted, one of the thalli is firstly fermented, then other thalli are inoculated on the basis of fermentation products and then fermentation is carried out, wherein only one of the thalli is inoculated at each stage.

Alternatively, in the production method, the culture for activating the fermentation product in step (1) may include:

putting the original strain into a liquid culture medium, and culturing until the OD value is 0.5-1.0; preferably, the cells are cultured until the cell density is 1X 10⁸CFU/ml above.

Optionally, for the preparation method, the processing of the dendrobe raw material in step (2) comprises:

pulverizing and sieving the dried dendrobium into powder, preferably pulverizing the powder to 60-100 meshes; and/or

Pulping fresh dendrobium, preferably selecting the mass volume ratio of dendrobium raw materials to water in the pulping process to be 1:100-1:1, and further preferably selecting the mass volume ratio to be 1:100-1: 5; and/or

The method c, extracting the dendrobium;

wherein the operation of the subsequent processing of step (5) comprises: centrifugation, filtration to give a clear liquid, and/or lyophilization or spray drying to give a powder product.

Optionally, in the method c, the reagent for extracting dendrobium nobile is one or more selected from water, lower alcohols, higher alcohols, polyols, esters, ketones and hydrocarbon solvents;

preferably, the lower alcohols include methanol, ethanol and propanol;

preferably, the higher alcohols include oleyl alcohol, stearyl alcohol, and octyldodecanol;

preferably, the esters include ethyl acetate, butyl acetate, methyl propionate, and glyceryl trioctanoate;

preferably, the ketones include acetone and methyl ethyl ketone;

preferably, the ethers include diethyl ether and isopropyl ether;

preferably, the hydrocarbon-based solvent includes n-hexane, toluene, and chloroform;

further preferably, the extraction reagent is a mixed solvent of water and ethanol, and the mass ratio of water: the ethanol is 1:1-25: 1; further preferred is a mixed solvent of water and glycerin, and the volume ratio of water: 1:1-20:1 of glycerol; further preferred is a mixed solvent of water and 1, 3-propanediol or water and 1, 3-butanediol, and the volume ratio of water: 1, 3-propanediol or water: the ratio of 1, 3-butanediol is 1:1-15: 1.

Optionally, for the preparation method, the performing enzymolysis on the dendrobe processed in the step (2) in the step (3) includes:

an enzymolysis agent: regulating the mass ratio of the total mass of the dendrobium raw materials to be 1:10000-1: 100; preferably 1: 1000;

the pH of the enzymolysis environment is 2-9, preferably 4-7;

the enzymolysis temperature is 30-100 ℃, preferably 30-60 ℃, and further preferably, for high-temperature amylase, the enzymolysis temperature is 60-100 ℃;

the enzymolysis time is 0.5-24h, preferably 2-6 h.

Optionally, for the method of making, wherein the B-mix simultaneous fermentation comprises: a combination of aspergillus niger and saccharomycetes, or a combination of aspergillus niger, lactobacillus and acetic acid bacteria, or a combination of saccharomycetes and lactobacillus, or a combination of yeast and acetic acid bacteria;

the step-by-step gradient fermentation comprises the following steps: firstly, lactobacillus is added to yeast and then acetic acid bacteria, or firstly, mould is added to yeast and then lactobacillus, or firstly, saccharomycetes is added to lactobacillus and then acetic acid bacteria.

Optionally, in the preparation method, the activated zymophyte in the step (1) is inoculated into the enzymolysis product in the step (3) in the step (4) for fermentation,

wherein the fermentation medium used in the fermentation comprises the enzymolysis product, a carbon source and a nitrogen source;

wherein, based on the total volume of the enzymolysis products obtained in the step (3), the volume ratio of the carbon source to the enzymolysis products is 1:2-1:1000, and the volume ratio of the nitrogen source to the enzymolysis products is 1:3-1: 1000;

preferably, the volume ratio of the carbon source to the enzymolysis product is 1:50, and the volume ratio of the nitrogen source to the enzymolysis product is 1: 100;

the carbon source is selected from one or more than two of glucose, fructose, sucrose, maltose, lactose and hydrolyzed starch, and is preferably glucose, sucrose and hydrolyzed starch;

the nitrogen source is selected from one or more of ammonium sulfate, ammonium chloride, soybean hydrolysate, beef extract, milk powder and yeast hydrolysate, preferably ammonium sulfate, ammonium chloride, hydrolyzed soybean protein and yeast extract.

Optionally, in the preparation method, the activated fermentation thallus in the step (1) is inoculated into the enzymolysis product in the step (3) in the step (4) for fermentation, wherein the addition amount of the activated fermentation thallus is 1:10000-1:100g/mL based on the total volume of the fermentation medium;

the preferable fermentation temperature is 25-45 ℃; preferably, the fermentation culture time is 0.5-10 days; preferably, the ventilation volume is 0-100L/min; preferably, the rotating speed is controlled to be 20-100 r/min;

preferably, the addition amount of the strains LGG and BB12 is 1X 10⁵CFU/ml×10¹⁰CFU/ml。

A herba Dendrobii fermented product is prepared by the above preparation method.

An external dendrobe leavening for skin care is characterized by being prepared by the preparation method;

wherein, the step (4) of inoculating the activated zymophyte in the step (1) into the enzymolysis product in the step (3) for fermentation comprises the following steps: adding an alcohol solvent into a fermentation medium in the fermentation process;

preferably, the alcoholic solvent includes at least one of ethanol, isopropanol, ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, pentylene glycol, and glycerin; preferably butylene glycol and glycerol;

the volume ratio of the alcohol solvent to the total volume of the fermentation medium is 1:1000-1: 5.

The dendrobium nobile fermentation product or the skin-care external dendrobium nobile fermentation product is applied to the fields of skin care products, cosmetics, medicines, health-care foods and foods.

The invention has the beneficial effects that:

(1) according to the technology disclosed by the invention, the dendrobium of different varieties and different parts are used as fermentation raw materials, and other natural products are added as little as possible, so that the dendrobium natural products can be more directly fermented, and the fermentation value of the dendrobium is reflected.

(2) The preparation method and the obtained dendrobe fermentation product use different microorganisms such as saccharomycetes, lactic acid bacteria, mould, acetic acid bacteria and the like.

(3) The Chinese fermentation process of the invention uses different fermentation modes such as single fermentation, mixed gradient fermentation and the like, and is matched with the use of an organic reagent, so that the Chinese fermentation process can more comprehensively embody the values of the fermented dendrobium in the aspects of medicine, skin care, health care and the like, and has good guiding and reference functions for the fermentation industry or the dendrobium industry.

Detailed Description

The invention aims to provide a dendrobe fermented product with oxidation resistance and immunity improvement and a preparation method and application thereof, the obtained dendrobe fermented product has good effects of oxidation resistance and tyrosinase activity inhibition, can improve intestinal tracts and improve the immunity of organisms by oral administration, and can be widely applied to various industries of foods, medicines, health-care foods and skin care products.

Specifically, the invention provides the following technical scheme:

in one aspect, the invention provides a dendrobe fermentation product, wherein the raw materials comprise: dendrobe, an enzymolysis agent and a fermentation microbial inoculum.

The dendrobium is used as a main raw material and is selected from one or more than two of dendrobium officinale, dendrobium nobile, dendrobium chrysotoxum, dendrobium fimbriatum and dendrobium.

In a preferred embodiment of the invention, the dendrobium raw material adopts dendrobium officinale.

The using parts of the dendrobium raw materials comprise dendrobium stems, dendrobium leaves, dendrobium flowers and a mixed state of the three

In a preferred embodiment of the present invention, the site of use is a stem of dendrobium.

The invention relates to a dendrobium raw material. The raw materials include herba Dendrobii naturally occurring form (including fresh state, semi-dry state and dry state) and corresponding extract thereof (extraction reagent including water, lower alcohols such as methanol, ethanol and propanol, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, polyhydric alcohols such as ethylene glycol, 1, 3-propanediol, 1, 2-pentanediol, 1, 3-butanediol and glycerol, esters such as ethyl acetate, butyl acetate, methyl propionate and tricaprylin, ketones such as acetone and methyl ethyl ketone, ethers such as diethyl ether and isopropyl ether, and hydrocarbon solvent such as n-hexane, toluene and chloroform, which can be used alone or in combination of two or more).

In a preferred embodiment of the invention, the extraction reagent comprises: a mixed solvent of water and ethanol, preferably the mass ratio (water: ethanol) is 1:1-25: 1; a mixed solvent of water and glycerol, preferably the volume ratio (water: glycerol) is 1:1-20: 1; the mixed solvent of water and 1, 3-propylene glycol or water and 1, 3-butanediol, preferably has a volume ratio (water: 1, 3-propylene glycol or water: 1, 3-butanediol) of 1:1-15: 1.

In a preferred embodiment of the invention, the method for processing the dendrobium raw material comprises the following steps: pulping fresh dendrobium, wherein the fresh dendrobium is selected from semi-dried and/or fully-dried dendrobium; the mass volume ratio of the dendrobium raw material to water in the pulping process is 1:100-1:1, and the preferable ratio is 1:100-1: 5; pulverizing to 60-100 mesh.

The enzymolysis agent used in the invention is selected from one or more than two of proteolytic enzyme, amylolytic enzyme, pectic lytic enzyme and cellulolytic enzyme, wherein the proteolytic enzyme comprises papain, bromelain, trypsin, pepsin and peptidase; the amylolytic enzyme comprises alpha-amylase, beta-amylase and high-temperature amylase; the pectic decomposition enzyme comprises pectin depolymerase, pectin lyase, and pectin demethoxylase; cellulolytic enzymes include cellulases, hemicellulases, mannanases, xylanases, ligninases and glucanases.

The fermentation bacteria agent used in the invention is selected from one or more than two of yeast, lactobacillus, aspergillus niger and acetic acid bacteria, wherein the yeast comprises saccharomyces cerevisiae, saccharomyces boulardii BLD, pichia pastoris, candida and sacculus complex yeast; lactic acid bacteria include lactobacillus pentosus, lactobacillus casei, bifidobacterium longum, bifidobacterium breve, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus rhamnosus, and commercial strains including LGG and BB 12.

On the other hand, the invention provides a preparation method of a dendrobe fermentation product, which comprises the following steps:

1) activating and culturing thalli;

2) processing a dendrobe raw material;

3) performing enzymolysis on the dendrobium processed in the step (2);

4) inoculating and fermenting the substances subjected to enzymolysis in the step (3);

5) and (5) carrying out subsequent treatment.

In a preferred embodiment of the present invention, the fermentation process of step (4) comprises: independent fermentation of various microorganisms, mixed synchronous fermentation, mixed use after independent fermentation and step-by-step gradient fermentation.

In a preferred embodiment of the invention, step (5) comprises centrifugation, filtration to give a clear liquid, and/or lyophilization or spray drying to give a powder product.

In addition, the invention also provides a skin-care external dendrobe fermentation product, which further comprises an alcohol solvent in the culture medium composition in the fermentation process in the step (4), wherein the ratio of the alcohol solvent to the total volume of the fermentation liquor is 1:1000-1:5 (volume ratio).

In a preferred embodiment of the invention, the fermentation is carried out after the completion of the enzymatic hydrolysis by inactivating the enzyme using a high temperature or by adjusting the pH.

In a preferred embodiment of the present invention, the dendrobium nobile fermentation product can be applied to the fields of skin care products, cosmetics, medicines, health foods and foods, wherein the edible dendrobium nobile fermentation product selects the natural dendrobium nobile raw material and the extract processed by an extraction reagent (such as ethanol or ethanol-water mixture) which can be used for food processing as a fermentation substrate, and a solvent other than water or a mixture of water and the solvent used for the dendrobium nobile extract for the skin care external use dendrobium nobile fermentation product fermentation is not limited thereto.

Information on the preservation of the strains

1. Saccharomyces boulardii BLD: saccharomyces boulardii Angel 1.27(Saccharomyces boulardi Angel 1.27) was deposited in China Center for Type Culture Collection (CCTCC) at 16/4/2012 with the deposit number of CCTCC NO: M2012116, and the deposit address: china, wuhan university, zip code: 430072; telephone: (027) -68754052. The strain has been disclosed in the granted patent publication No. CN 103374531B.

2. Acetic acid bacteria: the saccharomycete foal shaped bacillus AYC1.2(Komagataeibacter saccharolyticus AYC1.2) is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 7 and 19 months, the preservation number is CCTCC NO: M2019569, the preservation address is as follows: china, wuhan university, zip code: 430072; telephone: (027) -68754052.

3. Lactobacillus plantarum: lactobacillus plantarum RPC5(Lactobacillus plantarum RPC5) was deposited in the China Center for Type Culture Collection (CCTCC) at 19.7.2019 with the preservation number of CCTCC NO: M2019566, and the preservation address: china, wuhan university, zip code: 430072; telephone: (027) -68754052.

4. Candida spp: candida (C1.7) was deposited in the China Center for Type Culture Collection (CCTCC) in 2017, 12 and 11 months, with the deposit number of CCTCC NO: M2017782, and the deposit address: china, wuhan university, zip code: 430072; telephone: (027) -68754052.

5. Aspergillus niger: aspergillus niger d5.23(Aspergillus niger 5.23) is preserved in the China Center for Type Culture Collection (CCTCC) in 2017, 12 months and 04 days, the preservation number is CCTCC NO: M2017756, the preservation address is: china, wuhan university, zip code: 430072; telephone: (027) -68754052.

6. Lactobacillus pentosus: lactobacillus pentosus 001(Pediococcus pentosaceus 001) is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 10 months and 25 days, with the preservation number of CCTCC NO: M2017625, and the preservation address: china, wuhan university, zip code: 430072; telephone: (027) -68754052.

7. Buckling bag and laminating yeast: the sacculus-covering saccharomycete d6.16 (Saccharomyces fibuligera d6.16) is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 7 and 19 months, the preservation number is CCTCC NO: M2019570, the preservation address is: china, wuhan university, zip code: 430072; telephone: (027) -68754052.

8. And (3) saccharomyces cerevisiae: saccharomyces cerevisiae A3.12(Saccharomyces cerevisiae A3.12) is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 12 months and 11 days, with the preservation number of CCTCC NO: M2017781, and the preservation address: china, wuhan university, zip code: 430072; telephone: (027) -68754052.

9. Lactobacillus acidophilus: lactobacillus acidophilus 001(Lactobacillus acidophilus 001) is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 10 and 25 months, with the preservation number of CCTCC NO: M2017628, and the preservation address: china, wuhan university, zip code: 430072; telephone: (027) -68754052.

The reagents and instrumentation used in the examples of the invention were derived as follows:

TABLE 1 reagents and apparatus used in the examples

In the present invention, the commercial strain employed can be put into use without being activated.

The raw materials not listed in the above tables are all available from commercial sources.

The preparation of the fermentation product of dendrobium of the present invention will be further illustrated by the following examples.

Examples

Example 1

(1) Activating and culturing strains:

carrying out shake culture on Saccharomyces boulardii Angel 1.27(Saccharomyces boulardii Angel 1.27) with the preservation number of CCTCC NO: M2012116 and a ring of colonies in an inclined plane in a 500ml BD liquid culture medium, wherein the temperature of shake culture is 28 ℃, carrying out activation culture on a shake bed for 16h, and carrying out centrifugation, wherein the centrifugation rate is 5000r/min and the centrifugation time is 10min to obtain yeast;

wherein, the PDB liquid culture medium comprises the following components: 1L of water, 4.0g of potato extract and 20.0g of glucose, and adjusting the pH to 5.6 +/-0.2;

the OD value was 0.8 when the culture was completed.

The OD value testing method specifically comprises the following steps: in a spectrophotometer, the OD value was directly measured with pure water as a reference reagent in a visible light region a of 600 nm.

(2) Treating the dendrobium raw material:

cleaning fresh dendrobium officinale stems, pulping, adding water into the obtained dendrobium pulp for blending, and blending according to the mass-volume ratio of the fresh dendrobium to the water of 1:10 to obtain the dendrobium raw material.

(3) Enzymolysis:

heating the dendrobe raw material prepared in the step (2) to 80 ℃, adding high-temperature amylase according to the mass ratio of 1:10000 (based on the total mass of the dendrobe raw material after the preparation), stirring for enzymolysis reaction, cooling to 40 ℃ after the enzymolysis for 1h, adding pepsin according to the ratio of 1:10000 (based on the total mass of the dendrobe raw material after the preparation), and reacting for 4 h.

(4) Fermentation:

taking all products obtained after enzymolysis in the step (3), and adding nutrient substances by taking the products as a quality reference:

carbon source: according to the carbon source: adding glucose into an enzymolysis product in a mass ratio of 1: 10; nitrogen source: according to the nitrogen source: adding soybean hydrolysate into the enzymolysis product in the ratio of 1: 20; stirring, heating at 100 deg.C for sterilizing for 30min to obtain culture medium, and cooling to room temperature;

then, the product obtained in the step (1): adding the activated saccharomyces boulardii into the culture medium according to the mass-volume ratio of 1:1000 (based on the total mass of the culture medium), fermenting at the fermentation temperature of 28 ℃, the ventilation volume of 50L/min, the rotation speed of 100r/min, and fermenting and culturing for 2 days.

(5) Post-treatment

Filtering with a plate frame of 200-400nm after fermentation is finished, filtering with a 10000D membrane to obtain a clarified dendrobe fermentation product, and sterilizing at 130 ℃ for 3s to obtain the final clarified liquid of the dendrobe fermentation product.

Example 2

(1) Activating and culturing strains:

A. activation of saccharomyces cerevisiae: adopting saccharomyces cerevisiae: saccharomyces cerevisiae A3.12(Saccharomyces cerevisiae A3.12), picking a ring of colony in the inclined plane, putting the ring into PDB liquid culture medium, performing activated culture at 30 ℃ on a shaking table, finishing the culture when the shaking table is cultured for 20h at the rotating speed of 200r/min, and performing centrifugal separation to obtain the saccharomycete thallus.

The OD value was 0.7 when the culture was completed.

B. activating acetic acid bacteria: mixing acetic acid bacteria: a, selecting a loop of saccharomycete Alopectinatus AYC1.2 (Komagataibacterium saccharolyticum AYC1,2) with the preservation number of CCTCC NO: M2019569, performing activated shaking bed culture at 30 ℃ for 24h, performing centrifugal separation to obtain acetic acid bacteria thallus,

wherein, the components of the culture medium comprise: 1L of water, 1 percent of yeast extract, 1 percent of glucose, 1.5 percent of CaCO3 and 3 percent of absolute ethyl alcohol, and adjusting the pH value to 5.5;

C. activating lactobacillus plantarum: mixing the lactobacillus plantarum: lactobacillus plantarum RPC5(Lactobacillus plantarum RPC5) with preservation number of CCTCC NO: M2019566, selecting a ring, inoculating into corresponding MRS liquid culture medium, standing at 35 deg.C for 24 hr, centrifuging to obtain Lactobacillus thallus,

wherein the MRS liquid culture medium comprises the following components: 1L of water, 0.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 801.0mL of tween, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate and 0.25g of manganese sulfate, and the pH value is adjusted to 6.2-6.6.

(2) Treating the dendrobium raw material:

pulverizing dried stem of Dendrobium nobile, sieving with 60 mesh sieve, pulverizing flower of Dendrobium nobile, and mixing with the powder of Dendrobium nobile: mixing the dendrobium flowers at a ratio of 10:1, and then blending according to a mass-volume ratio of the dendrobium mixed powder to water of 1:200 to obtain the dendrobium raw material.

(3) Enzymolysis:

heating the dendrobe raw material modulated in the step (2) to 40 ℃, adding saccharifying enzyme according to the mass ratio of 1:1000 (based on the total mass of the modulated dendrobe raw material), stirring for enzymolysis, and performing enzymolysis for 2 hours;

and then adding cellulase according to the mass ratio of 1:10000 (based on the total mass of the modulated dendrobium raw material), and carrying out enzymolysis for 2 hours, wherein the temperature is kept unchanged to obtain an enzymolysis product.

(4) Fermentation of

Taking all products obtained by enzymolysis in the step (3), and adding nutrient substances by taking the products as a quality reference:

carbon source: according to the carbon source: adding sucrose into the enzymolysis product in a mass ratio of 1: 10; nitrogen source: according to the nitrogen source: adding yeast hydrolysate according to the mass ratio of 1:50 of the enzymolysis product, and adding the yeast hydrolysate into the enzymolysis product according to the nitrogen source: adding ammonium sulfate into the enzymolysis product in a mass ratio of 1: 200; stirring, heating at 120 deg.C, and sterilizing for 15min to obtain culture medium.

And (2) after the temperature is cooled to room temperature, adding the saccharomyces cerevisiae activated in the step (1) according to the mass-to-volume ratio of 1:1000 (calculated by the total mass of the culture medium), acetic acid bacteria according to the mass-to-volume ratio of 1:1000 (calculated by the total mass of the culture medium), and lactic acid bacteria according to the mass-to-volume ratio of 1:1000 (calculated by the total mass of the culture medium) respectively, then carrying out simultaneous fermentation, wherein the fermentation temperature is 30 ℃, the ventilation volume is 8L/min, the rotating speed is controlled at 20r/min, and the fermentation culture is carried out for 5 days.

(5) And (3) post-treatment:

after fermentation, firstly filtering by using a plate frame at 200-400nm, and then carrying out freeze drying treatment: and (3) placing the fermentation liquor in a material tray, performing normal freeze-drying process in a freeze dryer, and taking out the product after freeze-drying to obtain the dendrobium fermentation product freeze-dried powder.

Example 3

(1) Activating and culturing strains:

A. adopting candida: candida C1.7(Wickerhamomyces anomalus C1.7) with preservation number of CCTCC NO: M2017782, selecting a ring from the slant colony, placing the ring into a liquid culture medium, performing shake culture at 28 ℃ for 36h on a shaker, performing centrifugal separation to obtain yeast thallus,

wherein, the culture medium comprises the following components: 1L of water, 4.0g of potato extract and 20.0g of glucose, and adjusting the pH to 5.6 +/-0.2; (ii) a

The OD value at the end of the culture was 0.75 by detection.

B. Adopting Aspergillus niger: aspergillus niger d5.23(Aspergillus niger d5.23), with preservation number of CCTCC NO: M2017756, selecting a ring, inoculating into liquid culture medium, shake culturing at 30 deg.C for 24h to obtain Aspergillus niger thallus.

Wherein, the solid culture medium comprises the following components: 1L of water, 5g of peptone, 10g of glucose, 1g of monopotassium phosphate, 0.5g of magnesium sulfate and 0.1g of chloramphenicol, and the final pH is 5.6 +/-0.2.

C. Adopting lactobacillus pentosus: lactobacillus pentosus 001(Pediococcus pentosaceus 001) with CCTCC NO of M2017625, selecting a loop from the colony, inoculating into MRS liquid culture medium, standing and culturing at 35 deg.C for 24h, and centrifuging to obtain the thallus.

Wherein, the liquid culture medium comprises the following components: 1L of water, 0.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 801.0mL of tween, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate and 0.25g of manganese sulfate, and the pH value is adjusted to 6.2-6.6;

the OD value at the end of the culture was 0.6 by detection.

(2) Treating the dendrobium raw material: preparing the extract of Dendrobium nobile

Pulverizing dried stem of Dendrobium fimbriatum, sieving with 60 mesh sieve, pulverizing flower of Dendrobium nobile, and mixing with herba Dendrobii stem: mixing the dendrobium flower with the powder in a ratio of 3:1, and mixing the dendrobium flower with the powder: extracting 75% ethanol water solution at a mass-to-volume ratio of 1:10 at 60 deg.C for 6h, vacuum-pumping, vacuum-concentrating until the dry matter concentration reaches 20%, spraying powder at 120 deg.C and 90 deg.C to obtain herba Dendrobii extract.

(3) Enzymolysis:

and (3) mixing all the dendrobium extracts obtained in the step (2) with water according to the mass volume ratio of 1:10, adding the dendrobium into water to obtain a dendrobium raw material, and heating to 80 ℃;

high-temperature amylase is prepared by the following steps of (by mass ratio) mixing: adding high-temperature amylase into the dendrobium according to the proportion of 1:10000, stirring for enzymolysis, and performing enzymolysis for 30 min;

adjusting the temperature to 50 ℃, and mixing the following components in percentage by mass (based on the total mass of the dendrobium raw material): adding bromelain into the dendrobium raw material 1:1000, and hydrolyzing for 4 h;

then adding ligninase according to the mass ratio (calculated by the total mass of the dendrobium raw materials) of 1:10000, and hydrolyzing for 2h at the hydrolysis temperature of 55 ℃.

(4) Fermentation of

carbon source: according to the carbon source: adding hydrolyzed starch into the enzymolysis product according to the mass ratio of 1: 100; nitrogen source: according to the nitrogen source: adding ammonium chloride into the enzymolysis product at a mass ratio of 1:50, mixing and stirring the above materials uniformly, and heating and sterilizing at 80 ℃ for 60 min; the above material was equally distributed into three fermenters 1-3 of 5L volume.

After the temperature is cooled, respectively inoculating three strains obtained by the culture in the step (1) into three fermentation tanks: the candida is prepared by mixing candida: the mass-volume ratio of the culture medium is 1:500, and Aspergillus niger is added according to the weight ratio of Aspergillus niger: the mass-volume ratio of the culture medium is 1:10000, and the lactobacillus is prepared by mixing the following components in percentage by mass: adding culture medium with volume ratio of 1:1000 into respective fermentation tanks 1-3 for fermentation; wherein:

the fermentation temperature of the candida is 31 ℃, the ventilation volume is 20L/min, the rotating speed is controlled at 80r/min, and the fermentation culture is carried out for 2 days (24 h);

fermenting Aspergillus niger at 33 deg.C, ventilating amount of 65L/min, rotation speed of 50r/min, and fermenting and culturing for 3 days (36 h);

fermenting and culturing lactobacillus pentosus for 3 days (36h) at 36 deg.C, 5L/min ventilation and 10r/min rotation speed.

After the fermentation of the substances in the three fermentors is finished, the fermentation products in the three fermentors are mixed uniformly and heated at 90 ℃ for 60min to obtain a fermentation mixture.

(5) Post-treatment

Concentrating the fermentation mixture obtained in the step (4), and then vacuumizing and concentrating until the dry matter reaches 30%; and (4) drying in a tower, wherein the air inlet temperature is 115 ℃, the air outlet temperature is 95 ℃, and powder spraying is carried out to obtain the dendrobium fermentation product freeze-dried powder.

Example 4

(1) Activating and culturing strains:

A. and (3) adopting envelope-covering yeast: saccharomycopsis fibuligera d6.16 (Saccharomyces fibuligera d6.16) with preservation number of CCTCC NO of M2019570, placing a ring of colony in the inclined plane into liquid culture medium, performing shake culture at 28 deg.C for 36 hr, centrifuging to obtain saccharomycete thallus

Wherein, the components of the culture medium comprise: 1L of water, 4.0g of potato extract and 20.0g of glucose, and adjusting the pH to 5.6 +/-0.2; (ii) a

The OD value at the end of the culture was 0.76 by detection.

B. Mixing acetic acid bacteria: the culture medium is prepared by selecting a ring of saccharomycetous colal bacillus AYC1.2 (Komagataibacterium saccharolyticum AYC1.2) with the preservation number of CCTCC NO: M2019569, inoculating into a liquid culture medium, and performing activated shake culture at 30 ℃ for 24h to obtain acetic acid bacteria thallus;

wherein, the components of the culture medium comprise: adjusting the pH value to 5.5 by using 1L of water, 1% of yeast extract, 1% of glucose, 1.5% of CaCO3 and 3% of absolute ethyl alcohol;

the OD value at the end of the culture was 0.67 by detection.

(2) Preparation of dendrobe raw material

The stem of dendrobium chrysotoxum is dried, pulverized and sieved by a 100-mesh sieve to obtain dendrobium fine powder.

(3) Fermentation of

Adding all the dendrobium fine powder obtained in the step (2) into water according to a ratio of 1:50, mixing to obtain a dendrobium raw material, and adding nutrient substances by taking the total mass of the mixed dendrobium raw material as a reference:

carbon source: according to the carbon source: adding glucose into the dendrobium raw material in a mass ratio of 1: 100; nitrogen source: according to the nitrogen source: beef extract is added into the dendrobium according to the mass ratio of 1: 100. Mixing the above materials uniformly;

distributing the mixed materials into three 5L fermentation tanks, and heating and sterilizing at 80 deg.C for 60 min;

after the temperature is cooled to room temperature, respectively inoculating the two activated strains obtained in the step (1) and a commercial strain BB12 (the commercial strain is not required to be activated and can be directly used) into three fermentation tanks, wherein the specific inoculation mode is as follows:

inoculating the envelope-buckled compound yeast: the yeast is fermented at the temperature of 28-30 ℃ and the ventilation rate of 50-60L/min according to the mass-volume ratio of 1:1000, the rotating speed is controlled at 80r/min, and the fermentation time is 2 days;

inoculating acetic acid bacteria; adding into a fermentation tank according to the mass-to-volume ratio of 1:1000, at the temperature of 30-32 ℃, at the rotating speed of 100r/min, for 2 days, and supplementing sucrose at one time according to the ratio of 1:50 (the sucrose is sterilized at 120 ℃ for 15min in advance) based on the total mass of the culture medium;

inoculating commercial bacteria BB12 (commercial strains are not required to be activated, and can be used directly): fermenting at 36 deg.C and 5L/min of ventilation rate at 10r/min for 3 days at a ratio of 1: 1000.

The three fermentation products are mixed evenly without sterilization treatment.

(5) Post-treatment

And (4) directly freeze-drying the mixed fermentation product obtained in the step (4) to obtain the dendrobium fermentation product freeze-dried powder.

Example 5

Steps (1) to (2) are the same as in example 1;

(3) heating all the dendrobe raw materials prepared in the step (2) to 100 ℃, adding high-temperature amylase according to the ratio of 1:1000 (enzymolysis agent: the total volume of the prepared dendrobe raw materials), stirring for enzymolysis reaction, cooling to 40 ℃ after enzymolysis for 1h, adding pepsin according to the ratio of 1:1000, and hydrolyzing for 4 h.

(4) Taking the whole mixture obtained in the step (3) after enzymolysis, and adding nutrient substances by taking the mixture as a quality reference:

carbon source: adding glucose according to the proportion of 1: 10; nitrogen source: adding soybean hydrolysate according to the ratio of 1: 20; and adding glycerol according to the proportion of 1: 20; stirring, and sterilizing at 100 deg.C for 30 min.

And (2) after the temperature is cooled to room temperature, adding the activated saccharomyces boulardii obtained in the step (1) according to the mass-volume ratio of 1:1000 (based on the total mass after stirring) for fermentation, wherein the fermentation temperature is 28 ℃, the ventilation volume is 50L/min, the rotation speed is controlled at 100r/min, and the fermentation culture is carried out for 2 d.

Step (5) is the same as example 1, and finally the clear liquid of the dendrobium fermentation product is obtained.

Example 6

(1) Activating and culturing strains:

A. activating the envelope-buckled yeast: adopting saccharomycete d6.16 (Saccharomyces fibuligera d6.16) with preservation number of CCTCC NO of M2019570, putting the bacterial colony in the inclined plane into PDB liquid culture medium, activating at 28 ℃ on a shaking bed, culturing for 12h at the rotation speed of the shaking bed of 180r/min, centrifugally separating to obtain saccharomycete thallus,

wherein, the PDB liquid culture medium comprises the following components: 1L water, 4.0g potato extract, 20.0g glucose, and adjusting pH to 5.6 + -0.2

Detecting to obtain a fermentation thallus system with OD value of 0.8;

B. activating acetic acid bacteria: mixing acetic acid bacteria: the saccharomycete foal shaped bacillus AYC1.2 (Komagataibacter saccharolyticus AYC1.2) with preservation number of CCTCC NO: M2019569, selecting a ring, synchronously inoculating into liquid culture medium, activating shaking bed at 30 deg.C for 24h, centrifuging to obtain acetic acid bacteria thallus,

wherein the culture medium comprises the following components: adding 1L of water according to 10 yeast extract, 10 glucose, 15CaCO3 and 30 anhydrous ethanol, and adjusting pH to 5.5;

C. activating lactobacillus acidophilus: selecting Lactobacillus acidophilus 001(Lactobacillus acidophilus 001) with preservation number of CCTCC NO: M2017628, synchronously inoculating a ring of Lactobacillus acidophilus to MRS liquid culture medium, standing and culturing at 35 ℃ for 24h, and centrifuging to obtain Lactobacillus thallus;

wherein, the MRS culture medium comprises the following components: 1L of water, 0.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 801.0mL of Tween, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate and 0.25g of manganese sulfate, adjusting the pH to 6.2-6.6

(2) Treating the dendrobium raw material:

pulverizing dried Dendrobium officinale stems, sieving with a 60-mesh sieve, pulverizing herba Dendrobii flowers, and mixing the powder with the powder according to the weight ratio of herba Dendrobii stems: mixing the dendrobium flowers at a ratio of 5:1, and then blending according to the mass-volume ratio of the dendrobium mixed powder to water of 1: 100.

(3) Enzymolysis:

heating the dendrobe raw material modulated in the step (2) to 50 ℃, adding saccharifying enzyme according to a ratio of 1:1000, stirring for enzymolysis, and performing enzymolysis for 2 hours; and then adding cellulase according to the mass ratio of 1:1000 (enzyme to dendrobium raw material). Enzymolysis is carried out for 4 hours again, and the temperature is kept unchanged.

(4) Fermentation of

Taking the mixture obtained in the step (3) after enzymolysis, and adding nutrient substances by taking the total mass as a reference:

carbon source: adding sucrose according to the proportion of 1: 10; nitrogen source: adding yeast hydrolysate according to a ratio of 1:50, and adding ammonium sulfate according to a ratio of 1: 20; stirring, heating at 120 deg.C, and sterilizing for 15 min.

After the temperature is cooled to room temperature, carrying out aerobic fermentation on the saccharomyces cerevisiae activated in the step (1) for 24 hours at 30 ℃ according to the mass-to-volume ratio of 1:1000, controlling the ventilation volume to be 2L/min and the rotating speed to be 80r/min, and heating to 90 ℃ for 30min after the fermentation is finished;

adjusting pH to 6.0-6.5 when the temperature is reduced to 30 ℃, inoculating acetic acid bacteria, adding the acetic acid bacteria according to the mass volume ratio of 1:1000, carrying out aerobic fermentation at 30 ℃ for 36h, controlling the ventilation volume at 8L/min and the rotating speed at 20r/min, and heating to 90 ℃ for 30min after the fermentation is finished;

adjusting pH to 6.0-6.5 when the temperature is reduced to 35 deg.C, adding lactobacillus acidophilus at 35 deg.C for 24 hr, and performing anaerobic fermentation.

(5) And (3) post-treatment:

and after the fermentation is finished, filtering and clarifying by using a plate frame to obtain the dendrobium fermentation filtrate.

Comparative example 1

Steps (1) to (2) are the same as in example 1;

(5) heating the dendrobe raw material prepared in the step (2) to 100 ℃, adding high-temperature amylase according to the ratio of 1:10000 (based on the total mass of the prepared dendrobe raw material), stirring for enzymolysis reaction, cooling to 40 ℃ after enzymolysis for 1h, adding pepsin according to the ratio of 1:10000, and hydrolyzing for 4 h.

(6) Taking the whole mixture obtained in the step (3) after enzymolysis, and adding culture medium substances by taking the mixture as a mass standard:

carbon source: adding glucose according to the proportion of 1: 200; nitrogen source: adding soybean hydrolysate according to the proportion of 1: 200; and adding glycerol according to the proportion of 1: 20; stirring, and sterilizing at 100 deg.C for 30 min.

Comparative example 2

Steps (1) to (2) are the same as in example 1;

(7) heating all the dendrobium raw materials prepared in the step (2) to 50 ℃, adding an enzymolysis agent into high-temperature amylase according to the ratio of 1:1000 for enzymolysis, and adding a culture medium substance by taking the mass as a reference:

And (2) after the temperature is cooled to room temperature, adding the activated saccharomyces boulardii obtained in the step (1) according to the mass-volume ratio of 1:1000 (based on the total mass after stirring) for fermentation, wherein the fermentation temperature is 20 ℃, the ventilation volume is 50L/min, the rotation speed is controlled at 10r/min, and the fermentation culture is carried out for 2 d.

Comparative example 3

(1) The commercially available Lactobacillus plantarum LP90 (Shanghai purple stone Biotech Co., Ltd.) was used as it was without activation.

(2) Treating the dendrobium raw material:

(3) Enzymolysis:

heating all the dendrobium raw materials modulated in the step (2) to 50 ℃, adding saccharifying enzyme according to the ratio of 1:1000, stirring for enzymolysis, and carrying out enzymolysis for 2 hours; and then adding cellulase according to the mass ratio of 1:1000 (enzyme to dendrobium raw material). Enzymolysis is carried out for 4 hours again, and the temperature is kept unchanged.

(4) Fermentation of

Taking the mixture obtained in the step (3) after enzymolysis, and adding culture medium substances by taking the total mass as a reference:

carbon source: adding sucrose according to the proportion of 1: 10; nitrogen source: adding yeast hydrolysate according to a ratio of 1: 50; stirring, heating at 120 deg.C, and sterilizing for 15 min.

After the temperature is cooled to room temperature, adding the commercial lactobacillus plantarum according to the mass-to-volume ratio of 1:2000, and then carrying out aerobic fermentation, wherein the fermentation temperature is 35 ℃, the fermentation time is 48h, and the stirring speed is 10 r/min.

(5) And (3) post-treatment: and after the fermentation is finished, plate-frame filtering and clarifying, vacuum concentrating and drying until the dry matter is 30%, and spray drying to obtain the dendrobium fermentation liquor powder.

Comparative example 4

In this embodiment, the raw materials and operations in example 2 are adopted, but the enzymolysis in step (3) is not performed, and after the dendrobium raw material is obtained in step (2), nutrients required for fermentation are added by taking the dendrobium raw material as a quality standard, and fermentation is performed to finally obtain a fermentation product.

In order to verify the performance of the dendrobium fermentation product prepared by the invention, DPPH free radical clearance and tyrosinase inhibition rate tests are respectively carried out on the products before fermentation (enzymolysis products, if enzymolysis is not carried out, the dendrobium raw material) and after fermentation in the above examples and comparative examples, and the test results are summarized in the following table.

TABLE 2 summary of herba Dendrobii fermentation product Properties

As can be seen from the above table;

(1) compared with the dendrobe raw material, the dendrobe fermented product provided by the embodiments 1-6 of the invention has the free radical clearance rate promotion rate of 176-; compared with the dendrobium raw material, the dendrobium fermentation product in the comparative examples 1-4 has the advantages that the free radical clearance rate is increased by 96.8-194%, the tyrosinase inhibition rate is increased by 39.5-78.5%, and the free radical clearance rate and the tyrosinase inhibition rate of the product obtained in the comparative examples are both lower than the increase rate of the dendrobium fermentation product provided by the embodiment of the invention;

(2) in the dendrobe fermentation products provided in embodiments 1 to 6 of the invention, the increase rates of the free radical clearance rate and the tyrosinase inhibition rate of the dendrobe fermentation products provided in embodiments 2 and 6 are both about 300%, and are both more than 200%.

The performance test of the prepared dendrobe fermentation product comprises the following steps: the DPPH free radical clearance rate and the tyrosinase inhibition rate are tested by the following specific operations:

(1) evaluation of antioxidant Activity based on DPPH free radical scavenging ability

Preparing a DPPH standard solution: 0.003g of DPPH is weighed in a beaker, dissolved in a proper amount of absolute ethyl alcohol, ultrasonically treated for 5min, transferred to a volumetric flask to be constant volume of 50mL, and fully and uniformly mixed. Storing in dark. (3.5h used up)

Determination of the concentration of the sample solution:

the method comprises the following steps: DPPH3.0mL, the sample liquid is dripped, and the sample addition amount is recorded when the color of the solution basically fades.

Step two: setting 5-6 concentration gradients forwards by taking the sample adding amount in the step one as the maximum sample adding amount; supplementing to 3mL with distilled water; 3.0mL of DPPH solution was added; mixing uniformly; standing in dark for 30 min. Taking out and measuring the A value.

Settings on controls:

TABLE 3

Calculation for clearance:

the clearance S (%) [ (A0- (Ai-Aj))/A0 ]. multidot.100%

Plotted, IC50 values were calculated. Note that the R2 value of the standard curve needs to be above 0.99.

(2) Tyrosinase inhibitory activity test method

Material

Tyrosinase (reagent: 25 ku-20 ℃ preservation), L-tyrosine, disodium hydrogen phosphate, sodium dihydrogen phosphate, a thermostat, an ultraviolet spectrophotometer and a centrifuge.

Reagent preparation

Sodium phosphate buffer (1/15mol/L, pH 6.8)

1.000g of sodium dihydrogen phosphate and 1.186g of disodium hydrogen phosphate are accurately weighed, a small amount of deionized water is added for dissolution, the volume is determined to be 500mL, and the mixture is stored in a refrigerator at 4 ℃ for later use.

L-tyrosine solution (7.5mmol/L)

Accurately weighing 0.136g of L-tyrosine, firstly adding a plurality of drops of concentrated hydrochloric acid, adding about 50mL of deionized water, heating at 90 ℃ to completely dissolve, then adjusting the pH to about 7 by using a sodium hydroxide solution, adding deionized water to a constant volume to a 100mL brown volumetric flask, and storing in a dark place.

Preparation of tyrosinase liquid

Reagent preparation: dissolving the solid powder enzyme reagent in a small packaging bottle by using distilled water, sucking out the solid powder enzyme reagent by using a rubber head straw, transferring the solid powder enzyme reagent into a 250ml volumetric flask, continuously repeating the dissolving and transferring processes until the sucked enzyme liquid is changed from yellow brown to clear and transparent, and finally fixing the volume of 250ml in the volumetric flask to obtain 100u/ml enzyme liquid. 250ml of enzyme solution is subpackaged by a small centrifuge tube and stored at-20 ℃. The test piece is taken out as required in the experimental process and is used after being unfrozen.

Detection method

The total reaction system was 5 mL. The specific design is shown in Table l.

During the experiment, phosphate buffer solution, test solution with different concentration gradients and enzyme solution are sequentially added into a test tube, and water bath is carried out for 10min at 30 ℃. The substrate L-tyrosine was then added and the timer was started immediately. The absorbance at a wavelength of 475nm at 40min of the reaction was determined. During the determination, the inhibition rate of the test solution on tyrosinase is calculated by using the following formula with the corresponding negative control as a reference.

The inhibition rate was [ (A-B)/A ]. times.l 00%

Wherein, A is the light absorption value of the standard control, and B is the light absorption value of the test solution. Each experiment was done in 3 replicates. The high inhibition rate indicates that the inhibition intensity of the compound on tyrosinase activity is high.

TABLE 15mL test System design

Note: when the absorbance is measured by a spectrophotometer, the "test solution" and the "standard control" are respectively zeroed by the "negative control 1" and the "negative control 2".

Claims

1. A herba Dendrobii fermented product with oxidation resistance and immunity enhancing effect is prepared by fermenting herba Dendrobii, enzymolysis agent and zymophyte;

2. The fermented product of dendrobium nobile according to claim 1, wherein the dendrobium nobile in the raw material is selected from one or more than two of dendrobium officinale, dendrobium nobile, dendrobium chrysotoxum and dendrobium fimbriatum, preferably dendrobium officinale;

3. The fermented product of dendrobe of claim 1 or 2, wherein the proteolytic enzyme in the enzymolysis agent is selected from one or more of papain, bromelain, trypsin, pepsin and peptidase, preferably bromelain and/or pepsin;

4. The fermented product of dendrobe of any one of claims 1 to 3, wherein the yeast in the fermented thallus is selected from one or more of Saccharomyces cerevisiae, Bld, Pichia pastoris, Candida or Saccharomycopsis fibuligera; preferably selected from the group consisting of Bld, Candida or Babylonia, more preferably Candida or Babylonia;

5. A preparation method of the dendrobe leavening of any one of claims 1 to 4, which is characterized by comprising the following steps:

(1) activating and culturing the fermentation thalli;

(2) processing a dendrobe raw material;

(3) performing enzymolysis on the dendrobium processed in the step (2);

6. The preparation method according to claim 5, wherein the step (4) of inoculating the activated zymophyte of step (1) into the enzymolysis product of step (3) for fermentation comprises at least one of the following four fermentation methods:

7. The process according to claim 5 or 6, wherein the culture for activating the fermentation product in the step (1) comprises:

8. The preparation method according to any one of claims 5 to 7, wherein the dendrobe raw material treatment in the step (2) comprises:

The method c, extracting the dendrobium;

9. The method according to claim 8, wherein in the method c, the reagent for extracting dendrobe is one or more selected from water, lower alcohols, higher alcohols, polyols, esters, ketones and hydrocarbon solvents;

preferably, the lower alcohols include methanol, ethanol and propanol;

preferably, the ketones include acetone and methyl ethyl ketone;

preferably, the ethers include diethyl ether and isopropyl ether;

10. The preparation method according to any one of claims 5 to 9, wherein the step (3) of performing enzymatic hydrolysis on the dendrobium processed in the step (2) comprises:

the pH of the enzymolysis environment is 2-9, preferably 4-7;

the enzymolysis time is 0.5-24h, preferably 2-6 h.

11. The preparation method according to claim 6, wherein the B-mix simultaneous fermentation comprises: a combination of aspergillus niger and saccharomycetes, or a combination of aspergillus niger, lactobacillus and acetic acid bacteria, or a combination of saccharomycetes and lactobacillus, or a combination of yeast and acetic acid bacteria;

12. The method according to any one of claims 5 to 11, wherein the activated fermentation thallus of step (1) is inoculated into the enzymolysis product of step (3) for fermentation in step (4),

13. The preparation method according to claim 12, wherein the activated fermentation thallus of step (1) is inoculated into the enzymolysis product of step (3) for fermentation in step (4), and the addition amount of the activated fermentation thallus is 1:10000-1:100g/mL based on the total volume of the fermentation medium;

14. A dendrobe fermented product prepared by the preparation method of any one of claims 5 to 13.

15. An external dendrobium nobile fermented product for skin care, which is prepared by the preparation method of any one of claims 5 to 13;

16. The dendrobe fermented product of any one of claims 1 to 4 or claim 14 or the skin-care external dendrobe fermented product of claim 15, and the application of the dendrobe fermented product in the fields of skin care products, cosmetics, medicines, health foods and foods.