CN114196549B - Cordyceps militaris fungus chaff fermentation broth and preparation method thereof - Google Patents
Cordyceps militaris fungus chaff fermentation broth and preparation method thereof Download PDFInfo
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- OFEZSBMBBKLLBJ-BAJZRUMYSA-N cordycepin Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)C[C@H]1O OFEZSBMBBKLLBJ-BAJZRUMYSA-N 0.000 description 1
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
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- A61P3/00—Drugs for disorders of the metabolism
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- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses cordyceps militaris fungus chaff fermentation broth and a preparation method thereof. Inoculating cordyceps militaris fungus chaff into aspergillus oryzae for solid fermentation to obtain a first fermentation product; carrying out enzymolysis on the first fermentation product to obtain an enzymolysis liquid; extracting the enzymolysis liquid with water to obtain an extraction liquid; mixing the supernatant of the extract with Saccharomyces cerevisiae, and performing liquid fermentation to obtain Cordyceps militaris fungus chaff fermentation broth. The active ingredients in the cordyceps militaris fungus chaff fermentation broth can effectively remove OH ‑ 、O 2 ‑ And H 2 O 2 And has a certain inhibition effect on the activity of alpha-glucosidase, and has the functions of oxidation resistance and saccharification resistance.
Description
Technical Field
The invention relates to cordyceps militaris fungus chaff fermentation broth, and a preparation method and application thereof, and belongs to the technical field of microbial fermentation.
Background
Cordyceps militaris (Cordyceps millitaris), also known as Cordyceps militaris, belongs to the phylum of fungi, class Nuclear fungus, order of spherical shell, genus Cordyceps, is an abbreviation of Cordyceps militaris, and is a rare medicinal fungus and a modern rare Chinese herbal medicine in China. The Cordyceps militaris is rich in various active ingredients such as Cordyceps polysaccharide, nucleoside compounds, superoxide dismutase (SOD enzyme), vitamins and the like, has the effects of bacteriostasis, anti-inflammation, anti-tumor, antivirus, antioxidation and the like, and has been widely applied in the pharmaceutical industry. Along with the development of the cordyceps industry, the industrial production scale of the cordyceps is gradually enlarged, and the yield of the residual substrate (namely cordyceps militaris fungus chaff) cultured by the cordyceps militaris stroma after the cordyceps militaris fruiting body is harvested is also continuously increased. The cordyceps militaris fungus chaff contains various nutritional ingredients such as protein, polysaccharide, amino acid, cordycepic acid, cordycepin, ergosterol, galactomannan, uracil, adenine and the like as the cordyceps militaris fruiting body, and also contains a large amount of unused rice, wheat starch and the like, so that the cordyceps militaris fungus chaff has extremely high utilization value, but the cordyceps militaris fungus chaff is not fully developed and utilized due to the lack of effective and feasible utilization ways and technologies. Therefore, development of a utilization scheme for changing cordyceps militaris fungus chaff into a treasure is urgently needed.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the cordyceps militaris fungus chaff fermentation broth is provided, polysaccharide in the cordyceps militaris fungus chaff is obtained, secondary utilization of the cordyceps militaris fungus chaff is realized, and waste materials of the cordyceps militaris fungus chaff are changed into things of value.
In order to solve the problems, the invention provides the following technical scheme:
the invention provides a preparation method of cordyceps militaris fungus chaff fermentation broth, which comprises the following steps: inoculating cordyceps militaris fungus chaff into aspergillus oryzae for solid fermentation to obtain a first fermentation product; carrying out enzymolysis on the first fermentation product to obtain an enzymolysis liquid; extracting the enzymolysis liquid with water to obtain an extraction liquid; mixing the supernatant of the extract with Saccharomyces cerevisiae, and performing liquid fermentation to obtain Cordyceps militaris fungus chaff fermentation broth.
Preferably, the solid fermentation time is 48-72 h, and the solid fermentation temperature is 28-30 ℃.
Preferably, the mass ratio of the aspergillus oryzae to the cordyceps militaris fungus chaff is 0.0002-0.0004: 1.
preferably, the enzymolysis comprises the step of carrying out enzymolysis by using amylase contained in the first fermentation product, wherein the temperature of the enzymolysis is 40-50 ℃, the pH value of the enzymolysis is 6, and the enzymolysis time is 3-7 h.
Preferably, the extraction temperature is 85-100 ℃, and the extraction time is 1-3 h.
Preferably, the temperature of the liquid fermentation is 28-30 ℃, and the time of the liquid fermentation is 48-72 hours.
Preferably, the mass ratio of the saccharomyces cerevisiae to the cordyceps militaris fungus chaff is 0.004:1.
preferably, the preparation method further comprises: before inoculating aspergillus oryzae, mixing the cordyceps militaris fungus chaff with water for gelatinization treatment; when pasting, the ratio of the cordyceps militaris fungus chaff to the water is 1g: 2.4-4.8 mL.
The invention also provides the cordyceps militaris fungus chaff fermentation broth obtained by the preparation method.
The invention also provides application of the cordyceps militaris fungus chaff fermentation broth in preparing antioxidant and/or anti-saccharification food, health-care product or medicine.
According to the invention, by utilizing the characteristic of amylase production by aspergillus oryzae fermentation, macromolecular sugar such as starch in cordyceps militaris fungus chaff is decomposed into micromolecular sugar, and then the micromolecular sugar such as reducing sugar is converted into polysaccharide by saccharomyces cerevisiae fermentation, so that 11.94-12.21 g of polysaccharide is produced by fermenting every 100g of cordyceps militaris fungus chaff, the polysaccharide content in fermentation broth is improved, and the secondary utilization of cordyceps militaris fungus chaff is realized.
Drawings
FIG. 1 shows the fermentation broth pair OH of Cordyceps militaris fungus chaff of the invention - Is a cleaning effect of (a);
FIG. 2 shows the fermentation broth pair O of Cordyceps militaris and fungus chaff of the invention 2 - Is a cleaning effect of (a);
FIG. 3 shows the fermentation broth pair H of Cordyceps militaris fungus chaff of the invention 2 O 2 Is a cleaning effect of (a);
FIG. 4 shows the effect of the fermentation broth of Cordyceps militaris chaff on inhibiting alpha-glucosidase.
Detailed Description
The invention provides a preparation method of cordyceps militaris fungus chaff fermentation broth, which comprises the following steps:
inoculating Aspergillus oryzae with Cordyceps militaris bran, and performing solid fermentation to obtain a first fermentation product;
carrying out enzymolysis on the first fermentation product to obtain an enzymolysis liquid;
extracting the enzymolysis liquid with water to obtain an extraction liquid;
mixing the supernatant of the extract with Saccharomyces cerevisiae, and performing liquid fermentation to obtain Cordyceps militaris fungus chaff fermentation broth.
According to the invention, cordyceps militaris fungus chaff is inoculated with aspergillus oryzae for solid fermentation, so that a first fermentation product is obtained. The solid fermentation time of the invention is preferably 48 to 72 hours, more preferably 50 to 70 hours, even more preferably 55 to 65 hours, and most preferably 60 hours; the temperature of the solid fermentation is preferably 28-30 ℃; the mass ratio of the aspergillus oryzae to the cordyceps militaris fungus chaff is preferably 0.0002-0.0004: 1, more preferably 0.0004:1. The cordyceps militaris fungus chaff is preferably cordyceps militaris fungus chaff powder, and is further preferably dried cordyceps militaris fungus chaff powder. The particle size of the cordyceps militaris fungus chaff powder is preferably 80 meshes. The drying conditions are not particularly limited in the present invention, and a drying method well known in the art may be adopted. Through the solid fermentation, the invention has a plurality of enzymes such as amylase, protease, cellulase and the like which are generated by the aspergillus oryzae in a first fermentation product.
Before the cordyceps militaris fungus chaff is inoculated with the aspergillus oryzae, the cordyceps militaris fungus chaff is preferably mixed with water for gelatinization treatment, cooled and inoculated with the aspergillus oryzae for solid fermentation. When the cordyceps militaris fungus chaff is mixed with water for gelatinization treatment, the gelatinization temperature is preferably 121 ℃; the time of the gelatinization is preferably 15min. The mass volume ratio of the cordyceps militaris fungus chaff to the water is preferably 1g:2.4 mL-4.8 mL, more preferably 1g:3.0 mL-4.5 mL, more preferably 1g:3.5 mL-4.0 mL, most preferably 1g:3.6mL. The method of gelatinization of starch in the present invention is not particularly limited, and gelatinization methods well known in the art may be employed. When the cordyceps militaris fungus chaff powder is mixed with water to carry out starch gelatinization, sterilization is completed simultaneously. According to the invention, through pasting treatment of cordyceps militaris fungus chaff, water molecules can enter between starch microcrystal bundle structures, and finally starch comes out along with the water molecules, and starch is decomposed into saccharides by amylase produced by subsequent aspergillus oryzae.
The present invention is preferably cooled to 28 ℃ after gelatinization of the starch. The cooling mode is not strictly required, normal-temperature cooling can be performed in the invention, the growth of the aspergillus oryzae can be accelerated by cooling to the temperature, and the time of the aspergillus oryzae being fully paved with cordyceps militaris fungus chaff powder is shortened.
After the first fermentation product is obtained, the first fermentation product is subjected to enzymolysis to obtain an enzymolysis liquid. In the enzymolysis, the invention can decompose macromolecular sugar such as starch contained in cordyceps militaris fungus chaff into micromolecular sugar by directly utilizing a plurality of enzymes contained in the first fermentation product for enzymolysis without adding other enzymes. The enzymolysis temperature is preferably 40-50 ℃, more preferably 42-48 ℃, and even more preferably 45 ℃; the pH of the enzymolysis is preferably 6; the time for the enzymolysis is preferably 2 to 4 hours, more preferably 3 hours. In the enzymolysis process, the PBS buffer solution is preferably used for maintaining the pH of the enzymolysis process.
After the enzymatic hydrolysate is obtained, the enzymatic hydrolysate is preferably sterilized. The sterilization time of the invention is preferably 15min; the temperature of the sterilization is preferably 121 ℃.
The invention preferably uses water to extract the sterilized enzymolysis liquid to obtain the extracting liquid. In the invention, deionized water is preferably used when the sterilized enzymolysis liquid is extracted by water; the volume ratio of the water to the enzymolysis liquid is preferably 1-1. The temperature of the water according to the invention is preferably 100 ℃. The extraction time of the invention is preferably 2 hours; the number of extractions is preferably 1.
The invention preferably further comprises sterilization before the extraction, wherein the sterilization temperature is preferably 121 ℃; the sterilization time is preferably 15 minutes.
After the extracting solution is obtained, the extracting solution is preferably centrifuged to obtain the supernatant of the extracting solution. The rotation speed of the centrifugation is preferably 5000-8000 r/min, more preferably 6000-7000 r/min, and even more preferably 6500r/min; the time for the centrifugation is preferably 20 to 40 minutes, more preferably 25 to 35 minutes, and still more preferably 30 minutes.
After the supernatant of the extracting solution is obtained, the supernatant of the extracting solution is mixed with saccharomyces cerevisiae, and liquid fermentation is carried out to obtain cordyceps militaris fungus chaff fermentation broth. The temperature of the liquid fermentation is preferably 28-30 ℃; the time for the liquid fermentation is preferably 48 to 72 hours, more preferably 50 to 70 hours, still more preferably 55 to 65 hours, and most preferably 60 hours. The liquid fermentation mode of the invention is preferably shaking fermentation, and the speed of the shaking table is preferably 140rpm. The invention has no strict requirement on equipment used in the process of shaking table fermentation, and can be conventionally carried out.
In the present invention, when the supernatant of the extract is mixed with Saccharomyces cerevisiae, the Saccharomyces cerevisiae is preferably inoculated into the supernatant. The mass ratio of the saccharomyces cerevisiae inoculated by the invention to the cordyceps militaris fungus chaff is preferably 0.002-0.005:1, more preferably 0.003-0.005:1, and even more preferably 0.004:1. According to the invention, the Saccharomyces cerevisiae is used for carrying out liquid fermentation on the supernatant of the extracting solution, and the yeast uses small molecular sugar such as reducing sugar to generate polysaccharide, so that 11.94-12.21 g of polysaccharide is produced by fermenting every 100g of cordyceps militaris fungus chaff, the polysaccharide in the fungus chaff is extracted more efficiently, and the polysaccharide content in the fermenting solution is improved.
The cordyceps militaris fungus chaff fermentation broth provided by the invention contains high-concentration polysaccharide, so that the secondary utilization of the cordyceps militaris fungus chaff is realized, and the active ingredients in the fermentation broth can effectively remove OH - 、O 2 - And H 2 O 2 And has a certain inhibition effect on the activity of alpha-glucosidase, which indicates that the cordyceps militaris fungus chaff fermentation broth has the functions of antioxidation and anti-saccharification.
The invention has no special requirements on the source of the saccharomyces cerevisiae, and can be realized by adopting a mode well known in the field. The Saccharomyces cerevisiae of the present invention is preferably type YA200 or type YA300 Saccharomyces cerevisiae. The source of the Saccharomyces cerevisiae is not critical and can be obtained by purchase, preferably from Angel Yeast Co. The invention uses YA200 and YA300 type Saccharomyces cerevisiae to ferment, has higher clearance effect on DPPH, and the clearance rate of DPPH is as high as 92.63-99.08 percent, which is far higher than other types of yeast, such as YA600 type Saccharomyces cerevisiae and high wine type Saccharomyces cerevisiae.
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
Example 1
1) Drying, crushing and sieving Cordyceps militaris and fungus chaff with a 80-mesh sieve, accurately weighing 5g of Cordyceps militaris and fungus chaff powder in a triangular flask, adding 18mL of distilled water, and gelatinizing at 121 ℃ for 15min;
2) And (3) cooling to about 28 ℃, inoculating Aspergillus oryzae powder for solid fermentation, wherein the inoculum size of the Aspergillus oryzae is 0.002g, the fermentation time is 60h, and the fermentation temperature is 28 ℃ to obtain the fermentation fungus chaff.
3) Placing the fermentation fungus chaff into a centrifuge tube filled with 50mL of buffer solution for enzymolysis, wherein the enzymolysis temperature is 45 ℃, the enzymolysis pH is 6, the enzymolysis time is 3 hours, and the fermentation fungus chaff is sterilized for 15 minutes at 121 ℃ after the enzymolysis is finished;
4) After the sterilization is finished, 50mL of deionized water at 100 ℃ is added into the enzymolysis liquid, the enzymolysis liquid is extracted for one time, the extraction time is 2h, the centrifugation is carried out for 15min at 8000r/min, the supernatant is recovered, and the deionized water is added to the constant volume of 100mL;
5) Inoculating 0.02g of YA200 type Saccharomyces cerevisiae into the supernatant after volume fixing, fermenting at 30deg.C and 140rpm for 48 hr, sterilizing in water bath at 100deg.C for 30min after fermentation, and obtaining Cordyceps militaris fungus chaff fermentation broth.
Example 2
The difference from example 1 is that step 1) adds distilled water in a volume of 12mL.
Example 3
The difference from example 1 is that in step 1) the volume of distilled water added is 24mL.
Example 4
The procedure is as in example 1, except that the inoculum size of the Aspergillus oryzae in step 2) is 0.001g.
Example 5
The procedure is as in example 1, except that the inoculum size of the Aspergillus oryzae in step 2) is 0.0015g.
Example 6
The difference from example 1 is that the fermentation time in step 2) is 48h.
Example 7
The difference from example 1 is that the fermentation time in step 2) is 72h.
Example 8
The difference from example 1 is that the enzymolysis temperature in step 3) is 40 ℃.
Example 9
The difference from example 1 is that the enzymolysis temperature in step 3) is 50 ℃.
Example 10
The difference from example 1 is that the enzymolysis time in step 3) is 5 hours.
Example 11
The difference from example 1 is that the enzymolysis time in step 3) is 7h.
Example 12
The difference from example 1 is that in the case of the liquid fermentation in step 5), the fermentation time is 60 hours.
Example 13
The difference from example 1 is that in the case of the liquid fermentation in step 5), the fermentation time is 72 hours.
Example 14
The procedure is as in example 1, except that in step 5) 0.02g of Saccharomyces cerevisiae type YA300 is added.
Comparative example 1
The same as in example 1, except that only Aspergillus oryzae was used for fermentation, step 5) in example 1 was not performed, and Cordyceps militaris bran fermentation broth was obtained.
Comparative example 2
The difference from example 1 is that step 1) adds distilled water in a volume of 6mL.
Comparative example 3
The difference from example 1 is that step 1) adds distilled water in a volume of 30mL.
Comparative example 4
The procedure is as in example 1, except that the inoculum size of the Aspergillus oryzae in step 2) is 0.0005g.
Comparative example 5
The procedure is as in example 1, except that the inoculum size of the Aspergillus oryzae in step 2) is 0.0025g.
Comparative example 6
The difference from example 1 is that the fermentation time in step 2) is 12h.
Comparative example 7
The difference from example 1 is that the fermentation time in step 2) is 24h.
Comparative example 8
The difference from example 1 is that the fermentation time in step 2) is 36h.
Comparative example 9
The difference from example 1 is that the fermentation time in step 2) is 84h.
Comparative example 10
The difference from example 1 is that the fermentation time in step 2) is 96 hours.
Comparative example 11
The difference from example 1 is that the enzymolysis temperature in step 3) is 30 ℃.
Comparative example 12
The difference from example 1 is that the enzymolysis temperature in step 3) is 35 ℃.
Comparative example 13
The difference from example 1 is that step 3) has an enzymatic pH of 4.
Comparative example 14
The difference from example 1 is that step 3) has an enzymatic pH of 5.
Comparative example 15
The difference from example 1 is that step 3) has an enzymatic pH of 7.
Comparative example 16
The difference from example 1 is that step 3) has an enzymatic pH of 8.
Comparative example 17
The difference from example 1 is that the enzymolysis time in step 3) is 1h.
Comparative example 18
The difference from example 1 is that in the liquid fermentation in step 5) the access to Saccharomyces cerevisiae is 0.01g.
Comparative example 19
The difference from example 1 is that in the liquid fermentation in step 5) the access to Saccharomyces cerevisiae is 0.03g.
Comparative example 20
The difference from example 1 is that in the liquid fermentation in step 5) the access to Saccharomyces cerevisiae is 0.04g.
Comparative example 21
The difference from example 1 is that in the liquid fermentation in step 5) the access to Saccharomyces cerevisiae is 0.05g.
Comparative example 22
The difference from example 1 is that in the case of the liquid fermentation in step 5), the fermentation time is 24 hours.
Comparative example 23
The difference from example 1 is that in the case of the liquid fermentation in step 5), the fermentation time is 36h.
Comparative example 24
The procedure is as in example 1, except that in step 5) 0.02g of Saccharomyces cerevisiae type YA600 is added.
Comparative example 25
The procedure is as in example 1, except that in step 5) 0.02g of Saccharomyces cerevisiae with high alcohol content is added.
Test example 1
1mL of the cordyceps militaris fungus chaff fermentation broths obtained in the example 1 and the comparative example 1 are respectively taken, and the contents of active substances such as polysaccharide, flavone, polyphenol and the like in the fermentation broths obtained by fermentation of different strains are measured. The measurement results are shown in Table 1.
TABLE 1 active substance content of extract obtained by fermentation of different strains
As can be seen from Table 1, the polysaccharide content of the extract obtained by fermenting Aspergillus oryzae alone was 6.86%, and the polysaccharide content of the extract obtained by fermenting Aspergillus oryzae+yeast of the present invention was 12.21%, which was 5.35% higher than that obtained by fermenting Aspergillus oryzae alone. The invention selects Aspergillus oryzae as a strain for the first fermentation step, and selects Saccharomyces cerevisiae as a strain for the second fermentation step, so that the polysaccharide obtained by fermenting the cordyceps militaris fungus chaff has high content, and each 100g of cordyceps militaris fungus chaff is fermented to generate 12.21g of polysaccharide.
Test example 2
Taking 1g of the fungus chaff obtained in the step 2) of the examples 1-5 and the comparative examples 2-5 respectively, placing the fungus chaff into a centrifuge tube filled with 25mL of buffer solution, uniformly mixing and centrifuging to obtain supernatant. The pH and reducing sugar content of the supernatant were determined. The measurement results are shown in Table 2.
TABLE 2 influence of Cordyceps militaris chaff active substance content by different treatments
As can be seen from Table 2, the content of the reducing sugar obtained by fermenting the cordyceps militaris fungus chaff by utilizing aspergillus oryzae is the highest, and 1.73-4.48 g of reducing sugar is produced by fermenting every 100g of cordyceps militaris fungus chaff.
Test example 3
Sequentially taking 1g of the fungus chaff obtained in the step 2) of the Aspergillus oryzae obtained in the examples 1, 6-7 and the comparative examples 11-15, placing the fungus chaff into a centrifuge tube filled with 25mL of buffer solution, uniformly mixing and centrifuging to obtain supernatant. The supernatant amylase enzyme activity and alpha-amino nitrogen content were determined. The measurement results are shown in Table 3.
TABLE 3 influence of Cordyceps militaris chaff active substance content in different treatments
As can be seen from Table 3, the amylase activity in the fermentation process of the invention by means of fermenting the cordyceps militaris fungus chaff by using Aspergillus oryzae is high, which is as high as 32.383-58.636 mg/min/g.
Test example 4
1mL of the enzymatic hydrolysate obtained in step 3) of example 1, examples 8 to 11 and comparative examples 16 to 17 was taken in sequence, and the content of alpha-amino nitrogen, the content of reducing sugar and the content of flavone in the supernatant were measured. The measurement results are shown in Table 4.
TABLE 4 influence of Cordyceps militaris chaff active substance content in different treatments
As can be seen from Table 4, the content of the reducing sugar obtained by the enzymolysis treatment mode of the invention is high, and 3.14-4.41 g of reducing sugar is produced per 100g of cordyceps militaris fungus chaff by fermentation.
Test example 5
Sequentially taking 1mL of cordyceps militaris fungus chaff fermentation broth obtained in the step 5 of the example 1, the examples 12-13 and the comparative examples 18-22, and measuring the sugar degree value and the fungus concentration (OD) in the fermentation broth 580 ) pH and sugar degree. The measurement results are shown in Table 5.
TABLE 5 influence of Cordyceps militaris chaff active substance content in different treatments
As can be seen from Table 5, the content of reducing sugar obtained by the enzymolysis treatment mode of the invention is lower, and the content of reducing sugar in per 100g of cordyceps militaris fungus chaff is only 1.51-2.93 g after fermentation by using Saccharomyces cerevisiae, which indicates that more small molecular sugar such as reducing sugar can be converted into polysaccharide by using the fermentation mode of Saccharomyces cerevisiae.
Test example 6
1mL of the fermentation broths of cordyceps militaris fungus chaff obtained in the example 1, the example 16 and the comparative examples 24 to 25 are respectively taken, and the content of active substances such as total sugar, reducing sugar, polysaccharide, flavone and the like in the fermentation broths obtained by fermenting different types of saccharomyces cerevisiae is measured. The measurement results are shown in Table 6.
TABLE 6 active substance content of fermentation broths obtained by Yeast fermentation of different models
As can be seen from Table 6, the difference of polysaccharide content in fermentation broth obtained by inoculating yeasts of different types under the same conditions is not great, but the clearance effect of fermentation broth obtained by fermenting different types of yeasts on DPPH is remarkably different, the clearance effect of the fermentation broth on DPPH is far higher than that of the fermentation broth obtained by fermenting Saccharomyces cerevisiae of YA200 and YA300 types and that of the fermentation broth of Saccharomyces cerevisiae of YA600 type and Saccharomyces cerevisiae of high alcohol type by fermenting Saccharomyces cerevisiae of YA200 or YA300 type, the polysaccharide production effect is optimal, 11.94-12.21 g of polysaccharide is produced by fermenting per 100g of cordyceps militaris fungus chaff, and the clearance rate of DPPH is as high as 92.63% -99.08%.
Test example 7
Taking 1mL of fermentation liquor obtained after fermentation in the step 5) of the example 1 (the concentration of the fermentation liquor is the ratio of the volume of the fermentation liquor obtained by the mass of fungus chaff, namely 50 mg/mL), adding deionized water into the fermentation liquor, and carrying out gradient dilution on the fermentation liquor to obtain the Cordyceps militaris fungus chaff fermentation liquor with the concentration of 0.156mg/mL, 0.039mg/mL, 0.019mg/mL, 0.009mg/mL and 0.004mg/mL in sequence. Determination of the fermentation liquor pair OH with different concentrations - Is effective in scavenging. The measurement results are shown in Table 7 and FIG. 1.
TABLE 7 Cordyceps militaris chaff fermentation broths at different concentrations vs. OH - Is effective in scavenging
As can be seen from Table 7 and FIG. 1, the fermentation broth pair OH of Cordyceps militaris fungus chaff of the invention - The cleaning function of the fermentation liquid is related to the concentration of the cordyceps militaris fungus chaff fermentation liquid added into the reaction system, and when the concentration of the fermentation liquid is 0.156mg/mL, the OH is contained in the fermentation liquid - The clearance rate reaches 86.74 percent. Determination of its corresponding IC using SPSS software 50 The value was 0.043mg/mL.
Test example 8
Taking 1mL of fermentation liquor obtained after fermentation in the step 5) of the example 1 (the concentration of the fermentation liquor is the ratio of the volume of the fermentation liquor obtained by the mass of the fungus chaff, namely 50 mg/mL), adding deionized water into the fermentation liquor, and sequentially obtaining the Cordyceps militaris fungus chaff fermentation liquor with the concentration of 10mg/mL, 2.5mg/mL, 1.25mg/mL, 1mg/mL and 0.625 mg/mL. Determination of the fermentation liquor pair O with different concentrations 2 - Is effective in scavenging. The measurement results are shown in Table 8 and FIG. 2.
TABLE 8 Cordyceps militaris chaff fermentation broth pair O at different concentrations 2 - Is effective in scavenging
As can be seen from Table 8 and FIG. 2, the Cordyceps militaris fungus chaff fermentation broth pair O of the invention 2 - The clearing effect of the method is related to the concentration of the cordyceps militaris fungus chaff fermentation broth added into a reaction system, and the inhibition rate reaches 98.54% when the concentration of the fermentation broth is 10 mg/mL. Determination of its corresponding IC using SPSS software 50 The value was 1.711mg/mL.
Test example 9
Taking 1mL of fermentation liquor obtained after fermentation in the step 5) of the example 1, (the concentration of the fermentation liquor is the ratio of the volume of the fermentation liquor obtained by the mass of fungus chaff, namely 50 mg/mL), adding deionized water into the fermentation liquor, and sequentially obtaining the Cordyceps militaris fungus chaff fermentation liquor with the concentration of 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.312mg/mL, 0.156mg/mL, 0.078mg/mL and 0.039 mg/mL. Determination of different concentrations of fermentation liquor versus H 2 O 2 Is effective in scavenging. The measurement results are shown in Table 9 and FIG. 3.
TABLE 9 Cordyceps militaris chaff fermentation broth pair H at different concentrations 2 O 2 Is effective in scavenging
As can be seen from Table 9 and FIG. 3, the Cordyceps militaris fungus chaff fermentation broth H of the invention 2 O 2 The clearing effect of the method is related to the concentration of the cordyceps militaris fungus chaff fermentation broth added into the reaction system, and the inhibition rate reaches 86.84% when the concentration of the fermentation broth is 0.625 mg/mL. Determination of its corresponding IC using SPSS software 50 The value was 0.183mg/mL.
Test example 10
Taking 1mL of fermentation liquor obtained after fermentation in the step 5) of the example 1, (the concentration of the fermentation liquor is the ratio of the volume of the fermentation liquor obtained by the mass of the fungus chaff, namely 50 mg/mL), adding deionized water into the fermentation liquor, and sequentially obtaining the Cordyceps militaris fungus chaff fermentation liquor with the concentration of 10mg/mL, 7.5mg/mL, 5mg/mL, 2.5mg/mL, 1.25mg/mL and 0.625 mg/mL. The inhibition rate of the fermentation liquor with different concentrations to the alpha-glucosidase is measured. The measurement results are shown in Table 10 and FIG. 4.
TABLE 10 inhibition of alpha-glucosidase by Cordyceps militaris chaff fermentation broths at different concentrations
As can be seen from Table 10 and FIG. 4, the inhibition rate of the activity of alpha-glucosidase of the fermentation broth of Cordyceps militaris mushroom bran of the invention is related to the concentration of the fermentation extract of Cordyceps militaris mushroom bran added into the reaction system, the inhibition rate is increased with the increase of the concentration, the increase of the inhibition rate gradually becomes stable after the added extract is increased to a certain concentration, and the inhibition rate reaches 90.86% when the concentration of the fermentation broth is 10 mg/mL. This demonstrates that the active ingredients in the fermentation broth have a certain inhibitory effect on the alpha-glucosidase activity. Determination of its corresponding IC using SPSS software 50 The value was 2.144mg/mL.
The embodiment shows that the cordyceps militaris fungus chaff fermentation broth provided by the invention contains high-concentration polysaccharide, so that the secondary utilization of the cordyceps militaris fungus chaff is realized, and the active ingredients in the fermentation broth can effectively remove OH - 、O 2 - And H 2 O 2 And has a certain inhibition effect on the activity of alpha-glucosidase, which indicates that the cordyceps militaris fungus chaff fermentation broth has the functions of antioxidation and anti-saccharification.
Claims (14)
1. A preparation method of cordyceps militaris fungus chaff fermentation broth is characterized in that cordyceps militaris fungus chaff is inoculated with aspergillus oryzae for solid fermentation to obtain a first fermentation product; carrying out enzymolysis on the first fermentation product to obtain an enzymolysis liquid; extracting the enzymolysis liquid with water to obtain an extraction liquid; mixing the supernatant of the extracting solution with Saccharomyces cerevisiae, and performing liquid fermentation to obtain Cordyceps militaris fungus chaff fermentation broth;
the mass ratio of the aspergillus oryzae to the cordyceps militaris fungus chaff is 0.0002-0.0004:1;
the enzymolysis comprises the step of carrying out enzymolysis by using amylase contained in the first fermentation product, wherein the temperature of the enzymolysis is 40-50 ℃, and the pH value of the enzymolysis is 6; the enzymolysis time is 3-4 hours;
the mass ratio of the saccharomyces cerevisiae to the cordyceps militaris fungus chaff is 0.004:1
Before inoculating aspergillus oryzae, mixing the cordyceps militaris fungus chaff with water for gelatinization treatment; when pasting, the ratio of the cordyceps militaris fungus chaff to the water is 1 g:2.4-4.8 mL;
the solid fermentation time is 48-72 h, and the solid fermentation temperature is 28-30 ℃;
the extraction temperature is 85-100 ℃, and the extraction time is 1-3 hours;
the temperature of the liquid fermentation is 28-30 ℃, and the time of the liquid fermentation is 48-72 h.
2. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0004:1;
the enzymolysis temperature is 45 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 3.6mL;
the solid fermentation time is 60 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 48 hours.
3. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0004:1;
the enzymolysis temperature is 45 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 2.4mL;
the solid fermentation time is 60 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 48 hours.
4. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0004:1;
the enzymolysis temperature is 45 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 4.8mL;
the solid fermentation time is 60 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 48 hours.
5. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0002:1;
the enzymolysis temperature is 45 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 3.6mL;
the solid fermentation time is 60 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 48 hours.
6. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0003:1;
the enzymolysis temperature is 45 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 3.6mL;
the solid fermentation time is 60 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 48 hours.
7. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0004:1;
the enzymolysis temperature is 45 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 3.6mL;
the solid fermentation time is 48 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 48 hours.
8. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0004:1;
the enzymolysis temperature is 45 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 3.6mL;
the solid fermentation time is 72 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 48 hours.
9. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0004:1;
the enzymolysis temperature is 40 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 3.6mL;
the solid fermentation time is 60 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 48 hours.
10. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0004:1;
the enzymolysis temperature is 50 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 3.6mL;
the solid fermentation time is 60 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 48 hours.
11. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0004:1;
the enzymolysis temperature is 45 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 3.6mL;
the solid fermentation time is 60 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 60 hours.
12. The preparation method of claim 1, wherein the mass ratio of aspergillus oryzae to cordyceps militaris chaff is 0.0004:1;
the enzymolysis temperature is 45 ℃ and the enzymolysis time is 3 hours;
the ratio of the cordyceps militaris fungus chaff to water is 1g to 3.6mL;
the solid fermentation time is 60 hours, and the temperature is 28 ℃;
the extraction temperature is 100 ℃ and the extraction time is 2 hours;
the temperature of the liquid fermentation is 30 ℃ and the time is 72 hours.
13. The cordyceps militaris fungus chaff fermentation broth obtained by the preparation method of any one of claims 1-12.
14. Use of the cordyceps militaris fungus chaff fermentation broth of claim 13 in preparing antioxidant and/or anti-saccharification food, health product or medicine.
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