CN115074256B - Hericium erinaceus liquid fermentation medium and method for preparing triterpenes - Google Patents
Hericium erinaceus liquid fermentation medium and method for preparing triterpenes Download PDFInfo
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- 239000007788 liquid Substances 0.000 title claims abstract description 108
- 150000003648 triterpenes Chemical class 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 20
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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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- 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
- C12P33/00—Preparation of steroids
- C12P33/20—Preparation of steroids containing heterocyclic rings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention provides a hericium erinaceus liquid fermentation medium and a method for preparing triterpenes, wherein the hericium erinaceus liquid fermentation medium comprises the following components in percentage by mass: 2% -4% of glucose, 1% -3% of soluble starch, 0.5% -1.5% of yeast extract, 0.05% -0.15% of monopotassium phosphate, 0.04% -0.1% of magnesium sulfate heptahydrate and the balance of water. According to the invention, through optimizing the liquid fermentation conditions of the edible fungi, the fermentation condition which enables the growth speed of the hericium erinaceus to be the fastest is found. The invention also increases the content of triterpene which is the secondary metabolite of the hericium erinaceus and increases the nutritional value and the economic value of the hericium erinaceus by optimizing the triterpene extraction conditions.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a hericium erinaceus liquid fermentation medium and a method for preparing triterpenes.
Background
Hericium erinaceus is Hericium, large-scale edible and pharmaceutical fungi. Fruiting bodies, large meat, look like the head of a monkey, so called "Hericium erinaceus". Hericium erinaceus is one of eight major "delicacies" in China, "delicacies hericium erinaceus, sea bird's nest", and is four famous dishes in parallel with bear's hands, sea cucumbers and shark fins, and has tender meat, delicious taste, rich nutrition, and good color, smell and taste. In addition, the hericium erinaceus is also a traditional and valuable Chinese medicinal material in China, and has the functions of nourishing and building body, helping digestion and benefiting five viscera. Modern researches have shown that it contains active ingredients such as polypeptide, polysaccharide, fat and protein, etc., and has certain curative effects on digestive tract tumor, gastric ulcer, duodenal ulcer, gastritis, abdominal distention, etc. The economic value of the hericium erinaceus is very high, such as a hericium erinaceus health-care beverage, a hericium erinaceus biscuit, a hericium erinaceus rice thin and the like.
The liquid fermentation technology is one of modern biotechnology, and is characterized by that it utilizes carbon source, nitrogen source, inorganic salt and other trace elements necessary for edible and medicinal fungus in the course of growth and cultivation as culture medium, and adopts the processes of sterilizing, inoculating strain, introducing sterile air and stirring so as to provide oxygen required for respiratory metabolism of edible fungus body, controlling proper external condition and making mass culture and propagation of mycelium. The industrial large-scale fermentation culture is fermentation production, and is also called submerged culture or submerged culture. The obtained fermentation broth contains thallus, nutritional components decomposed by thallus and not decomposed, and metabolites produced by thallus. Compared with the solid plate culture of edible fungi, the liquid fermentation has many advantages, such as rapid mycelium growth, and in the liquid culture, the nutrient components of the liquid culture medium are uniformly distributed, which is beneficial to the full contact and absorption of fungus nutrients. The mycelium cells can grow in the culture medium under the conditions of optimal temperature, pH, oxygen and carbon nitrogen ratio, and can timely discharge metabolic waste gas generated by respiration, so that metabolism is vigorous, mycelium growth and division are rapid, and a large number of mycelium and metabolic products with physiological activities such as polysaccharide, polypeptide and the like can be accumulated in a short time; the production period is short, and the time for obtaining a large amount of mycelium and physiologically active substances by liquid fermentation culture of edible and medicinal fungi is generally only about 10 days, while the time for solid culture is 20-30 days.
The carbon source is the carbon source for the growth of the hericium erinaceus, is also the energy source for the metabolic activity of the hericium erinaceus, and is a raw material for producing various metabolic substances. Glucose is the predominant carbon source and is also the most commonly used carbon source. However, too much glucose is added to adversely affect the growth of hyphae due to the glucose effect. The nitrogen source is an important nutritional component of the mycelium of the hericium erinaceus and is also an indispensable raw material for synthesizing protein and nucleic acid from the hericium erinaceus. The potassium dihydrogen phosphate and the magnesium sulfate heptahydrate serve as inorganic salts and play a role in maintaining the osmotic pressure of the culture solution and the cell morphology.
The temperature has great influence on the liquid fermentation of the hericium erinaceus and is complicated. Firstly, the growth speed of the mycelium pellets of the hericium erinaceus is influenced by the temperature, and along with the rise of the temperature, the growth and the reproduction of the hericium erinaceus are accelerated due to the participation of enzymes. According to the kinetics of the enzymatic reaction, the temperature is increased, the enzymatic reaction is enhanced, the respiration intensity is increased, and the growth speed of the hericium erinaceus is increased. However, as the temperature increases, the enzyme inactivation speed is increased, the fermentation period is shortened, and the thalli die; secondly, the temperature affects the yield of hericium erinaceus metabolites; finally, the temperature influences the physical properties of the liquid fermentation broth, and the biosynthesis of the hericium erinaceus is indirectly influenced by changing the physical properties of the fermentation broth, so that the absorption of the hericium erinaceus to nutrient substances in the fermentation broth is also influenced.
The pH has great influence on the fermentation of the hericium erinaceus liquid, and firstly, the pH also influences the enzymatic reaction of the hericium erinaceus, strong acid and strong alkali easily deactivate the hericium erinaceus, so that the growth speed of the hericium erinaceus is reduced; secondly, the pH influences the charge state of the cell membrane of the hericium erinaceus, so that the permeability of the cell membrane is changed, further the absorption of the hericium erinaceus to nutrient substances is influenced, the growth speed of the hericium erinaceus is influenced, and the generation of metabolites is also influenced; in addition, pH also affects the morphology of Hericium erinaceus; finally, pH has a significant impact on some biosynthetic pathways of hericium erinaceus. Experiments prove that the hericium erinaceus can only absorb organic components in fermentation liquor in a medium acidity environment.
The rotation speed of the shaking table can change the dissolved oxygen amount, the dissolved oxygen influences the growth of mycelium by participating in the physiology, biochemistry and process of the liquid fermentation of the hericium erinaceus, the too small rotation speed can influence the dissolved oxygen to further influence the growth of mycelium, and the too large rotation speed can force the mycelium to form blocks and form clusters to inhibit the growth of mycelium.
Triterpene components are terpenoid compounds with basic parent nucleus composed of 30 carbon atoms, exist in free form or in the form of being combined with sugar into glycoside or ester, and have various biochemical activities. Triterpene is also an important active substance in Hericium erinaceus, and has anticancer, antibacterial, antiinflammatory, antiviral, cholesterol reducing, and human body benefiting effects.
Therefore, the quick development of the hericium erinaceus is desired, the industrial production is realized, the liquid fermentation medium suitable for the hericium erinaceus is researched, the most suitable fermentation conditions are found, and the growth of the mycelium of the hericium erinaceus can be accelerated. The optimal triterpene extraction method is found, so that the yield of triterpene products can be increased, and the economic benefit of hericium erinaceus is improved.
Disclosure of Invention
The invention aims to: aiming at the defects of the prior art, the invention provides the hericium erinaceus liquid fermentation medium for effectively accelerating the growth of bacteria and accelerating the generation of bacterial metabolites.
The invention also solves the technical problem of providing a method for producing mycelium pellets by fermentation of hericium erinaceus.
The invention also solves the technical problem of providing a method for preparing hericium erinaceus triterpene.
In order to solve the first technical problem, the invention discloses a hericium erinaceus liquid fermentation medium which comprises the following components in percentage by mass: 2% -4% of glucose, 1% -3% of soluble starch, 0.5% -1.5% of yeast extract, 0.05% -0.15% of monopotassium phosphate, 0.04% -0.1% of magnesium sulfate heptahydrate and the balance of water. In some embodiments, the hericium erinaceus liquid fermentation medium comprises the following components in percentage by mass: glucose 3%, soluble starch 2%, yeast extract 1%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.06% and the balance of water.
The hericium erinaceus liquid fermentation medium disclosed by the invention is suitable for all hericium erinaceus in the prior art, including but not limited to a hericium erinaceus primary test tube strain (https:// m.tb.cn/h.ft114 lesm=b 79355 tk= Xvjk29O0 vbZ) for Wu Hanzhou Yulin edible fungus research.
In some embodiments, the hericium erinaceus liquid fermentation culture is sterilized based on sterilization in a sterilization pot at 115 ℃ for 20 min.
In order to solve the second technical problem, the invention discloses a method for producing mycelium pellets by fermentation of hericium erinaceus, which comprises the steps of inoculating a strain obtained after activation of hericium erinaceus into the liquid fermentation medium for fermentation to obtain the mycelium pellets of hericium erinaceus.
In some embodiments, the activating medium in the activating comprises the following components in weight percent: glucose 2%, bran water 10%, peptone 0.4%, yeast extract 0.3%, agar 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B 1 0.001.001%, and water in balance. In some embodiments, the bran water is prepared as follows: adding 80-120g/500mL of crude bran into water, boiling, preserving heat for 10-50min, and filtering with one-seven layers of gauze to obtain filtrate, namely bran water. In some embodiments, the bran water is prepared as follows: adding the crude bran into water at the dosage of 100g/500mL, boiling, preserving heat for 30min, and filtering with four layers of gauze to obtain filtrate, namely bran water.
In some embodiments, the activation is to cut out 0.5cm fungus blocks of the hericium erinaceus under a sterile environment, inoculate the fungus blocks in an activation culture medium, and culture the fungus blocks in an incubator at 26 ℃ for 12-14 days to obtain the activated fungus.
In some embodiments, the strain is inoculated into the liquid fermentation medium in an amount of 1 to 5 pieces per 100 mL; in some embodiments, the species is inoculated into the liquid fermentation medium at a rate of 0.5 x 0.5cm 3/block x 3/100 mL.
In some embodiments, the initial pH of the fermentation is between 5.0 and 6.0, the temperature of the fermentation is between 25 and 29 ℃, and the rotational speed of the shaker during the fermentation is between 150 and 210r/min. In some embodiments, the initial pH of the fermentation is 5.5, the temperature of the fermentation is 27 ℃, and the rotational speed of the shaker during the fermentation is 180r/min.
In some embodiments, the fermentation time is 8-12 days; in some embodiments, the fermentation time is 10 days.
In order to solve the third technical problem, the invention discloses a method for preparing hericium erinaceus triterpenes, which comprises the steps of dissolving mycelium spheres prepared by the method in an ethanol water solution, carrying out ultrasonic extraction and centrifuging, wherein the obtained supernatant is an extracting solution containing the hericium erinaceus triterpenes.
In some embodiments, the volume concentration of ethanol in the ethanol solution is 40% -80%; in some embodiments, the volume concentration of ethanol in the ethanol solution is 80%.
In some embodiments, the feed solution ratio of mycelium pellets to aqueous ethanol is 1g:15-30mL; in some embodiments, the feed ratio of mycelium pellets to aqueous ethanol is 1g:30ml.
In some embodiments, the temperature of the ultrasonic extraction is 40-80 ℃; in some embodiments, the temperature of the ultrasonic extraction is 80 ℃.
In some embodiments, the time of the ultrasonic extraction is 10-50 minutes; in some embodiments, the time of the ultrasonic extraction is 30 minutes.
In some embodiments, the frequency of the ultrasonic extraction is 30-50khz; in some embodiments, the frequency of the ultrasonic extraction is 40khz.
In some embodiments, the rotational speed of the centrifugation is 3000-5000r/min; in some embodiments, the rotational speed of the centrifugation is 4000r/min.
In some embodiments, the centrifugation is for a period of 5-15 minutes; in some embodiments, the centrifugation is for 10 minutes.
In some embodiments, the triterpene content is determined by ultraviolet spectrophotometry, which comprises the following steps: firstly, preparing triterpenoid oleanolic acid into oleanolic acid solution with the concentration of 0.2mg/ml, respectively sucking 0,0.1,0.2,0.3,0.4,0.5ml and 25ml of colorimetric tubes, evaporating the solvent in a boiling water bath, adding 0.5ml of vanillin-glacial acetic acid solution with the concentration of 50g/L and 1ml of perchloric acid solution, mixing, then carrying out water bath for 20min at 60 ℃, cooling with cold water for 15min, and then adding 3ml of glacial acetic acid. And (3) reacting for 10min at room temperature, and finally measuring the light absorption value at 547nm, wherein the light absorption value is the ordinate and the mass of oleanolic acid is the abscissa, and a standard curve is made. The triterpene content can be known by adding corresponding reagents into the triterpene extract according to the method, measuring under the same conditions, and substituting the triterpene extract into a standard curve.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
The invention provides a culture medium for liquid fermentation of hericium erinaceus, and fermentation conditions enabling the growth speed of hericium erinaceus to be the fastest are found through optimization of the fermentation conditions of edible fungi.
According to the invention, the triterpene extraction conditions are optimized, so that the content of the triterpene serving as a secondary metabolite of the hericium erinaceus is increased, and the nutritional value and the economic value of the hericium erinaceus are increased.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 shows the growth of Hericium erinaceus on an activation medium.
FIG. 2 shows the growth of Hericium erinaceus in fermentation media.
FIG. 3 shows the drying of Hericium erinaceus mycelium.
FIG. 4 shows the grinding of Hericium erinaceus mycelium.
FIG. 5 shows the extract of Hericium erinaceus triterpenes.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
The Hericium erinaceus inclined plane strain (Hericium erinaceus) is a Hericium erinaceus primary test tube strain purchased from the research of Wuhan Zhou Yulin edible fungi (https:// m.tb.cn/h.ft 114lesm=b 79355 tk= Xvjk29O0 vbZ).
In examples 1 to 3, the external fermentation conditions were identical in each example, the initial pH was 5.5, and the culture was carried out on a shaker at 27℃and 180r/min for 10 days
Example 1: mycelium pellet prepared by liquid fermentation of hericium erinaceus
(1) Activating a parent strain: cutting Hericium erinaceus slant strain preserved in refrigerator under aseptic condition to obtain 0.5cm pieces, inoculating onto activating culture medium, and culturing at 26deg.C incubator for 12-14 days to obtain primary strain (figure 1). The mass content of each component in the activation medium is as follows: glucose 2%, bran water 10%, peptone 0.4%, yeast extract 0.3%, agar 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B 1 0.001.001%, and the balance water, wherein the bran water is prepared by boiling 100g of crude bran with 500ml of pure water, maintaining the temperature for 30min, and filtering on four layers of gauze.
(2) Sterilization of liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises 2 weight percent of glucose, 1 weight percent of soluble starch, 0.5 weight percent of yeast extract, 0.05 weight percent of monopotassium phosphate, 0.04 weight percent of magnesium sulfate heptahydrate, the balance of pure water and the pH value of 5.5. Placing the liquid fermentation culture medium into a 250mL triangular flask according to the amount of 100mL, placing the triangular flask into a sterilizing pot at 115 ℃, preserving heat for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of hericium erinaceus: cutting off 0.5cm by 0.5cm of the primary strain obtained in the step (1), inoculating 3 pieces of fungus blocks per bottle under aseptic condition in the liquid fermentation culture medium in the step (2), and culturing for 10 days on a shaker at 27 ℃ and 180r/min (figure 2) to form mycelium spheres, thereby obtaining fermentation liquor containing the mycelium spheres.
Dry weight of hyphae dry weight method: filtering mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, spreading the filtered mycelium pellets on the flat plate, putting the flat plate into a 65 ℃ oven for drying (figure 3), weighing again, subtracting the weight of the flat plate from the total weight to obtain mycelium dry weight, and storing the filtered mycelium liquid in a refrigerator for standby.
Example 2: mycelium pellet prepared by liquid fermentation of hericium erinaceus
(1) Activating a parent strain: cutting Hericium erinaceus inclined plane strain preserved in refrigerator under aseptic environment to obtain 0.5cm bacterial pieces, inoculating onto activating culture medium, and culturing at 26deg.C for 12-14 days to obtain primary strain. The mass content of each component in the activation medium is as follows: glucose 2%, bran water 10%, peptone 0.4%, yeast extract 0.3%, agar 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B10.001% and water the balance, wherein the bran water is prepared by adding 100g of crude bran into 500ml of pure water, boiling, preserving heat for 30min, and filtering on four layers of gauze.
(2) Sterilization of liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of monopotassium phosphate, 0.06% of magnesium sulfate heptahydrate, and the balance of pure water, wherein the pH is 5.5. Placing the liquid fermentation culture medium into a 250mL triangular flask according to the amount of 100mL, placing the triangular flask into a sterilizing pot at 115 ℃, preserving heat for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of edible fungi: cutting off 0.5cm of the primary strain obtained in the step (1), 0.5cm of the primary strain is inoculated into the liquid fermentation culture medium in the step (2), 3 fungus blocks are inoculated per bottle under the aseptic condition, and the culture is carried out on a shaking table for 10 days at the temperature of 27 ℃ and the speed of 180r/min to form mycelium spheres, so that fermentation liquor containing the mycelium spheres is obtained.
Dry weight of hyphae dry weight method: filtering mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, spreading the filtered mycelium pellets on the flat plate, putting the flat plate into a 65 ℃ oven for drying, weighing again, subtracting the weight of the flat plate from the total weight to obtain mycelium dry weight, and storing the filtered mycelium liquid in a refrigerator for standby.
Example 3: mycelium pellet prepared by liquid fermentation of hericium erinaceus
(1) Activating a parent strain: cutting Hericium erinaceus inclined plane strain preserved in refrigerator under aseptic environment to obtain 0.5cm bacterial pieces, inoculating onto activating culture medium, and culturing at 26deg.C for 12-14 days to obtain primary strain. The mass content of each component in the activation medium is as follows: glucose 2%, bran water 10%, peptone 0.4%, yeast extract 0.3%, agar 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B10.001% and water the balance, wherein the bran water is prepared by adding 100g of crude bran into 500ml of pure water, boiling, preserving heat for 30min, and filtering on four layers of gauze.
(2) Sterilization of liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises, by weight, 4% of glucose, 3% of soluble starch, 1.5% of yeast extract, 0.15% of monopotassium phosphate, 0.1% of magnesium sulfate heptahydrate, and the balance of pure water, wherein the pH is 5.5. Placing the liquid fermentation culture medium into a 250mL triangular flask according to the amount of 100mL, placing the triangular flask into a sterilizing pot at 115 ℃, preserving heat for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of edible fungi: cutting off 0.5cm of the primary strain obtained in the step (1), 0.5cm of the primary strain is inoculated into the liquid fermentation culture medium in the step (2), 3 fungus blocks are inoculated per bottle under the aseptic condition, and the culture is carried out on a shaking table for 10 days at the temperature of 27 ℃ and the speed of 180r/min to form mycelium spheres, so that fermentation liquor containing the mycelium spheres is obtained.
Dry weight of hyphae dry weight method: filtering mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, spreading the filtered mycelium pellets on the flat plate, putting the flat plate into a 65 ℃ oven for drying, weighing again, subtracting the weight of the flat plate from the total weight to obtain mycelium dry weight, and storing the filtered mycelium liquid in a refrigerator for standby.
In examples 4 to 6, the fermentation medium in each example was uniform in composition, and the medium composition was glucose 3%, soluble starch 2%, yeast extract 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.06%, and the balance pure water
Example 4: mycelium pellet prepared by liquid fermentation of hericium erinaceus
(1) Activating a parent strain: cutting Hericium erinaceus inclined plane strain preserved in refrigerator under aseptic environment to obtain 0.5cm bacterial pieces, inoculating onto activating culture medium, and culturing at 26deg.C for 12-14 days to obtain primary strain. The mass content of each component in the activation medium is as follows: glucose 2%, bran water 10%, peptone 0.4%, yeast extract 0.3%, agar 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B10.001% and water the balance, wherein the bran water is prepared by adding 100g of crude bran into 500ml of pure water, boiling, preserving heat for 30min, and filtering on four layers of gauze.
(2) Sterilization of liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of monopotassium phosphate, 0.06% of magnesium sulfate heptahydrate, and the balance of pure water, wherein the pH is 5.0. Placing the liquid fermentation culture medium into a 250mL triangular flask according to the amount of 100mL, placing the triangular flask into a sterilizing pot at 115 ℃, preserving heat for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of edible fungi: cutting off 0.5cm of the primary strain obtained in the step (1), 0.5cm of the primary strain is inoculated into the liquid fermentation culture medium in the step (2), 3 fungus blocks are inoculated per bottle under aseptic conditions, and the culture is carried out on a shaking table for 10 days at 25 ℃ and 150r/min to form mycelium spheres, so that fermentation liquor containing the mycelium spheres is obtained.
Dry weight of hyphae dry weight method: filtering mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, spreading the filtered mycelium pellets on the flat plate, putting the flat plate into a 65 ℃ oven for drying, weighing again, subtracting the weight of the flat plate from the total weight to obtain mycelium dry weight, and storing the filtered mycelium liquid in a refrigerator for standby.
Example 5: mycelium pellet prepared by liquid fermentation of hericium erinaceus
(1) Activating a parent strain: cutting Hericium erinaceus inclined plane strain preserved in refrigerator under aseptic environment to obtain 0.5cm bacterial pieces, inoculating onto activating culture medium, and culturing at 26deg.C for 12-14 days to obtain primary strain. The mass content of each component in the activation medium is as follows: glucose 2%, bran water 10%, peptone 0.4%, yeast extract 0.3%, agar 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B10.001% and water the balance, wherein the bran water is prepared by adding 100g of crude bran into 500ml of pure water, boiling, preserving heat for 30min, and filtering on four layers of gauze.
(2) Sterilization of liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of monopotassium phosphate, 0.06% of magnesium sulfate heptahydrate, and the balance of pure water, wherein the pH is 5.5. Placing the liquid fermentation culture medium into a 250mL triangular flask according to the amount of 100mL, placing the triangular flask into a sterilizing pot at 115 ℃, preserving heat for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of edible fungi: cutting off 0.5cm of the primary strain obtained in the step (1), 0.5cm of the primary strain is inoculated into the liquid fermentation culture medium in the step (2), 3 fungus blocks are inoculated per bottle under the aseptic condition, and the culture is carried out on a shaking table for 10 days at the temperature of 27 ℃ and the speed of 180r/min to form mycelium spheres, so that fermentation liquor containing the mycelium spheres is obtained.
Dry weight of hyphae dry weight method: filtering mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, spreading the filtered mycelium pellets on the flat plate, putting the flat plate into a 65 ℃ oven for drying, weighing again, subtracting the weight of the flat plate from the total weight to obtain mycelium dry weight, and storing the filtered mycelium liquid in a refrigerator for standby.
Example 6: mycelium pellet prepared by liquid fermentation of hericium erinaceus
(1) Activating a parent strain: cutting Hericium erinaceus inclined plane strain preserved in refrigerator under aseptic environment to obtain 0.5cm bacterial pieces, inoculating onto activating culture medium, and culturing at 26deg.C for 12-14 days to obtain primary strain. The mass content of each component in the activation medium is as follows: glucose 2%, bran water 10%, peptone 0.4%, yeast extract 0.3%, agar 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B10.001% and water the balance, wherein the bran water is prepared by adding 100g of crude bran into 500ml of pure water, boiling, preserving heat for 30min, and filtering on four layers of gauze.
(2) Sterilization of liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of monopotassium phosphate, 0.06% of magnesium sulfate heptahydrate, and the balance of pure water, wherein the pH is 5.5. Placing the liquid fermentation culture medium into a 250mL triangular flask according to the amount of 100mL, placing the triangular flask into a sterilizing pot at 115 ℃, preserving heat for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of edible fungi: cutting off 0.5cm of the primary strain obtained in the step (1), 0.5cm of the primary strain is inoculated into the liquid fermentation culture medium in the step (2), 3 fungus blocks are inoculated per bottle under the aseptic condition, and the culture is carried out on a shaking table for 10 days at 29 ℃ and 210r/min to form mycelium spheres, so that fermentation liquor containing the mycelium spheres is obtained.
Dry weight of hyphae dry weight method: filtering mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, spreading the filtered mycelium pellets on the flat plate, putting the flat plate into a 65 ℃ oven for drying, weighing again, subtracting the weight of the flat plate from the total weight to obtain mycelium dry weight, and storing the filtered mycelium liquid in a refrigerator for standby.
Examples 7 to 9 are the preparation of Hericium erinaceus triterpenes, and the mycelium pellet used is the mycelium pellet prepared in example 2
The triterpene content was measured by ultraviolet spectrophotometry in the following examples: the triterpene standard oleanolic acid is firstly prepared into an oleanolic acid solution with the concentration of 0.2mg/ml (0.2 mg oleanolic acid is dissolved in 1ml of methanol solution), 0,0.1,0.2,0.3,0.4,0.5ml and 25ml of colorimetric tubes are respectively absorbed, the solvent is evaporated in a boiling water bath, 0.5ml of vanillin-glacial acetic acid solution with the concentration of 50g/L (50 g of vanillin is dissolved in 1L of glacial acetic acid solution) and 1ml of perchloric acid solution are added, the mixture is mixed, the water bath is carried out for 20min at 60 ℃, and 3ml of glacial acetic acid is added after cooling for 15 min. And (3) reacting for 10min at room temperature, and finally measuring the light absorption value at 547nm, wherein the light absorption value is the ordinate and the mass of oleanolic acid is the abscissa, and a standard curve is made. And adding the triterpene extract extracted in each example into corresponding reagents according to the method, measuring under the same conditions, taking the standard curve, and calculating the triterpene content of the hericium erinaceus.
Example 7: ultrasonic-assisted extraction of triterpenes with ethanol
The dried mycelium pellets were ground (FIG. 4), dissolved in 40% ethanol, and the dissolved solution was extracted on an ultrasonic cleaner for 30min at an ultrasonic frequency of 40khz at 40℃and a feed-to-liquid ratio of mycelium pellets to ethanol of 1g:10ml. The extract was then centrifuged at 4000r/min for 10min in a centrifuge and the supernatant was collected as the triterpene extract (FIG. 5).
Example 8: ultrasonic-assisted extraction of triterpenes with ethanol
Grinding the dried mycelium pellet, dissolving with 60% ethanol, extracting the dissolved solution on an ultrasonic cleaner for 30min, wherein the ultrasonic frequency is 40khz, the temperature is 60 ℃, and the feed liquid ratio of mycelium pellet to ethanol is 1g:15ml. And centrifuging the extract on a centrifuge at 4000r/min for 10min, and collecting supernatant, wherein the supernatant is the triterpene extract.
Example 9: ultrasonic-assisted extraction of triterpenes with ethanol
Grinding the dried mycelium pellet, dissolving with 80% ethanol, extracting the dissolved solution on an ultrasonic cleaner for 30min, wherein the ultrasonic frequency is 40khz, the temperature is 80 ℃, and the feed liquid ratio of mycelium pellet to ethanol is 1g:30ml. And centrifuging the extract on a centrifuge at 4000r/min for 10min, and collecting supernatant, wherein the supernatant is the triterpene extract.
The results of the above examples are shown in tables 1 to 3.
Table 1: hericium erinaceus liquid fermentation result (external fermentation conditions are consistent)
Table 2: hericium erinaceus liquid fermentation results (consistent medium composition)
Table 3: preparation of triterpene from Hericium erinaceus metabolite
| Triterpene production (mg/L broth) | Triterpene yield (%) | |
| Example 7 | 21.27 | 0.19 |
| Example 8 | 31.36 | 0.28 |
| Example 9 | 33.57 | 0.30 |
As can be seen from Table 1, when the medium composition was glucose 3%, soluble starch 2%, yeast extract 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.06% and the balance being pure water (example 2), hericium erinaceus grew at the fastest rate; when the nutritional ingredients were further increased (example 3), there was little effect on the growth of Hericium erinaceus, probably because the glucose content was too high, resulting in glucose effects.
As can be seen from Table 2, the fermentation conditions were that the pH of the fermentation medium was 5.5, the shaking culture temperature was 27℃and the rotational speed was 180r/min (example 5), the liquid fermentation of Hericium erinaceus was best, the mycelium growth was fastest, the mycelium dry weight was also greatest, and the change of the external conditions greatly affected the growth of Hericium erinaceus.
It can be concluded that the fermentation conditions are a liquid fermentation medium with an initial pH of 5.5, a fermentation temperature of 27 ℃, a shaking table rotation speed of 180r/min, most suitable for liquid fermentation of hericium erinaceus and the maximum dry weight of hyphae, wherein the liquid fermentation medium comprises, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of monopotassium phosphate, 0.06% of magnesium sulfate heptahydrate and the balance of pure water.
It can be seen from Table 3 that the triterpene extraction conditions and the triterpene yields and yields were different. When the ethanol concentration is 80%, the feed liquid ratio of the hericium erinaceus mycelium pellets to ethanol is 1:30g/ml, and the extraction temperature is 80 ℃ (example 9), the triterpene yield is the highest, and the yield is the highest.
In conclusion, glucose and soluble starch are used as a composite carbon source, so that the glucose effect is relieved, the nutrition of the liquid fermentation broth is increased, and the growth speed of the hericium erinaceus is increased; the yeast extract is used as a nitrogen source, is rich in nutrition, contains essential amino acids, nucleotides, trace elements and the like required by the growth of the hericium erinaceus, and can greatly accelerate the growth of the hericium erinaceus. In addition, the invention optimizes the content of each component of the culture medium and finds the most suitable condition for liquid fermentation of the hericium erinaceus; experiments prove that, for the liquid fermentation of the hericium erinaceus, the temperature is controlled at 25-29 ℃, and the mycelium growth speed is high; experiments prove that, for the liquid fermentation of the hericium erinaceus, the pH is controlled to be 5.0-6.0, and the mycelium growth speed is high; according to experimental verification, the rotating speed of the shaking table is 150-210r/min, and the mycelium grows fastest.
The invention provides a hericium erinaceus liquid fermentation medium, a method for preparing triterpenes, and a method for preparing the hericium erinaceus liquid fermentation medium. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (2)
1. A method for producing mycelium pellets by fermentation of hericium erinaceus, which is characterized by comprising the following steps:
(1) Activating a parent strain: cutting Hericium erinaceus inclined plane strain preserved in refrigerator under aseptic environment to obtain 0.5cm bacterial pieces, inoculating onto activating culture medium, and culturing at 26deg.C incubator for 12-14 days to obtain primary strain; the mass content of each component in the activation medium is as follows: glucose 2%, bran water 10%, peptone 0.4%, yeast extract 0.3%, agar 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B10.001% and water the rest, wherein the bran water is prepared by adding 500ml of pure water into 100g of crude bran, boiling, preserving heat for 30min, and filtering on four layers of gauze;
(2) Sterilization of liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of monopotassium phosphate, 0.06% of magnesium sulfate heptahydrate, and the balance of pure water, wherein the pH is 5.5; placing the liquid fermentation culture medium into a 250mL triangular flask according to the amount of 100mL, placing the triangular flask into a sterilizing pot at 115 ℃, preserving heat for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying;
(3) Liquid fermentation of edible fungi: cutting off 0.5cm of the primary strain obtained in the step (1), 0.5cm of the primary strain is inoculated into the liquid fermentation culture medium in the step (2), 3 fungus blocks are inoculated per bottle under the aseptic condition, and the culture is carried out on a shaking table for 10 days at the temperature of 27 ℃ and the speed of 180r/min to form mycelium spheres, so that fermentation liquor containing the mycelium spheres is obtained.
2. A method for preparing a hericium erinaceus triterpene, comprising:
(1) Activating a parent strain: cutting Hericium erinaceus inclined plane strain preserved in refrigerator under aseptic environment to obtain 0.5cm bacterial pieces, inoculating onto activating culture medium, and culturing at 26deg.C incubator for 12-14 days to obtain primary strain; the mass content of each component in the activation medium is as follows: glucose 2%, bran water 10%, peptone 0.4%, yeast extract 0.3%, agar 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B10.001% and water the rest, wherein the bran water is prepared by adding 500ml of pure water into 100g of crude bran, boiling, preserving heat for 30min, and filtering on four layers of gauze;
(2) Sterilization of liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of monopotassium phosphate, 0.06% of magnesium sulfate heptahydrate, and the balance of pure water, wherein the pH is 5.5; placing the liquid fermentation culture medium into a 250mL triangular flask according to the amount of 100mL, placing the triangular flask into a sterilizing pot at 115 ℃, preserving heat for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying;
(3) Liquid fermentation of edible fungi: cutting off 0.5cm of the primary strain obtained in the step (1) and 0.5cm of the primary strain, inoculating the primary strain to the liquid fermentation culture medium in the step (2), inoculating 3 pieces of fungus blocks per bottle under aseptic conditions, and culturing for 10 days on a shaking table at the temperature of 27 ℃ and the speed of 180r/min to form mycelium spheres, thereby obtaining fermentation liquor containing the mycelium spheres;
(4) Dissolving the mycelium pellet in ethanol solution, performing ultrasonic extraction, and centrifuging to obtain supernatant, which is an extracting solution containing Hericium erinaceus triterpene;
the volume concentration of the ethanol in the ethanol solution is 60% -80%; the feed liquid ratio of the mycelium pellets to the ethanol water solution is 1g:15-30mL; the temperature of the ultrasonic extraction is 60-80 ℃.
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| CN101971762A (en) * | 2010-02-09 | 2011-02-16 | 中国农业科学院农业资源与农业区划研究所 | Liquid submerged fermentation culture method for hericium erinaceus |
| CN104082037A (en) * | 2014-07-10 | 2014-10-08 | 乳山市华隆生物科技有限公司 | Method for postprocessing of hericium erinaceus fermentation mycelium solution and preparing mycelium powder through biological enzyme |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101971762A (en) * | 2010-02-09 | 2011-02-16 | 中国农业科学院农业资源与农业区划研究所 | Liquid submerged fermentation culture method for hericium erinaceus |
| CN104082037A (en) * | 2014-07-10 | 2014-10-08 | 乳山市华隆生物科技有限公司 | Method for postprocessing of hericium erinaceus fermentation mycelium solution and preparing mycelium powder through biological enzyme |
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| 猴头菇三萜提取工艺优化及其抗氧化活性分析;李英迪等;食品工业科技;153-159 * |
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