CN115074256A - 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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Abstract
The invention provides a hericium erinaceus liquid fermentation culture medium and a method for preparing triterpenes, wherein the hericium erinaceus liquid fermentation culture medium comprises the following components in percentage by mass: 2 to 4 percent of glucose, 1 to 3 percent of soluble starch, 0.5 to 1.5 percent of yeast extract, 0.05 to 0.15 percent of monopotassium phosphate, 0.04 to 0.1 percent of magnesium sulfate heptahydrate and the balance of water. According to the invention, the fermentation conditions for the hericium erinaceus to grow at the fastest speed are found by optimizing the fermentation conditions of the edible fungus liquid. The invention also optimizes the triterpene extraction conditions, increases the content of secondary metabolite triterpene of the hericium erinaceus, and increases the nutritional value and economic value of the hericium erinaceus.
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 a large-scale edible fungus of the Hericium genus. The fruit body is large in meat quality and looks like the head of a monkey, so the monkey is called as a hericium erinaceus. The hericium erinaceus is one of eight Chinese "mountain delicacies", the mountain delicacies hericium erinaceus and the seafood cubilose are listed as four famous vegetables together with the bear paw, the sea cucumber and the shark fin, and are tender in meat, delicious, tasty, rich in nutrition, superior in color, fragrance and taste. In addition, the hericium erinaceus is also a traditional precious Chinese medicinal material in China, and has the functions of nourishing, building body, promoting digestion and benefiting five internal organs. Modern researches show that the traditional Chinese medicine composition contains active ingredients such as polypeptide, polysaccharide, fat, protein and the like, and has certain curative effects on digestive tract tumors, gastric ulcer, duodenal ulcer, gastritis, abdominal distension and the like. The economic value of the hericium erinaceus is high, such as hericium erinaceus health-care beverage, hericium erinaceus biscuits, hericium erinaceus rice and the like.
The liquid fermentation technology is one of the modern biotechnology, and is a process of dissolving some trace elements such as carbon source, nitrogen source, inorganic salt and the like and other nutrient substances necessary for edible and medicinal fungi in the growth process in pure water to be used as a culture medium, inoculating the strains after sterilization, introducing sterile air and stirring to provide oxygen required by respiratory metabolism of edible fungi, controlling proper external conditions, and carrying out mass culture and propagation of hyphae. The industrialized large-scale fermentation culture is fermentation production, also called submerged culture or submerged culture. The obtained fermentation liquid contains thallus, nutrient components decomposed and not decomposed by thallus, and metabolite produced by thallus. Compared with solid plate culture of edible fungi, liquid fermentation has many advantages, such as rapid growth of mycelium, and uniform distribution of nutrients in liquid culture medium, and is beneficial to full contact and absorption of fungi nutrients. Hypha cells can grow in a culture medium under the conditions of optimal temperature, pH, oxygen and carbon-nitrogen ratio, and metabolic waste gas generated by respiration can be discharged in time, so that metabolism is vigorous, hypha grows and splits quickly, and a large amount of mycelium, polysaccharide, polypeptide and other metabolites with physiological activity can be accumulated in a short time; the production cycle is short, the time for obtaining a large amount of mycelium and physiologically active substances through liquid fermentation culture of edible and medicinal fungi is only about 10 days generally, and the solid culture time is 20-30 days.
The carbon source is a carbon source for the growth of the hericium erinaceus and is also an energy source for the metabolic activity of the hericium erinaceus, and is a raw material for generating various metabolic substances. Glucose is the most predominant carbon source and is also the most commonly used carbon source. However, the addition of too much glucose is disadvantageous in the growth of hyphae due to the glucose effect. The nitrogen source is an important nutrient component of the hericium erinaceus mycelium and is also an indispensable raw material for synthesizing protein and nucleic acid by the hericium erinaceus. The potassium dihydrogen phosphate and magnesium sulfate heptahydrate are used as inorganic salts to maintain the osmotic pressure of the culture solution and the cell morphology.
The temperature has great influence on the fermentation of the hericium erinaceus liquid and is complicated. Firstly, the temperature influences the growth speed of hericium erinaceus mycelium pellets, and with the temperature rise, the growth and the propagation speed of the hericium erinaceus are accelerated because enzymes participate in the growth metabolism and the propagation of the hericium erinaceus. According to the kinetics of the enzymatic reaction, the temperature is increased, the enzymatic reaction is enhanced, and the respiration intensity is increased, so that the growth speed of the hericium erinaceus is increased. But with the rise of temperature, the speed of enzyme inactivation is accelerated, the fermentation period is shortened, and thalli die; secondly, temperature affects the yield of hericium erinaceus metabolites; finally, the temperature influences the physical properties of the liquid fermentation broth, indirectly influences the biosynthesis of the hericium erinaceus by changing the physical properties of the fermentation broth, and also influences the absorption of the hericium erinaceus on nutrient substances in the fermentation broth.
The pH has great influence on the liquid fermentation of the hericium erinaceus, firstly, the pH also influences the enzymatic reaction of the hericium erinaceus, strong acid and strong alkali easily inactivate the hericium erinaceus and reduce the growth speed of the hericium erinaceus; secondly, the pH influences the charge state of the hericium erinaceus cell membrane, causes the permeability change of the cell membrane, further influences the absorption of the hericium erinaceus on nutrient substances, influences the growth speed of the hericium erinaceus, and influences the generation of metabolites; in addition, pH also affects the morphology of hericium erinaceus; finally, pH has a significant impact on certain biosynthetic pathways of hericium erinaceus. Experiments prove that the hericium erinaceus can absorb organic components in the fermentation liquor only in a medium-acidity environment.
The shaking table rotational speed can change dissolved oxygen volume, and dissolved oxygen influences the growth of mycelium through participating in the physiology and biochemistry and the in-process of hericium erinaceus liquid fermentation, and the rotational speed undersize can influence dissolved oxygen and then influence the growth of hypha, and the rotational speed is too big then can force the hypha to become the piece, thereby the agglomeration suppresses the hypha and grows.
The triterpene component is a terpenoid compound with a basic parent nucleus composed of 30 carbon atoms, exists in a free form or a form combined with sugar into glycoside or ester, and has various biochemical activities. Triterpenes are also important active substances in hericium erinaceus, can be used for resisting cancers, bacteria and viruses, resisting inflammation and viruses and reducing cholesterol, and are beneficial to human bodies.
Therefore, the hericium erinaceus is required to be developed rapidly, industrial production is realized, a liquid fermentation culture medium suitable for the hericium erinaceus is researched, the most suitable fermentation condition is found, and growth of hericium erinaceus hyphae can be accelerated. An optimal triterpene extraction method is found, the yield of triterpene products can be increased, and the economic benefit of the hericium erinaceus is improved.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of the prior art and provides a hericium erinaceus liquid fermentation culture medium to effectively accelerate the growth of bacteria and the generation of bacteria metabolites.
The invention also aims to solve the technical problem of providing a method for producing mycelium spheres by fermenting hericium erinaceus.
The invention also aims to solve the technical problem of providing a method for preparing the hericium erinaceus triterpene.
In order to solve the first technical problem, the invention discloses a hericium erinaceus liquid fermentation culture medium which comprises the following components in percentage by mass: 2 to 4 percent of glucose, 1 to 3 percent of soluble starch, 0.5 to 1.5 percent of yeast extract, 0.05 to 0.15 percent of monopotassium phosphate, 0.04 to 0.1 percent 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: 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 water.
The hericium erinaceus liquid fermentation culture medium is suitable for all hericium erinaceus in the prior art, and comprises but is not limited to hericium erinaceus first-level test tube strains (in the research of Wuhanzhou jade unicorn edible fungi) ((https://m.tb.cn/h.ft114lesm＝ b79355tk＝Xvjk29O0vbZ)。
In some embodiments, the hericium erinaceus liquid fermentation culture medium is sterilized in a sterilization pot, and incubated at 115 ℃ for 20 min.
In order to solve the second technical problem, the invention discloses a method for producing mycelium spheres by hericium erinaceus fermentation, which comprises the step of inoculating a strain obtained by activating hericium erinaceus into the liquid fermentation culture medium for fermentation to obtain the hericium erinaceus mycelium spheres.
In some embodiments, the activation medium in the activating comprises the following components in weight percent: 2% of glucose, 10% of bran water, 0.4% of peptone, 0.3% of yeast extract, 2% of agar, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate and vitamin B 1 0.001% and the balance of water. In some embodiments, the bran water is prepared as follows: adding the crude bran into water with the dosage of 80-120g/500mL, boiling, preserving the heat for 10-50min, and filtering by using one-seven layers of gauze to obtain filtrate, namely bran water. In some embodiments, the bran water is prepared as follows: crude product is treatedAdding 100g/500mL bran into water, boiling, keeping the temperature for 30min, and filtering with four layers of gauze to obtain filtrate, namely bran water.
In some embodiments, the activation is performed by inoculating a piece of hericium erinaceus cut 0.5cm by 0.5cm in a sterile environment into an activation medium, and culturing the piece of hericium erinaceus in an incubator at 26 ℃ for 12 to 14 days to obtain an activated strain.
In some embodiments, the seed is inoculated into the liquid fermentation medium at an amount of 1-5 pieces per 100 mL; in some embodiments, the seed is at 0.5 x 0.5cm 3 Piece 3 pieces/100 mL of the amount inoculated into the liquid fermentation medium.
In some embodiments, the initial pH of the fermentation is 5.0-6.0, the fermentation temperature is 25-29 ℃, and the rotation speed of the shaker during the fermentation is 150-210 r/min. In some embodiments, the initial pH of the fermentation is 5.5, the temperature of the fermentation is 27 ℃, and the rotation speed of the shaker during the fermentation is 180 r/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 triterpene, which comprises the steps of dissolving mycelium spheres prepared by the method in an ethanol water solution, carrying out ultrasonic extraction and centrifuging, and obtaining supernate, namely the extracting solution containing the hericium erinaceus triterpene.
In some embodiments, the ethanol solution has a concentration of 40% to 80% by volume ethanol; in some embodiments, the ethanol solution has a concentration of 80% ethanol by volume.
In some embodiments, the feed-to-liquid ratio of the mycelium spheres to the aqueous ethanol solution is 1g:15-30 mL; in some embodiments, the feed-to-liquid ratio of the mycelium spheres to the aqueous ethanol solution is 1g to 30 mL.
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 min; in some embodiments, the time of the ultrasound extraction is 30 min.
In some embodiments, the frequency of the ultrasonic extraction is 30-50 khz; in some embodiments, the frequency of the ultrasonic extraction is 40 khz.
In some embodiments, the rotational speed of the centrifugation is 3000-; in some embodiments, the rotation speed of the centrifuge is 4000 r/min.
In some embodiments, the time of centrifugation is 5-15 min; in some embodiments, the time of centrifugation is 10 min.
In some embodiments, the triterpene content is determined by uv spectrophotometry, which comprises the following steps: firstly, preparing a triterpene standard oleanolic acid into an 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 colorimetric tubes, evaporating a 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, carrying out water bath at 60 ℃ for 20min, cooling with cold water for 15min, and then adding 3ml of glacial acetic acid. Reacting at room temperature for 10min, and measuring absorbance at 547nm, and making standard curve with oleanolic acid quality as abscissa and absorbance as ordinate. Adding the triterpene extractive solution into corresponding reagent according to the above method, measuring under the same conditions, and fitting into standard curve to obtain triterpene content.
Has the advantages 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 the fermentation conditions which enable the growth speed of the hericium erinaceus to be fastest are found by optimizing the fermentation conditions of edible fungus liquid.
According to the invention, the triterpene extraction conditions are optimized, so that the content of the secondary metabolite triterpene 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.
FIG. 1 shows the growth of Hericium erinaceum on an activated medium.
FIG. 2 shows the growth of Hericium erinaceus in a fermentation medium.
FIG. 3 shows oven-dried Hericium erinaceus mycelia.
FIG. 4 shows the milling of Hericium erinaceum mycelia.
FIG. 5 shows the triterpene extract from Hericium erinaceum.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
The hericium erinaceus slant strain (hericium erinaceus) is a hericium erinaceus first-grade test tube strain (https:// m.tb.cn/h.ft114lesm ═ b79355tk ═ Xvjk29O0vbZ) purchased from the study of edible fungi of the pericarp of wuhan zhou.
In examples 1 to 3, the external fermentation conditions were the same in each example, the initial pH was 5.5, and the mixture was cultured on a shaker at 27 ℃ and 180r/min for 10 days
Example 1: preparation of mycelium ball by liquid fermentation of hericium erinaceus
(1) Activation of mother strain: cutting Hericium Erinaceus slant strain in refrigerator under aseptic environment to obtain 0.5cm × 0.5cm block, inoculating on activation culture medium, and culturing in constant temperature box at 26 deg.C for 12-14 days to obtain first-stage strain (FIG. 1). The mass contents of the components in the activation medium are as follows: 2% of glucose, 10% of bran water, 0.4% of peptone, 0.3% of yeast extract, 2% of agar, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate and vitamin B 1 0.001%, and water in balance, wherein the bran water is prepared by adding 100g crude bran into about 500ml pure water, boiling, keeping 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 components of the liquid fermentation culture medium comprise, by weight, 2% of glucose, 1% of soluble starch, 0.5% of yeast extract, 0.05% of monopotassium phosphate, 0.04% of magnesium sulfate heptahydrate and the balance of pure water, and the pH value is 5.5. Putting the liquid fermentation medium into a 250mL triangular flask according to the amount of 100mL, putting the triangular flask into a sterilizing pot, sterilizing at 115 ℃ for 20min, and drying in a 65 ℃ oven after sterilization.
(3) Liquid fermentation of hericium erinaceus: and (2) cutting off 0.5cm x 0.5cm of the primary strain obtained in the step (1), inoculating 3 fungus blocks into the liquid fermentation medium obtained in the step (2) under an aseptic condition, and culturing on a shaking table at 27 ℃ and 180r/min for 10 days (figure 2) to form mycelium spheres, thereby obtaining a fermentation liquid containing the mycelium spheres.
Dry weight measurement by hypha dry weight method: and (3) filtering the mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, flatly paving the filtered mycelium pellets on the flat plate, drying the mycelium pellets in a 65-DEG C oven (shown in figure 3), weighing again, wherein the weight of the flat plate subtracted from the total weight is the dry weight of the mycelium, and storing the filtered bacterial liquid in a refrigerator for later use.
Example 2: preparation of mycelium ball by liquid fermentation of hericium erinaceus
(1) Activation of mother strain: cutting Hericium Erinaceus slant strain in refrigerator under aseptic environment to obtain 0.5cm × 0.5cm block, inoculating on activating culture medium, and culturing in constant temperature box at 26 deg.C for 12-14 days to obtain first-stage strain. The mass contents of the components in the activation medium are 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 in balance, wherein the bran water is prepared by adding about 500ml pure water into 100g of crude bran, boiling, keeping the temperature for 30min, and filtering on four layers of gauze.
(2) And (3) sterilizing a liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the components of the liquid fermentation culture medium comprise, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.06% of magnesium sulfate heptahydrate and the balance of pure water, and the pH value 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 the temperature for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of edible fungi: and (3) cutting off the primary strain obtained in the step (1) by 0.5cm x 0.5cm, inoculating the cut primary strain into the liquid fermentation medium obtained in the step (2), inoculating 3 fungus blocks into each bottle under an aseptic condition, and culturing on a shaking table at the temperature of 27 ℃ and the speed of 180r/min for 10 days to form mycelium spheres, thereby obtaining the fermentation liquid containing the mycelium spheres.
Dry weight measurement by hypha dry weight method: and (4) filtering the mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, flatly paving the filtered mycelium pellets on the flat plate, drying the mycelium pellets in a 65-DEG C oven, weighing again, and storing the filtered bacterial liquid in a refrigerator for later use, wherein the weight of the filtered mycelium pellets is subtracted from the total weight of the mycelium pellets to obtain the dry weight of the mycelium.
Example 3: mycelium sphere prepared by hericium erinaceus liquid fermentation
(1) Activation of mother strain: cutting Hericium Erinaceus slant strain in refrigerator under aseptic environment to obtain 0.5cm × 0.5cm block, inoculating on activating culture medium, and culturing in constant temperature box at 26 deg.C for 12-14 days to obtain first-stage strain. The mass contents of the components in the activation medium are 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 in balance, wherein the bran water is prepared by adding about 500ml pure water into 100g of crude bran, boiling, keeping the temperature for 30min, and filtering on four layers of gauze.
(2) And (3) sterilizing a liquid fermentation medium: preparing a liquid fermentation culture medium, wherein the components of the liquid fermentation culture medium comprise, 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, and the pH value 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 the temperature for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of edible fungi: and (3) cutting off the primary strain obtained in the step (1) by 0.5cm x 0.5cm, inoculating the cut primary strain into the liquid fermentation medium obtained in the step (2), inoculating 3 fungus blocks into each bottle under an aseptic condition, and culturing on a shaking table at the temperature of 27 ℃ and the speed of 180r/min for 10 days to form mycelium spheres, thereby obtaining the fermentation liquid containing the mycelium spheres.
Dry weight measurement by hypha dry weight method: and (4) filtering the mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, flatly paving the filtered mycelium pellets on the flat plate, drying the mycelium pellets in a 65-DEG C oven, weighing again, and storing the filtered bacterial liquid in a refrigerator for later use, wherein the weight of the filtered mycelium pellets is subtracted from the total weight of the mycelium pellets to obtain the dry weight of the mycelium.
In examples 4 to 6, the fermentation medium components were identical in each example, and the medium components were glucose 3%, soluble starch 2%, yeast extract 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.06%, and pure water in balance
Example 4: preparation of mycelium ball by liquid fermentation of hericium erinaceus
(1) Activation of mother strain: cutting Hericium Erinaceus slant strain in refrigerator under aseptic environment to obtain 0.5cm × 0.5cm block, inoculating on activating culture medium, and culturing in constant temperature box at 26 deg.C for 12-14 days to obtain first-stage strain. The mass contents of the components in the activation medium are 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 in balance, wherein the bran water is prepared by adding about 500ml pure water into 100g of crude bran, boiling, keeping 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 components of the liquid fermentation culture medium comprise, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.06% of magnesium sulfate heptahydrate and the balance of pure water, and the pH value 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 the temperature for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of edible fungi: and (3) cutting off the primary strain obtained in the step (1) by 0.5cm x 0.5cm, inoculating the cut primary strain into the liquid fermentation medium obtained in the step (2), inoculating 3 fungus blocks into each bottle under aseptic conditions, and culturing for 10 days on a shaking bed at the temperature of 25 ℃ and at the speed of 150r/min to form mycelium spheres, thereby obtaining the fermentation liquid containing the mycelium spheres.
Dry weight measurement by hypha dry weight method: and (4) filtering the mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, flatly paving the filtered mycelium pellets on the flat plate, drying the mycelium pellets in a 65-DEG C oven, weighing again, and storing the filtered bacterial liquid in a refrigerator for later use, wherein the weight of the filtered mycelium pellets is subtracted from the total weight of the mycelium pellets to obtain the dry weight of the mycelium.
Example 5: preparation of mycelium ball by liquid fermentation of hericium erinaceus
(1) Activation of mother strain: cutting Hericium Erinaceus slant strain in refrigerator under aseptic environment to obtain 0.5cm × 0.5cm block, inoculating on activating culture medium, and culturing in constant temperature box at 26 deg.C for 12-14 days to obtain first-stage strain. The mass contents of the components in the activation medium are 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 in balance, wherein the bran water is prepared by adding about 500ml pure water into 100g of crude bran, boiling, keeping 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 components of the liquid fermentation culture medium comprise, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.06% of magnesium sulfate heptahydrate and the balance of pure water, and the pH value is 5.5. Putting the liquid fermentation medium into a 250mL triangular flask according to the amount of 100mL, putting the triangular flask into a sterilization pot, sterilizing at 115 ℃ for 20min, and putting the triangular flask into a 65 ℃ oven for drying after sterilization.
(3) Liquid fermentation of edible fungi: and (3) cutting off the primary strain obtained in the step (1) by 0.5cm x 0.5cm, inoculating the cut primary strain into the liquid fermentation medium obtained in the step (2), inoculating 3 fungus blocks into each bottle under an aseptic condition, and culturing on a shaking table at the temperature of 27 ℃ and the speed of 180r/min for 10 days to form mycelium spheres, thereby obtaining the fermentation liquid containing the mycelium spheres.
Dry weight measurement by hypha dry weight method: and (4) filtering the mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, flatly paving the filtered mycelium pellets on the flat plate, drying the mycelium pellets in a 65-DEG C oven, weighing again, and storing the filtered bacterial liquid in a refrigerator for later use, wherein the weight of the filtered mycelium pellets is subtracted from the total weight of the mycelium pellets to obtain the dry weight of the mycelium.
Example 6: preparation of mycelium ball by liquid fermentation of hericium erinaceus
(1) Activation of mother strain: cutting Hericium Erinaceus slant strain in refrigerator under aseptic environment to obtain 0.5cm × 0.5cm block, inoculating on activating culture medium, and culturing in constant temperature box at 26 deg.C for 12-14 days to obtain first-stage strain. The mass contents of the components in the activation medium are 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 in balance, wherein the bran water is prepared by adding about 500ml pure water into 100g of crude bran, boiling, keeping 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 components of the liquid fermentation culture medium comprise, 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, and the pH value of the liquid fermentation culture medium 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 the temperature for 20min for sterilization, and placing the sterilized liquid fermentation culture medium into a 65 ℃ oven for drying.
(3) Liquid fermentation of edible fungi: and (3) cutting off the primary strain obtained in the step (1) by 0.5cm x 0.5cm, inoculating the cut primary strain into the liquid fermentation medium obtained in the step (2), inoculating 3 fungus blocks into each bottle under an aseptic condition, and culturing on a shaking table at 29 ℃ for 10 days at 210r/min to form mycelium spheres, thereby obtaining the fermentation liquid containing the mycelium spheres.
Dry weight measurement by hypha dry weight method: and (4) filtering the mycelium pellets obtained in the step (3) by using single-layer filter cloth, weighing a clean flat plate, flatly paving the filtered mycelium pellets on the flat plate, drying the mycelium pellets in a 65-DEG C oven, weighing again, and storing the filtered bacterial liquid in a refrigerator for later use, wherein the weight of the filtered mycelium pellets is subtracted from the total weight of the mycelium pellets to obtain the dry weight of the mycelium.
Examples 7 to 9 are the preparation of triterpene from Hericium erinaceum, and the mycelium pellet used was the mycelium pellet prepared in example 2
In the following examples, the triterpene content was determined by uv spectrophotometry, specifically: firstly, preparing a triterpene standard oleanolic acid into an oleanolic acid solution with the concentration of 0.2mg/ml (0.2mg oleanolic acid is dissolved in 1ml methanol solution), respectively sucking 0,0.1,0.2,0.3,0.4,0.5ml and 25ml colorimetric tubes, evaporating a solvent in boiling water bath, adding 0.5ml vanillin-glacial acetic acid solution with the concentration of 50g/L (50g vanillin is dissolved in 1L glacial acetic acid solution) and 1ml perchloric acid solution, mixing, carrying out water bath at 60 ℃ for 20min, cooling with cold water for 15min, and then adding 3ml glacial acetic acid. Reacting at room temperature for 10min, and measuring absorbance at 547nm, and making standard curve with oleanolic acid quality as abscissa and absorbance as ordinate. And adding corresponding reagents into the triterpene extracting solution extracted in each embodiment according to the method, measuring under the same condition, substituting into a standard curve, and calculating the content of the hericium erinaceus triterpene.
Example 7: ultrasonic-assisted extraction of triterpenes from ethanol
Grinding the dried mycelium pellet (figure 4), dissolving with 40% ethanol, and extracting the dissolved solution in an ultrasonic cleaning instrument at 40khz under the condition of ultrasonic frequency of 40khz and temperature of 40 deg.C, wherein the ratio of mycelium pellet to ethanol is 1g:10 ml. Then centrifuging the extractive solution at 4000r/min for 10min, and collecting supernatant, which is triterpene extractive solution (FIG. 5).
Example 8: ultrasonic-assisted extraction of triterpenes from ethanol
Grinding the dried mycelium pellet, dissolving with 60% ethanol, and extracting the dissolved solution in an ultrasonic cleaning instrument at ultrasonic frequency of 40khz and temperature of 60 deg.C for 30min, wherein the ratio of mycelium pellet to ethanol is 1g:15 ml. And then centrifuging the extracting solution on a centrifuge at the rotating speed of 4000r/min for 10min, and collecting supernatant, wherein the supernatant is the triterpene extracting solution.
Example 9: ultrasonic-assisted extraction of triterpenes from ethanol
Grinding the dried mycelium pellets, dissolving the mycelium pellets in 80% ethanol, and extracting the dissolved solution on an ultrasonic cleaning instrument for 30min at the ultrasonic frequency of 40khz and the temperature of 80 ℃ with the ratio of the mycelium pellets to the ethanol being 1g to 30 ml. And then centrifuging the extracting solution on a centrifuge at the rotating speed of 4000r/min for 10min, and collecting supernatant, wherein the supernatant is the triterpene extracting solution.
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 result (culture medium with consistent content of components)
Table 3: preparation of triterpene as metabolite of Hericium erinaceus
| Triterpene yield (mg/L fermentation liquor) | 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 components were glucose 3%, soluble starch 2%, yeast extract 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.06%, and pure water in balance (example 2), the growth rate of Hericium erinaceus was the fastest; when the nutrient content was further increased (example 3), the effect on the growth of Hericium erinaceum was not large, probably because the glucose effect was produced due to the excessively high glucose content.
As can be seen from Table 2, the fermentation conditions were that the pH of the fermentation medium was 5.5, the temperature of shake cultivation was 27 ℃ and the rotation speed was 180r/min (example 5), the liquid fermentation of Hericium erinaceus was the best, the mycelia grew the fastest, the dry weight of mycelia was also the greatest, and the change in external conditions had a great influence on the growth of Hericium erinaceus.
Therefore, it can be concluded that the liquid fermentation medium comprises, by weight, 3% of glucose, 2% of soluble starch, 1% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.06% of magnesium sulfate heptahydrate, and the balance of pure water, wherein the fermentation conditions are that the initial pH is 5.5, the fermentation temperature is 27 ℃, the rotation speed of a shaking table is 180r/min, the liquid fermentation is most suitable for the liquid fermentation of hericium erinaceus, and the dry weight of mycelia is maximum.
As can be seen from Table 3, the yield of triterpene varied from triterpene extraction condition to triterpene extraction condition. When the ethanol concentration is 80%, the feed-liquid ratio of the hericium erinaceus mycelium pellets to the ethanol is 1:30g/ml, and the extraction temperature is 80 ℃ (example 9), the triterpene yield is highest and the yield is also highest.
In conclusion, the invention utilizes glucose and soluble starch as composite carbon sources, thus eliminating the glucose effect, increasing the nutrition of the liquid fermentation liquor and increasing the growth speed of the hericium erinaceus; the yeast extract is used as a nitrogen source, is rich in nutrition, is rich in 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 content of each component of the culture medium is optimized, and the condition most suitable for liquid fermentation of the hericium erinaceus is found; experiments prove that for liquid fermentation of hericium erinaceus, the temperature is controlled to be 25-29 ℃, and the growth speed of mycelia is high; experiments prove that for liquid fermentation of hericium erinaceus, the pH is controlled to be 5.0-6.0, and the growth speed of mycelia is high; according to the experimental verification, the rotation speed of the shaking table is 150-210r/min, and the mycelium grows fastest.
The present invention provides a method and a medium for liquid fermentation of hericium erinaceus and a method for preparing triterpenes, and a method and a means for implementing the method and the means are numerous, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (10)
1. The hericium erinaceus liquid fermentation culture medium is characterized by comprising the following components in percentage by mass: 2 to 4 percent of glucose, 1 to 3 percent of soluble starch, 0.5 to 1.5 percent of yeast extract, 0.05 to 0.15 percent of monopotassium phosphate, 0.04 to 0.1 percent of magnesium sulfate heptahydrate and the balance of water.
2. The hericium erinaceus liquid fermentation culture medium according to claim 1, characterized by comprising the following components in percentage by mass: 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 water.
3. A method for producing mycelium spheres by fermentation of hericium erinaceus is characterized in that a strain obtained by activating the hericium erinaceus is inoculated into the liquid fermentation medium according to claim 1 or 2 and fermented to obtain the hericium erinaceus mycelium spheres.
4. The method according to claim 3, wherein the activation medium in the activation comprises the following components in percentage by weight: 2% of glucose, 10% of bran water, 0.4% of peptone, 0.3% of yeast extract, 2% of agar, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate and vitamin B 1 0.001% and the balance of water.
5. The method as claimed in claim 4, wherein said bran water is prepared as follows: adding the crude bran into water with the dosage of 80-120g/500mL, boiling, preserving the heat for 10-50min, and filtering by using one-seven layers of gauze to obtain filtrate, namely bran water.
6. The method of claim 3, wherein the bacterial species is selected from the group consisting of 0.2-0.8 x 0.2-0.8 3 And/piece 1-5 pieces/100 mL, inoculating into liquid fermentation medium.
7. The method as claimed in claim 3, wherein the initial pH of the fermentation is 5.0-6.0, the fermentation temperature is 25-29 ℃, and the rotation speed of the rocking platform during the fermentation is 150-210 r/min.
8. The method of claim 3, wherein the initial pH of the fermentation is 5.5, the temperature of the fermentation is 27 ℃, and the rotation speed of the rocking platform during the fermentation is 180 r/min.
9. A method for preparing triterpene from Hericium erinaceus is characterized in that mycelium spheres prepared by the method of any one of claims 3 to 6 are dissolved in ethanol solution, ultrasonic extraction and centrifugation are carried out, and the obtained supernatant is an extracting solution containing triterpene from Hericium erinaceus.
10. The method according to claim 9, wherein the volume concentration of ethanol in the ethanol solution is 40-80%; the feed-liquid ratio of the mycelium spheres to the ethanol aqueous solution is 1g:15-30 mL; the temperature of ultrasonic extraction is 40-80 ℃.
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