CN114214387B - Method for preparing schizophyllan and ergothioneine by co-fermentation of two strains - Google Patents
Method for preparing schizophyllan and ergothioneine by co-fermentation of two strains Download PDFInfo
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Abstract
The invention provides a method for preparing schizophyllan and ergothioneine by co-fermentation of two strains. The method comprises the following steps: inoculating the fermentation seed liquid of the schizophyllum commune and the fermentation seed liquid of the mushroom fungus to a co-fermentation culture medium for co-fermentation culture to obtain fermentation liquid containing schizophyllan and ergothioneine. The method of the invention co-ferments the schizophyllum commune and the bacterial strain for preparing the ergothioneine, finds that the co-fermentation of the two bacterial strains can play a synergistic role, can obviously improve the yield of the ergothioneine, and the obtained co-fermentation solution has obvious capacity of resisting oxidation and removing free radicals.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing schizophyllan and ergothioneine through co-fermentation of double strains.
Background
Ergothioneine (EGT), chemically 2-mercaptohistidine trimethyl inner salt, is a rare natural chiral amino acid. Ergothioneine has unique physiological functions in the aspects of oxidation resistance, free radical removal, metal ion chelation, ultraviolet radiation damage prevention, cancer inhibition and the like, has more superior physiological functions in certain aspects than natural antioxidants such as glutathione and the like, and is widely applied to the fields of food additives, health care products, cosmetics and the like at present. At present, most of the production methods of ergothioneine adopt a single edible fungus liquid fermentation mode, and then the products after fermentation are subjected to post-treatment to finally obtain the ergothioneine. However, the ergothioneine obtained by the existing production method has low yield and cannot meet the increasing market demand.
Schizophyllum commune polysaccharide (SPG) is water-soluble polysaccharide obtained by fermenting Schizophyllum commune, and has various physiological activities of inhibiting tumor, resisting bacteria, diminishing inflammation, resisting radiation, improving immunity, etc. At present, the production method of schizophyllan mostly adopts a single edible fungus liquid fermentation mode, and then the fermented product is subjected to post-treatment to finally obtain the schizophyllan. However, the schizophyllan obtained by fermentation is difficult to re-dissolve and is not beneficial to biological absorption, and the application of the schizophyllan is greatly limited.
CN109939027A discloses a method for preparing a cosmetic stock solution containing ergothioneine by fermenting hericium erinaceus, which comprises the following steps: (1) inoculating hericium erinaceus hypha into a liquid seed culture medium for culture to obtain a seed solution; (2) inoculating the seed liquid into a fermentation culture medium for fermentation, adding an ergothioneine precursor substance, and fermenting to the end point to obtain a fermentation liquid; and step 3: and treating the fermentation liquor containing the mycelium to obtain a fermentation stock solution containing ergothioneine as a cosmetic stock solution containing the ergothioneine. The method is a widely applied ergothioneine production method at present, a single edible fungus liquid fermentation mode is adopted, and a product after fermentation is subjected to post-treatment, so that a stock solution product contains about 300mg/L of ergothioneine.
CN112195215A discloses a method for producing ergothioneine by combined fermentation of pleurotus citrinopileatus matsutake mycelia, which comprises the following steps: inoculating Armillaria matsutake mycelium into cultured Pleurotus citrinopileatus seed solution, supplementing a certain amount of seed culture medium, and culturing for 2-3 days; inoculating seed liquid containing Pleurotus Citrinopileatus Sing and Armillaria matsutake obtained by culturing into fermentation medium at an inoculation amount of 5-20% of the volume of the fermentation medium, adding precursor, fermenting for 2-4 days, adding small amount of precursor, and continuing to ferment for 3-4 days; and treating the fermented liquid to obtain solution containing ergothioneine. The pleurotus citrinopileatus and the agaricus blazei are selected as the production strains of the ergothioneine, the aim of improving the ergothioneine yield is achieved in a combined fermentation mode, the ergothioneine content in the final fermentation liquid can reach about 880mg/L, and the ergothioneine yield has a space for improving.
CN107557407A discloses a method for regulating and controlling molecular weight of Schizophyllum commune fermentation product Schizophyllum commune polysaccharide, which comprises inoculating and culturing Schizophyllum commune slant strain in PDA culture medium test tube slant to obtain fermentation seed solution; inoculating the seed culture solution into a fermentation tank for fermentation, respectively controlling the fermentation conditions, discharging materials after the fermentation is finished, and removing mycelia; and (4) carrying out post-treatment to obtain the refined schizophyllan. The method adopts a single edible fungus liquid fermentation mode, so that fermentation conditions need to be respectively controlled during the fermentation of the schizophyllum commune, and the preparation process is complex.
CN113337545A discloses a Schizophyllum commune fermentation product, a preparation method thereof, a skin care product and a Schizophyllum commune culture medium. The preparation method of the Schizophyllum commune fermentation product comprises the following steps: culturing Schizophyllum commune in a Schizophyllum commune culture medium, and collecting a fermentation product; wherein the schizophyllum commune culture medium contains kudzu root. Adding radix Puerariae powder into the culture medium to culture Schizophyllum commune, and collecting the fermentation product. The invention improves the antioxidation level of the schizophyllum commune fermentation product by a method of containing the kudzuvine root in the schizophyllum commune culture medium.
Therefore, there is a need to develop a method for preparing schizophyllan and ergothioneine by co-fermentation of two bacterial species.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for preparing schizophyllan and ergothioneine by double-strain co-fermentation, which co-ferments schizophyllan and a strain for preparing ergothioneine, finds that the double-strain co-fermentation can exert a synergistic effect, can obviously improve the yield of the ergothioneine, and obtains a co-fermentation solution with obvious antioxidant and free radical scavenging capabilities.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a process for the preparation of schizophyllan and ergothioneine by co-fermentation of two bacterial species, said process comprising the steps of: inoculating the fermentation seed liquid of the schizophyllum commune and the fermentation seed liquid of the mushroom fungus to a co-fermentation culture medium for co-fermentation culture to obtain fermentation liquid containing schizophyllan and ergothioneine.
In the invention, the schizophyllum commune and the bacterial strain for preparing the ergothioneine are co-fermented, and the co-fermentation of the two bacterial strains is found, so that not only can no competition phenomenon occur, but also the synergistic effect can be exerted, thereby leading the ergothioneine to be prepared in a large amount from the mycelium, ensuring the product quality of the ergothioneine and obviously improving the yield of the ergothioneine; and the preparation process is simple, and is particularly suitable for large-scale production of ergothioneine.
Particularly, as the finally obtained fermentation liquor contains schizophyllan and ergothioneine which are matched with each other, the schizophyllan and the ergothioneine have synergistic effect, the composition can further improve the scavenging effect on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals, can enhance the antioxidant capacity of skin tissues, reduce the free radical content of the skin tissues and play a role in delaying skin photoaging.
Preferably, the co-fermentation culture medium consists of the following components in percentage by mass: 0.5-5% of glucose, 0.5-3% of tryptone, 0.1-3% of yeast extract, 0.1-0.5% of yeast extract, 0.01-0.15% of magnesium sulfate, 0.05-0.2% of potassium dihydrogen phosphate and the balance of water.
In the invention, a proper culture medium is selected, so that the Schizophyllum commune and the mushroom can be subjected to co-fermentation culture, the two strains can exert a synergistic effect, and the yield of the ergothioneine is further improved.
The glucose content may be 0.5 to 5%, for example, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, 5%, etc., preferably 3 to 5%, based on 100% by mass of the co-fermentation medium.
The content of tryptone may be, for example, 0.5 to 3%, preferably 2 to 3%, based on 100% by mass of the co-fermentation medium, such as 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, and the like.
The content of the yeast extract is 0.1 to 3%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, etc., preferably 2 to 3%, based on 100% by mass of the co-fermentation medium.
The content of the yeast extract is 0.1 to 0.5%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or the like, based on 100% by mass of the co-fermentation medium.
The content of magnesium sulfate may be, for example, 0.01 to 0.15%, 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.15% or the like, based on 100% by mass of the co-fermentation medium.
The content of potassium dihydrogen phosphate may be 0.05 to 0.2%, for example, 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, etc., based on 100% by mass of the co-fermentation medium.
Preferably, the co-fermentation medium has a pH of 5.5-7.0, and may be, for example, 5.5, 5.6, 5.8, 6.0, 6.2, 6.5, 6.7, 6.9, 7.0, and the like.
Preferably, the mass percentage of the fungus body in the seed liquid for Schizophyllum commune fermentation is 5-30%, for example, 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, etc.
Preferably, the mass percentage of the fungus body in the fermentation seed liquid of the mushroom fungus is 5 to 30%, for example, 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, etc.
Preferably, the amount of inoculum of the fermentation seed liquid of Schizophyllum commune in the co-fermentation medium is 2-20 wt%, and may be, for example, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, etc.
Preferably, the amount of inoculation of the fermentation seed liquid of mushroom in the co-fermentation medium is 5-20 wt%, and may be, for example, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, etc.
Preferably, the specific steps of the co-fermentation culture are as follows: inoculating the fermentation seed liquid of the Schizophyllum commune and the fermentation seed liquid of the mushroom to a shake flask filled with a co-fermentation culture medium, and placing the shake flask in a constant-temperature shaking table for shake culture.
Preferably, the temperature of the shaking culture is 28-30 ℃, such as 28 ℃, 28.5 ℃, 29 ℃, 29.5 ℃, 30 ℃ and the like, the rotation speed of the shaking culture is 100-150r/min, such as 100r/min, 110r/min, 120r/min, 140r/min, 150r/min and the like, and the time of the shaking culture is 3-5 days, such as 3 days, 3.5 days, 4 days, 4.5 days, 5 days and the like.
Preferably, the co-fermentation culture further comprises sonication and/or centrifugation.
Preferably, the power of the ultrasound is 400-1000, such as 400W, 500W, 600W, 700W, 800W, 900W, 1000W, etc., and the time of the ultrasound is 5-20min, such as 5min, 6min, 8min, 10min, 12min, 14min, 16min, 18min, 20min, etc.
Preferably, the rotation speed of the centrifugation is 8000-10000r/min, such as 8000r/min, 8500r/min, 9000r/min, 9500r/min and the like, and the centrifugation time is 5-10min, such as 5min, 6min, 7min, 8min, 9min, 10min and the like.
Preferably, the Schizophyllum commune fermented seed liquid is prepared by the following preparation method:
(a) inoculating Schizophyllum commune to a PDA culture medium for activation culture to obtain activated seeds;
(b) inoculating the activated seeds obtained in the step (a) into a liquid culture medium for seed culture to obtain a fermentation seed liquid of the schizophyllum commune.
Preferably, in step (a), the temperature of the activation culture is 24-26 ℃, for example, 24 ℃, 25.5 ℃, 25 ℃, 25.5 ℃, 26 ℃ and the like, and the time of the activation culture is 2-5 days, for example, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 5 days and the like.
Preferably, in the step (b), the liquid culture medium consists of the following components in percentage by mass: 0.5-2% of glucose, 0.1-2% of yeast extract, 0.01-0.15% of magnesium sulfate, 0.05-0.2% of potassium dihydrogen phosphate and the balance of water.
The content of glucose may be, for example, 0.5 to 2%, for example, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, or the like, based on 100% by mass of the liquid medium.
The content of the yeast extract is 0.1 to 2% by mass of the liquid medium, and may be, for example, 0.1%, 0.2%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, or the like.
The content of magnesium sulfate may be, for example, 0.01 to 0.15%, 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.15% or the like, based on 100% by mass of the liquid medium.
The content of monopotassium phosphate is 0.05-0.2%, for example, 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, etc., based on 100% by mass of the liquid medium.
Preferably, in step (b), the seed culture is performed in a constant temperature shaking table, the temperature of the culture is 24-26 ℃, such as 24 ℃, 25.5 ℃, 25 ℃, 25.5 ℃, 26 ℃ and the like, the rotation speed of the culture is 150-200r/min, such as 150r/min, 160r/min, 170r/min, 180r/min, 190r/min, 200r/min and the like, and the time of the culture is 3-5 days, such as 3 days, 3.5 days, 4 days, 4.5 days, 5 days and the like.
Preferably, the fermented seed liquid of the mushroom is prepared by the following preparation method: inoculating the mycelium of mushroom fungus into a seed culture medium for seed culture to obtain the fermentation seed liquid of the mushroom fungus.
Preferably, the mushroom fungus includes any one or a combination of at least two of hericium erinaceus, pleurotus ostreatus, ganoderma lucidum, pleurotus eryngii, phoenix mushroom, pleurotus citrinopileatus, matsutake, oyster mushroom, and agaric.
Preferably, the seed culture medium consists of the following components in percentage by mass: 1-10% of cane sugar, 3-5% of bean cake powder, 1-3% of corn flour, 10.1-0.3% of vitamin B, 0.01-0.15% of magnesium sulfate, 0.05-0.2% of potassium dihydrogen phosphate and the balance of water.
The sucrose content may be 1 to 10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc., based on 100% by mass of the seed medium.
The content of the soybean cake meal is 3 to 5%, for example, 3%, 3.5%, 4%, 4.5%, 5%, etc., based on 100% by mass of the seed medium.
The corn meal content is 1-3%, for example, 1%, 1.5%, 2%, 2.5%, 3%, etc., based on 100% by mass of the seed medium.
The content of vitamin B1 may be 0.1 to 0.3%, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, or the like, based on 100% by mass of the seed medium.
The content of magnesium sulfate may be, for example, 0.01 to 0.15%, 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.15% or the like, based on 100% by mass of the seed medium.
The content of potassium dihydrogen phosphate is 0.05-0.2%, for example, 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, etc., based on 100% by mass of the seed medium.
Preferably, the seed culture is performed in a constant temperature shaking table, the temperature of the culture is 23-25 ℃, for example, 23 ℃, 23.5 ℃, 24 ℃, 24.5 ℃, 25 ℃ and the like, the rotation speed of the culture is 160-180r/min, for example, 160r/min, 165r/min, 170r/min, 175r/min, 180r/min and the like, and the time of the culture is 1-3 days, for example, 1 day, 1.5 days, 2 days, 2.5 days, 3 days and the like.
Preferably, the ergothioneine content in the fermentation broth containing schizophyllan and ergothioneine is above 900mg/L, for example 900mg/L, 920mg/L, 940mg/L, 960mg/L, 980mg/L, 1000mg/L, 1100mg/L, 1200mg/L, 1400mg/L, 1600mg/L, 2000mg/L, etc., preferably 950-.
Preferably, the schizophyllan and ergothioneine-containing fermentation broth has a schizophyllan content of 9g/L or more, for example, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, 16g/L, 18g/L, 20g/L, etc., preferably 9 to 12 g/L.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention co-ferments the schizophyllum commune and the bacterial strain for preparing the ergothioneine, finds that the co-fermentation of the two bacterial strains does not cause the competition phenomenon, and can also play a role in synergy, so that the ergothioneine is prepared in a large amount from the mycelium, the product quality of the ergothioneine is ensured, and the yield of the ergothioneine is obviously improved; the preparation process is simple, and is particularly suitable for large-scale production of ergothioneine;
(2) the fermentation liquor finally obtained by the invention contains schizophyllan and ergothioneine, the schizophyllan and the ergothioneine are mutually matched, so that the synergistic effect is achieved, the scavenging effect of the composition on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals can be further improved, the oxidation resistance of skin tissues can be enhanced, the free radical content of the skin tissues can be reduced, and the effect of delaying skin photoaging can be achieved.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.
The following examples and comparative examples, where no specific techniques or conditions are indicated, can be performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products commercially available from a formal channel or may be prepared by the prior art, without reference to the manufacturer.
Preparation example 1
The preparation example provides a Schizophyllum commune fermentation seed liquid, which is prepared by the following preparation method:
(a) inoculating Schizophyllum commune into PDA culture medium, activating and culturing at 24 deg.C for 3 days to obtain activated seed, and storing at 4 deg.C for seed preservation;
wherein the PDA culture medium comprises the following components in percentage by mass: 20% of potato, 2% of glucose and 2% of agar, and the balance of water; sterilizing the prepared PDA culture medium at 121 deg.C for 20 min;
(b) inoculating the activated seeds obtained in the step (a) into a shake flask filled with a liquid culture medium, placing the shake flask in a constant-temperature shaking table for shake culture at the temperature of 24 ℃, the rotating speed of the culture is 150r/min, and the culture time is 3 days, so as to obtain a zymolysis seed liquid of schizophyllum commune with the thallus mass content of 10%;
wherein the liquid culture medium comprises the following components in percentage by mass: 1% of glucose, 1% of yeast extract, 0.05% of magnesium sulfate, 0.1% of monopotassium phosphate and the balance of water; the prepared liquid culture medium is sterilized at 121 deg.C for 20 min.
Preparation example 2
The preparation example provides a Schizophyllum commune fermentation seed liquid, which is prepared by the following preparation method:
(a) inoculating Schizophyllum commune into PDA culture medium, activating and culturing at 25 deg.C for 4 days to obtain activated seed, and storing at 4 deg.C for seed preservation;
wherein the PDA culture medium comprises the following components in percentage by mass: 20% of potato, 2% of glucose and 2% of agar, and the balance of water; sterilizing the prepared PDA culture medium at 121 deg.C for 20 min;
(b) inoculating the activated seeds obtained in the step (a) into a shake flask filled with a liquid culture medium, placing the shake flask in a constant-temperature shaking table for shake culture at the temperature of 25 ℃, the rotating speed of the culture of 180r/min and the culture time of 4 days to obtain a zymolysis seed liquid of the schizophyllum commune with the thallus mass content of 18%;
wherein the liquid culture medium comprises the following components in percentage by mass: 1% of glucose, 1% of yeast extract, 0.05% of magnesium sulfate, 0.1% of potassium dihydrogen phosphate and the balance of water; the prepared liquid culture medium is sterilized at 121 deg.C for 20 min.
Preparation example 3
The preparation example provides a Schizophyllum commune fermentation seed liquid, which is prepared by the following preparation method:
(a) inoculating Schizophyllum commune to PDA culture medium, activating and culturing at 26 deg.C for 5 days to obtain activated seed, and storing at 4 deg.C for seed preservation;
wherein the PDA culture medium comprises the following components in percentage by mass: 20% of potato, 2% of glucose and 2% of agar, and the balance of water; sterilizing the prepared PDA culture medium at 121 deg.C for 20 min;
(b) inoculating the activated seeds obtained in the step (a) into a shake flask filled with a liquid culture medium, and placing the shake flask in a constant-temperature shaking table for shake culture at the temperature of 26 ℃, the rotating speed of the culture of 200r/min and the culture time of 5 days to obtain a schizophyllum fermentation seed liquid with the thallus mass content of 25%;
wherein the liquid culture medium comprises the following components in percentage by mass: 1% of glucose, 1% of yeast extract, 0.05% of magnesium sulfate, 0.1% of potassium dihydrogen phosphate and the balance of water; after the preparation of the liquid medium, the liquid medium was sterilized at 121 ℃ for 20 min.
Preparation example 4
The preparation example provides a mushroom fermented seed liquid, which is prepared by the following preparation method: inoculating the mycelium of the hericium erinaceus into a shake flask filled with a liquid culture medium, and placing the shake flask in a constant-temperature shaking table for shake culture; culturing at 23 deg.C at a rotation speed of 160r/min for 1 day to obtain fermented seed solution of Hericium erinaceus with thallus content of 12%;
wherein the seed culture medium comprises the following components in percentage by mass: 5% of cane sugar, 4% of bean cake powder, 2% of corn flour, 10.2% of vitamin B, 0.1% of magnesium sulfate, 0.05% of potassium dihydrogen phosphate and the balance of water; after the preparation of the seed culture medium, the seed culture medium is sterilized at 121 ℃ for 20 min.
Preparation example 5
The preparation example provides a mushroom fermented seed liquid, which is prepared by the following preparation method: inoculating the mycelium of Pleurotus ostreatus into a shake flask containing liquid culture medium, and shake culturing in a constant temperature shaking table; culturing at 24 deg.C at 170r/min for 2 days to obtain fermented seed solution of Pleurotus ostreatus with thallus mass content of 23%;
wherein the seed culture medium comprises the following components in percentage by mass: 6% of cane sugar, 3% of bean cake powder, 1% of corn flour, 10.3% of vitamin B, 0.1% of magnesium sulfate, 0.05% of potassium dihydrogen phosphate and the balance of water; the prepared seed culture medium is sterilized at 121 deg.C for 20 min.
Preparation example 6
The preparation example provides a mushroom fermented seed liquid, which is prepared by the following preparation method: inoculating agaricus blazei murill mycelium into a shake flask filled with a liquid culture medium, and placing the shake flask in a constant-temperature shaking table for shake culture; culturing at 23 deg.C at rotation speed of 180r/min for 3 days to obtain fermented seed solution of Armillaria matsutake with thallus mass content of 30%;
the seed culture medium comprises the following components in percentage by mass: 6% of cane sugar, 3% of bean cake powder, 1% of corn flour, 10.3% of vitamin B, 0.1% of magnesium sulfate, 0.05% of potassium dihydrogen phosphate and the balance of water; the prepared seed culture medium is sterilized at 121 deg.C for 20 min.
Example 1
This example provides a method for preparing schizophyllan and ergothioneine by co-fermentation of two bacterial species, comprising the following steps:
(1) inoculating the fermented seed liquid of the schizophyllum commune provided in preparation example 1 and the fermented seed liquid of the mushroom provided in preparation example 4 into a shake flask filled with a co-fermentation culture medium, and placing the shake flask in a constant-temperature shaking table for shake culture to obtain a fermentation liquid;
wherein the co-fermentation culture medium comprises the following components in percentage by mass: 3% of glucose, 3% of tryptone, 3% of yeast extract, 0.3% of yeast extract, 0.1% of magnesium sulfate, 0.05% of potassium dihydrogen phosphate and the balance of water, wherein the pH value is 6.0;
wherein the inoculation amount of the fermentation seed liquid of the schizophyllum commune is 10 wt%, and the inoculation amount of the fermentation seed liquid of the mushroom is 10 wt%; the temperature of the shake culture is 28 ℃, the rotating speed of the shake culture is 120r/min, and the time of the shake culture is 4 days;
(2) and (2) firstly carrying out ultrasonic treatment on the fermentation liquor obtained in the step (1) at the power of 800W for 15min, then centrifuging at the rotating speed of 9000r/min for 8min, and filtering out hyphae to obtain the fermentation liquor containing schizophyllan and ergothioneine.
Example 2
This example provides a method for preparing schizophyllan and ergothioneine by co-fermentation of two bacterial species, comprising the following steps:
(1) inoculating the fermented seed liquid of the schizophyllum commune provided in preparation example 2 and the fermented seed liquid of the mushroom provided in preparation example 5 into a shake flask filled with a co-fermentation culture medium, and placing the shake flask in a constant-temperature shaking table for shake culture to obtain a fermentation liquid;
wherein the co-fermentation culture medium comprises the following components in percentage by mass: 5% of glucose, 2% of tryptone, 2% of yeast extract, 0.2% of yeast extract, 0.1% of magnesium sulfate, 0.06% of potassium dihydrogen phosphate and the balance of water, wherein the pH value is 6.5;
wherein the inoculation amount of the fermentation seed liquid of the schizophyllum commune is 12 wt%, and the inoculation amount of the fermentation seed liquid of the mushroom is 8 wt%; the temperature of the shake culture is 28 ℃, the rotating speed of the shake culture is 110r/min, and the time of the shake culture is 4 days;
(2) and (2) performing ultrasonic treatment on the fermentation liquor obtained in the step (1) for 5min under the power of 1000W, centrifuging at the rotating speed of 8000r/min for 10min, and filtering to remove hyphae to obtain the fermentation liquor containing schizophyllan and ergothioneine.
Example 3
This example provides a method for preparing schizophyllan and ergothioneine by co-fermentation of two bacterial species, comprising the following steps:
(1) inoculating the fermented seed liquid of the Schizophyllum commune provided in preparation example 3 and the fermented seed liquid of the mushroom provided in preparation example 6 into a shake flask filled with a co-fermentation culture medium, and placing the shake culture medium in a constant-temperature shaking table for shake culture to obtain a fermentation liquid;
wherein the co-fermentation culture medium comprises the following components in percentage by mass: glucose 4%, tryptone 2.5%, yeast extract 0.1%, magnesium sulfate 0.08%, potassium dihydrogen phosphate 0.06%, and balance water, with pH 5.8;
wherein the inoculation amount of the fermentation seed liquid of the schizophyllum commune is 8 wt%, and the inoculation amount of the fermentation seed liquid of the mushroom is 12 wt%; the temperature of the shake culture is 30 ℃, the rotating speed of the shake culture is 100r/min, and the time of the shake culture is 3 days;
(2) and (2) performing ultrasonic treatment on the fermentation liquor obtained in the step (1) for 20min under the power of 400W, centrifuging for 5min at the rotating speed of 10000r/min, and filtering out hyphae to obtain the fermentation liquor containing schizophyllan and ergothioneine.
Example 4
This example provides a process for the preparation of schizophyllan and ergothioneine by two-strain co-fermentation, differing from example 1 only in that the inoculum size of the fermented seed liquid of schizophyllan was 15 wt%, the inoculum size of the fermented seed liquid of mushroom was 5 wt%, and the other steps were the same as example 1.
Example 5
This example provides a process for the preparation of schizophyllan and ergothioneine by two-strain co-fermentation, differing from example 1 only in that the inoculum size of the fermented seed liquid of schizophyllan was 5 wt%, the inoculum size of the fermented seed liquid of mushroom was 15 wt%, and the other steps were the same as example 1.
Example 6
This example provides a process for the preparation of schizophyllan and ergothioneine by co-fermentation of two bacterial species, differing from example 1 only in that the co-fermentation medium consists of, in mass percent: 3% of glucose, 3% of peptone, 4% of yeast extract, 0.1% of magnesium sulfate, 0.05% of potassium dihydrogen phosphate and the balance of water; the other steps are the same as in example 1.
Example 7
This example provides a process for the preparation of schizophyllan and ergothioneine by co-fermentation of two bacterial species, differing from example 1 only in that the co-fermentation medium consists of, in mass percent: 3% of glucose, 3% of peptone, 4% of yeast extract, 0.1% of magnesium sulfate, 0.05% of potassium dihydrogen phosphate and the balance of water; the other steps are the same as in example 1.
Example 8
This example provides a method for preparing schizophyllan and ergothioneine by co-fermentation of two strains, which differs from example 1 only in that the temperature of shaking culture is 26 ℃ and the rotation speed of shaking culture is 180 r/min.
Example 9
This example provides a method for preparing schizophyllan and ergothioneine by co-fermentation of two strains, which is different from example 1 only in that the temperature of shake culture is 32 ℃, the rotation speed of shake culture is 80r/min, and the time of shake culture is 2 days.
Comparative example 1
The present comparative example provides a method for preparing schizophyllan by single strain fermentation, comprising in particular the steps of:
(1) inoculating the fermentation seed liquid of the schizophyllum commune provided by the preparation example 1 into a shake flask filled with a fermentation medium, and placing the shake flask into a constant-temperature shaking table for shake culture to obtain fermentation liquid;
wherein the fermentation medium comprises the following components in percentage by mass: 3% of glucose, 3% of tryptone, 3% of yeast extract, 0.3% of yeast extract, 0.1% of magnesium sulfate, 0.05% of potassium dihydrogen phosphate and the balance of water, wherein the pH value is 6.0;
wherein the inoculation amount of the zymolysis seed liquid of the schizophyllum commune is 15 wt%; the temperature of the shake culture is 28 ℃, the rotating speed of the shake culture is 120r/min, and the time of the shake culture is 7 days;
(2) and (2) centrifuging the fermentation liquor obtained in the step (1) for 5min at the rotating speed of 10000r/min, and filtering out hyphae to obtain the fermentation liquor containing schizophyllan.
Comparative example 2
This comparative example provides a process for the preparation of ergothioneine by fermentation of a single species, the process comprising the steps of:
(1) inoculating the fermented seed liquid of the mushroom provided in preparation example 4 into a shake flask containing a fermentation medium, and placing the shake flask in a constant-temperature shaking table for shake culture to obtain a fermentation liquid;
wherein the fermentation medium comprises the following components in percentage by mass: 3% of glucose, 3% of tryptone, 3% of yeast extract, 0.3% of yeast extract, 0.1% of magnesium sulfate, 0.05% of potassium dihydrogen phosphate and the balance of water, wherein the pH value is 6.0;
wherein the inoculation amount of the fermentation seed liquid of the mushroom is 15 wt%; the temperature of the shaking culture is 28 ℃, the rotating speed of the shaking culture is 120r/min, and the time of the shaking culture is 5 days;
(2) and (2) performing ultrasonic treatment on the fermentation liquor obtained in the step (1) for 3min under the power of 300W, centrifuging for 8min at the rotating speed of 9000r/min, and filtering out hyphae to obtain the fermentation liquor containing ergothioneine.
Test example 1
Determination of the content of ergothioneine and schizophyllan
Test samples: examples 1-9, the schizophyllan-and-ergothioneine-containing fermentation broths provided in comparative example 1, the ergothioneine-containing fermentation broths provided in comparative example 2;
the test method comprises the following steps: performing HPLC determination on a standard substance of schizophyllan, an ergothioneine standard substance and a sample to be detected, comparing a sample chromatogram with a standard solution chromatogram, and determining an ergothioneine peak in the sample according to retention time; drawing a standard curve by using the concentration of the ergothioneine standard and the corresponding peak area, and quantifying by using an external standard method under the condition that the sample introduction amount of the ergothioneine standard is the same as that of the sample to calculate the content of the ergothioneine and schizophyllan in the sample;
the specific test results are shown in table 1:
TABLE 1
As can be seen from the test data in Table 1, the fermentation broth containing schizophyllan and ergothioneine prepared by the invention has the ergothioneine content of more than 900mg/L and the schizophyllan content of more than 9 g/L. The invention shows that the schizophyllum commune and the bacterial strain for preparing the ergothioneine are co-fermented, and the co-fermentation of the two bacterial strains is found, so that not only can no competition phenomenon occur, but also the synergistic effect can be exerted, thereby leading the ergothioneine to be prepared in a large amount from the mycelium, ensuring the product quality of the ergothioneine and obviously improving the yield of the ergothioneine; and the preparation process is simple, and is particularly suitable for large-scale production of ergothioneine.
Test example 2
Molecular weight and viscosity measurements of Schizophyllan
Test samples: examples 1-9, the schizophyllan and ergothioneine-containing fermentation broths provided in comparative example 1;
the test method comprises the following steps: concentrating the fermentation liquor to 1/4 of the original volume, adding 85 vol% ethanol with 5 times of the volume for precipitation, centrifuging at 10000rpm to remove supernatant, collecting the precipitate, dissolving the precipitate again with distilled water, and filtering ergothioneine, protein and other micromolecule products by adopting an ultrafiltration membrane with the molecular weight of 5kDa to obtain wet-cracked pleat polysaccharide; freeze drying the obtained wet cracked pleat polysaccharide at-20 deg.C for 24 hr; measuring the molecular weight of the obtained schizophyllan by adopting high-efficiency gel permeation chromatography;
the test conditions were: a chromatographic column: 7.8X 300mm (Ultrahydrogel TM 120, 250, 1000, supplied by Waters Corporation); column temperature: 35 ℃; mobile phase: high purity water; the flow rate is 0.6 mL/min; sample injection amount: 50 mu L of the solution;
the specific test results are shown in table 2:
TABLE 2
| Sample (I) | Average molecular weight of polysaccharide (kDa) |
| Example 1 | 1023 |
| Example 2 | 948 |
| Example 3 | 923 |
| Example 4 | 1150 |
| Example 5 | 1024 |
| Example 6 | 1168 |
| Example 7 | 1147 |
| Example 8 | 1155 |
| Example 9 | 1073 |
| Comparative example 1 | 1357 |
As can be seen from the test data in Table 2, the molecular weight of schizophyllan was below 1200kDa in the schizophyllan and ergothioneine-containing fermentation broth of the present invention. The invention shows that the schizophyllum commune and the bacterial strain for preparing the ergothioneine are co-fermented, and the co-fermentation of the two bacterial strains is found, so that the ergothioneine is prepared in a large amount from the mycelium, and the schizophyllum commune with relatively lower molecular weight and better water solubility can be obtained, thus being more beneficial to the biological absorption and the in vivo biological activity, and greatly expanding the application of the schizophyllum commune.
Test example 3
Determination of antioxidant Activity
Test samples: examples 1-9, the schizophyllan-and-ergothioneine-containing fermentation broths provided in comparative example 1, the ergothioneine-containing fermentation broths provided in comparative example 2;
the test method comprises the following steps:
1. evaluation of the ability to scavenge DPPH free radicals
DPPH, also known as 1, 1-diphenyl-2-trinitrophenylhydrazine, is a very stable free radical with a nitrogen center, and the stability of DPPH is mainly caused by steric hindrance of 3 benzene rings with resonance stabilization, so that unpaired electrons on the nitrogen atom in the middle cannot play the role of electron pairing. The absolute ethyl alcohol solution of the compound is purple, has maximum absorption at the wavelength of 517nm, and the absorbance and the concentration are in a linear relation. When the free radical scavenger is added into the solution, DPPH can be combined or replaced, so that the number of free radicals is reduced, the absorbance is reduced, the color of the solution is lightened, and the capability of scavenging the free radicals can be evaluated;
the specific experimental steps are as follows: taking Trolox solution as a positive control, and determining the scavenging effect of Trolox on DPPH free radicals at different concentrations. The experimental result is expressed by a TEAC value, and the unit of the experimental result is mu mol Trolox equivalent/100 mL, namely the antioxidant capacity of the mu mol Trolox is equivalent to that of a 100mL sample; respectively sucking 2mL of sample solution (or absolute ethyl alcohol) and 2mL of LDPPH solution into a test tube with a plug, and uniformly mixing; reacting for 30min in a dark place, and measuring the light absorption value at the wavelength of 517 nm;
DPPH radical scavenging ratio (%) - [1- (A) 1 -A 2 )/A 0 ]×100％；
In the formula: a. the 0 DPPH was added, representing no added sample; a. the 1 Represents the addition of sample or absolute ethanol, followed by DPPH; a. the 2 Representing addition of sample or notAqueous ethanol, no DPPH added;
2. evaluation of ABTS +. radical scavenging ability
ABTS is oxidized into green ABTS +. under the action of proper oxide, when antioxidant substances are added into the reaction, the generation of ABTS +. is inhibited, and the total antioxidant capacity of the sample can be measured and calculated by measuring the absorbance at 734 nm;
the specific experimental steps are as follows: and (3) determining the scavenging effect of Trolox on ABTS +. free radicals under different concentrations by taking Trolox solution as a positive control. The experimental result is expressed by a TEAC value, and the unit of the experimental result is mu mol Trolox equivalent/100 mL, namely the antioxidant capacity of the mu mol Trolox is equivalent to that of a 100mL sample; taking 7mmol/L ABTS solution, and diluting the solution by 60 times with 10mmol/L PBS (pH 7.4) to obtain ABTS working solution; the sample solution (or PBS) was added to 190. mu.L of LABTS working solution (or PBS) and the solution was allowed to stand at 30 ℃ for 6min with shaking for 10 seconds, and the absorbance A at 734nm was measured.
ABTS +. radical scavenging rate (%) [1- (a) 1 -A 2 )/A 0 ]×100％；
In the formula: a. the 0 ABTS was added, representing no sample added; a. the 1 Represents addition of sample or PBS, addition of ABTS; a. the 2 Represents addition of sample or PBS, no ABTS;
the specific test results are shown in table 3:
TABLE 3
As can be seen from the test data in Table 3, the stock solution of the fermentation broth prepared by the method has a DPPH radical scavenging rate of over 90%, and the DPPH radical scavenging rate can still reach over 67% even after the stock solution is diluted to a concentration of 10 vol%; the stock solution of the fermentation liquor has the clearance rate of ABTS +. free radicals over 90 percent, and even if the stock solution is diluted to the concentration of 10vol percent, the clearance rate of ABTS +. free radicals can still reach over 67 percent; the finally obtained fermentation liquor contains schizophyllan and ergothioneine, and the schizophyllan and the ergothioneine are matched with each other to have a synergistic effect, so that the scavenging effect of the composition on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals can be further improved, the antioxidant capacity of skin tissues can be enhanced, and the free radical content of the skin tissues can be reduced.
The applicant states that the present invention is illustrated by the above examples for the preparation of schizophyllan and ergothioneine by co-fermentation of two strains, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be practiced by relying on the above examples. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (23)
1. A process for the preparation of schizophyllan and ergothioneine by co-fermentation of two species, comprising the steps of: inoculating the fermentation seed liquid of the schizophyllum commune and the fermentation seed liquid of the mushroom fungus to a co-fermentation culture medium for co-fermentation culture to obtain fermentation liquid containing schizophyllan and ergothioneine;
the mushroom fungus comprises any one or combination of at least two of Hericium erinaceus, Pleurotus ostreatus or Armillaria matsutake.
2. The method for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 1, wherein the co-fermentation medium consists of the following components in percentage by mass: 0.5-5% of glucose, 0.5-3% of tryptone, 0.1-3% of yeast extract, 0.1-0.5% of yeast extract, 0.01-0.15% of magnesium sulfate, 0.05-0.2% of potassium dihydrogen phosphate and the balance of water.
3. The process for the preparation of schizophyllan and ergothioneine by co-fermentation of two species as claimed in claim 1, wherein the co-fermentation medium has a pH of from 5.5 to 7.0.
4. The method for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 1, wherein the mass percentage of thallus in the seed liquid of Schizophyllum commune is 5-30%.
5. The method for preparing schizophyllan and ergothioneine by double-strain co-fermentation of claim 1, wherein the mass percentage of the thallus in the fermented seed liquid of mushroom is 5-30%.
6. The process for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 1, wherein the amount of the inoculum of the fermentation seed liquid of schizophyllan is 2-20 wt% in the co-fermentation medium.
7. The process for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 1, wherein the inoculum size of the fermentation seed liquid of mushroom is 5-20 wt% in said co-fermentation medium.
8. The method for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 1, wherein the co-fermentation culture comprises the following specific steps: inoculating the fermentation seed liquid of the Schizophyllum commune and the fermentation seed liquid of the mushroom to a shake flask filled with a co-fermentation culture medium, and placing the shake flask in a constant-temperature shaking table for shake culture.
9. The method for preparing schizophyllan and ergothioneine through double-strain co-fermentation as claimed in claim 8, wherein the temperature of shake culture is 28-30 ℃, the rotation speed of shake culture is 100-150r/min, and the time of shake culture is 3-5 days.
10. The process for the preparation of schizophyllan and ergothioneine by co-fermentation of two strains according to claim 1, wherein the co-fermentation further comprises sonication and/or centrifugation after culturing.
11. The method for preparing schizophyllan and ergothioneine through double-strain co-fermentation according to claim 10, wherein the power of the ultrasound is 400-1000W, and the time of the ultrasound is 5-20 min.
12. The method for preparing schizophyllan and ergothioneine by double-strain co-fermentation as claimed in claim 10, wherein the centrifugation speed is 8000-10000r/min, and the centrifugation time is 5-10 min.
13. The method for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 1, wherein the fermentation seed liquid of schizophyllan is prepared by the following preparation method:
(a) inoculating Schizophyllum commune to a PDA culture medium for activation culture to obtain activated seeds;
(b) inoculating the activated seeds obtained in the step (a) into a liquid culture medium for seed culture to obtain a fermentation seed liquid of the schizophyllum commune.
14. The process for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 13, wherein the temperature of the activation culture in step (a) is 24-26 deg.C and the time of the activation culture is 2-5 days.
15. The process for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 13, wherein in step (b), the liquid medium consists of the following components in percentage by mass: 0.5-2% of glucose, 0.1-2% of yeast extract, 0.01-0.15% of magnesium sulfate, 0.05-0.2% of potassium dihydrogen phosphate and the balance of water.
16. The method for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 13, wherein in step (b), the seed culture is performed in a constant temperature shaking table at a temperature of 24-26 deg.C and a rotation speed of 150-.
17. The method for preparing schizophyllan and ergothioneine through double-strain co-fermentation according to claim 1, wherein the fermented seed liquid of mushroom is prepared by the following preparation method: inoculating mycelium of mushroom into seed culture medium, and performing seed culture to obtain fermented seed liquid of mushroom.
18. The method for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 17, wherein the seed medium consists of, in mass percent: 1-10% of cane sugar, 3-5% of bean cake powder, 1-3% of corn flour, 10.1-0.3% of vitamin B, 0.01-0.15% of magnesium sulfate, 0.05-0.2% of potassium dihydrogen phosphate and the balance of water.
19. The method for preparing schizophyllan and ergothioneine through double-strain co-fermentation as claimed in claim 17, wherein the seed culture is carried out in a constant temperature shaking table at 23-25 deg.C and at a rotation speed of 160-180r/min for 1-3 days.
20. The method for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 1, wherein the ergothioneine content in the fermentation broth containing schizophyllan and ergothioneine is 900mg/L or more.
21. The method for preparing schizophyllan and ergothioneine through double-strain co-fermentation as claimed in claim 20, wherein the ergothioneine content in the fermentation broth containing schizophyllan and ergothioneine is 950-1050 mg/L.
22. The method for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 1, wherein the schizophyllan and ergothioneine-containing fermentation broth has a schizophyllan content of 9g/L or more.
23. The method for preparing schizophyllan and ergothioneine by co-fermentation of two strains according to claim 22, wherein the schizophyllan and ergothioneine-containing fermentation broth has a schizophyllan content of 9-12 g/L.
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