A kind of extract rich in ergothioneine and preparation method thereof

Abstract

The present invention relates to the field of microbial fermentation technology, and in particular to an extract rich in thioneine and a preparation method thereof. The extract is prepared by inoculating flat mushroom seed liquid in a fermentation medium containing a polygonatum hydrolysate and fermenting and culturing, and the composition of the fermentation medium includes the following components: 50-200g / L of polygonatum hydrolysate, 20-60g / L of an inorganic nitrogen source, 0.1-5.0g / L of an inorganic salt, and 0.001-0.005g / L of vitamins. The present invention has the advantages of high thioneine content, good antioxidant, anti-aging and anti-aging effects, and low cost.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to an extract rich in ergothioneine and a preparation method thereof. Background Art

[0002] Ergothioneine (EGT) is a compound discovered in 1909. It has received widespread attention in the cosmetics market due to its excellent anti-inflammatory and antioxidant effects.

[0003] As a kind of mushroom, Psalliota campestris is widely used in the biosynthesis of EGT by mycelium submerged fermentation technology because it is rich in EGT. This technology is the mainstream direction of low-cost large-scale fermentation production of EGT today.

[0004] Currently, a variety of edible fungi deep fermentation technologies have been used in the production of ergothioneine, but this fermentation technology has the main problems of relatively single effect of extracting ingredients and too low EGT fermentation concentration.

[0005] For example, the existing technology (Tang Baosha, Wu Li, Weng Minjie, et al. Optimization of culture medium for deep fermentation biosynthesis of ergothioneine by Pleurotus eryngii 528 [J]. Journal of Fujian Agricultural Sciences, 2021, 36(6):7.) investigated the addition of synthetic precursors while adding carbon and nitrogen source combinations in different proportions, but the EGT concentration was only 20.50±1.80 mg / L.

[0006] The existing technology (Mei Baoliang, Liu Qi, Jiang Wenxia, ​​et al. Research on nutritional factors to enhance the biosynthesis of ergothioneine [J]. Food Research and Development, 2015 (15).) investigated the effects of various precursors on the production of ergothioneine and found that methionine can significantly increase the content of ergothioneine, but its final content is only 192.4 mg / L.

[0007] Chinese patent application CN 115895923 A discloses a method for preparing a flat mushroom extract, which uses flat mushroom and Candida lipolytica to prepare an extract rich in polypeptides and EGT, and the preparation method includes the following steps: activating flat mushroom and Candida lipolytica strains and then implanting them into a liquid culture medium to obtain seed liquid; inoculating the obtained seed liquid into a fermentation medium for fermentation; after the fermentation is completed, the fermentation liquid can be obtained after separation treatment. However, the invention only measures the antioxidant activity of the prepared fermentation liquid and does not develop new functions, and the addition of a large amount of precursors has a high cost.

[0008] In summary, the current development and utilization of deep fermentation technology for preparing EGT from edible fungi mainly has the following problems: (1) A large amount of precursors need to be added to the current culture system, which results in a high fermentation cost; (2) Active substances or nutrients need to be added multiple times in the current culture system, which results in a complicated process; (3) The active substances obtained in the current culture system have a single effect; and (4) The EGT fermentation concentration is too low.

[0009] Polygonatum sibiricu is a perennial herbaceous plant rhizome of the genus Polygonatum in the family Liliaceae. It was first seen in the "Ming Yi Bie Lu". It is sweet and flat in nature, and enters the spleen, lung, and kidney meridians. It has the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs, and nourishing the kidneys. It is a traditional Chinese medicine and food resource. Modern pharmacological studies have shown that the main active ingredients of Polygonatum sibiricu are Polygonatum sibiricu polysaccharides and Polygonatum sibiricu oligosaccharides, and their content is mostly above 10%. Polygonatum sibiricu polysaccharides and Polygonatum sibiricu oligosaccharides have anti-tumor, anti-aging, immunomodulatory, and anti-oxidative damage effects.

[0010] At present, there is no research on the application of Polygonatum sibiricum raw materials or its related substances to the deep fermentation technology of edible fungi for the preparation of EGT. Summary of the invention

[0011] The object of the present invention is to overcome the deficiencies in the prior art and provide an extract rich in ergothioneine with high EGT content, good anti-aging and anti-aging effects and low cost, and a preparation method thereof. The present invention replaces the organic carbon and nitrogen source required for the fermentation of flat mushrooms with a polygonatum hydrolyzate, significantly improves the ergothioneine yield of mycelium through fermentation regulation, and the obtained fermented liquid has significant anti-aging and anti-aging capabilities as an extract.

[0012] The present invention is achieved through the following technical solutions:

[0013] A method for preparing an extract rich in ergothioneine is prepared by inoculating agaricus serrata seed liquid into a fermentation medium containing polygonatum hydrolysate and fermenting the medium. The fermentation medium comprises the following components: 50-200 g / L polygonatum hydrolysate, 20-60 g / L inorganic nitrogen source, 0.1-5.0 g / L inorganic salt and 0.001-0.005 g / L vitamin.

[0014] The enzymatic hydrolysate of polygonatum used in the present invention contains a large number of nutrients, such as a large number of monosaccharides, oligosaccharides and amino acids. Among them, monosaccharides and oligosaccharides can provide energy for flat mushrooms, promote growth and development, and a large number of amino acids such as histidine and methionine can help the production of EGT. At the same time, the unconsumed polygonatum oligosaccharides are retained in the subsequent extraction process, thereby enhancing the anti-aging and anti-aging effects of the extract and broadening the use of the fermentation liquid.

[0015] Adding Polygonatum sibiricum enzymatic hydrolysate to the Agaricus cycad culture medium under the process conditions of the preparation method can not only increase the yield of EGT, but also expand other uses of the fermentation product.

[0016] Preferably, the fermentation medium comprises the following components: 50-200 g / L of polygonatum sibiricum hydrolysate, 20-60 g / L of ammonium chloride, 0.1-3.0 g / L of potassium dihydrogen phosphate, 0.1-1.5 g / L of magnesium sulfate, and 0.001-0.005 g / L of vitamin B12.

[0017] Preferably, the inorganic nitrogen source in the fermentation medium is one or more of ammonium nitrate, ammonium chloride and ammonium sulfate.

[0018] Preferably, the inorganic salt in the fermentation medium is one or more of sodium nitrate, sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, zinc sulfate, zinc chloride, and manganese chloride.

[0019] Preferably, the vitamins in the fermentation medium are one or more of VB1, VB3, VB6, and VB12.

[0020] Preferably, after the fermentation and culturing is completed, the product is crushed, boiled and filtered in sequence to obtain a fermentation liquid which is an extract rich in ergothioneine.

[0021] Preferably, the preparation method of the polygonatum hydrolysate comprises: adding the polygonatum raw material into water, then adding a hydrolase for enzymolysis, boiling and filtering after the enzymolysis is completed, and the obtained filtrate is the polygonatum hydrolysate.

[0022] More preferably, the raw material of Polygonatum sibiricum includes one or more of Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red. and Polygonatum cyrtonema Hua.

[0023] More preferably, the amount of the polygonatum raw material added is 5-40% of the mass of water.

[0024] More preferably, the amount of the hydrolase added is 0.001-0.5% of the volume of water. Because the added hydrolase is liquid, it is added according to the volume percentage of water. The hydrolase can also be solid, in which case it is added according to the mass percentage of water.

[0025] More preferably, the enzymatic hydrolysis conditions are: keeping warm at 37-50°C for 3-8h.

[0026] More preferably, the hydrolase comprises one or more of α-amylase, pectinase, cellulase, acid protease, neutral protease, alkaline protease and papain.

[0027] Preferably, the ratio of polygonatum polysaccharide and polygonatum oligosaccharide in the polygonatum hydrolysate accounts for 30-60% of the total mass of the polygonatum hydrolysate.

[0028] Preferably, the inoculation amount of the Agaricus rufa seed liquid is 5-40% by volume.

[0029] Preferably, the fermentation temperature is 20-30° C. More preferably, the fermentation time is 7-21 days.

[0030] Preferably, stirring is performed during the fermentation culture process, and the stirring speed is 120-220 r / min.

[0031] Preferably, the preparation of the flatland mushroom seed liquid includes two processes: S1 strain culture and S2 seed liquid culture;

[0032] More preferably, step S1 specifically comprises: inoculating the mycelium of Agaricus rufa on a PDA solid culture medium for cultivation to obtain Agaricus rufa strains containing the culture medium.

[0033] Further preferably, the culture temperature in step S1 is 20-30°C and the culture time is 7-14 days;

[0034] More preferably, step S2 specifically comprises: inoculating the Agaricus spp. containing culture medium into a seed liquid culture medium for seed liquid culture to obtain Agaricus spp. seed liquid.

[0035] Preferably, the temperature of the seed solution culture in step S2 is 20-30°C, stirring is performed during the culture process, and the stirring speed is 50-180 r / min. Preferably, the time of the seed solution culture in step S2 is 7-21 days.

[0036] Further preferably, the composition of the seed liquid culture medium includes the following components: 20.0-50.0 g / L of carbon source, 10.0-30.0 g / L of nitrogen source and 0.1-5.0 g / L of inorganic salt.

[0037] More preferably, the composition of the seed liquid culture medium in step S2 includes the following components: soluble starch 10.0-20.0 g / L, glucose 10.0-30.0 g / L, yeast powder 10.0-30.0 g / L, potassium dihydrogen phosphate 0.1-1.0 g / L and magnesium sulfate 0.1-1.0 g / L.

[0038] More preferably, the preparation method of the extract rich in ergothioneine comprises:

[0039] S1: inoculating the mycelium of Agaricus rufipogon on a PDA solid culture medium and culturing at 20-30° C. for 7-14 days to obtain Agaricus rufipogon spawn containing the culture medium;

[0040] S2: Inoculating the Agaricus spp. containing the culture medium into a seed liquid culture medium for seed liquid culture, wherein the culture temperature is 20-30° C., the culture time is 7-21 days, stirring is performed during the culture process, and the stirring speed is 50-180 r / min, to obtain Agaricus spp. seed liquid; the composition of the seed liquid culture medium includes the following components: soluble starch 10.0-20.0 g / L, glucose 10.0-30.0 g / L, yeast powder 10.0-30.0 g / L, potassium dihydrogen phosphate 0.1-1.0 g / L and magnesium sulfate 0.1-1.0 g / L;

[0041] S3: Inoculate the flat mushroom seed liquid with a volume fraction of 5-40% into a fermentation medium containing polygonatum hydrolysate at 20-30°C for 7-21 days, wherein the fermentation medium comprises the following components: 20-100 g / L polygonatum hydrolysate, 20-60 g / L ammonium chloride, 0.1-5.0 g / L inorganic salt and 0.001-0.005 g / L vitamin B1, and stir during the fermentation process at a speed of 120-220 r / min;

[0042] The preparation method of the enzymatic hydrolysate of polygonatum comprises: taking a polygonatum raw material according to 5-40% of the water mass, adding a hydrolase according to 0.001-0.5% of the water volume for enzymatic hydrolysis, wherein the conditions for enzymatic hydrolysis are: keeping warm at 37-50° C. for 3-8 hours, wherein the hydrolase comprises one or more of α-amylase, pectinase, cellulase, acid protease, neutral protease, alkaline protease and papain, and boiling and filtering after the enzymatic hydrolysis is completed, and the obtained filtrate is the enzymatic hydrolysate of polygonatum;

[0043] S4: After the fermentation and cultivation is completed, the fermentation liquid is crushed, boiled and filtered in sequence, and the obtained fermentation liquid is an extract rich in ergothioneine.

[0044] The present invention also relates to an extract rich in ergothioneine prepared by the above preparation method.

[0045] The beneficial effects of the present invention are:

[0046] (1) The present invention selects Agaricus rutaecarpa as the production strain of ergothioneine. Under the preparation process conditions of the present invention, the addition of Polygonatum sibiricum hydrolysate not only does not cause inhibition, but also helps to increase the EGT content in the fermentation product.

[0047] (2) The present invention not only increases the EGT content by 126% to 555 mg / L, but also increases the fibroblast proliferation to 31.5% and the collagen content to 103.7%. It can be used as a raw material for functional skin care products and has good application prospects. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0049] In the following examples, the reagents and equipment used are conventional commercial products unless otherwise specified, and are purchased from Shanghai Titan Technology Co., Ltd.

[0050] The preparation process of Agaricus fowleri block in the following examples is as follows: Agaricus fowleri mycelium is inoculated on PDA solid culture medium and cultured at 25° C. for 13 days to obtain Agaricus fowleri containing culture medium, which is the “Agaricus fowleri block (with culture medium)” in the following examples.

[0051] The specific preparation process of the enzymolysis product of polygonatum in the following embodiment is as follows: take polygonatum (Polygonatum sibiricumRed.) and add it to water according to 25% of the water mass, then add hydrolase according to 0.25% of the water volume for enzymolysis, and the conditions of enzymolysis are: keep warm at 40°C for 5h, and the hydrolase is a mixed enzyme of α-amylase, pectinase, cellulase, acid protease, neutral protease, alkaline protease and papain, wherein each enzyme system accounts for 40% of the added enzyme mass percentage: cellulase accounts for 10%, α-amylase and pectinase each account for 10%, acid protease, neutral protease, alkaline protease and papain each account for 10%. After the enzymolysis is completed, boil and filter, and the obtained filtrate is the polygonatum enzymolysis product.

[0052] The ergothioneine solution fermentation broth prepared in the following examples is also called Agaricus rutaecarpa extract.

[0053] Example 1

[0054] (1) Take 4cm 2 Four Agaricus fowleri blocks (with culture medium) were inoculated into the seed liquid culture medium and cultured at 20°C and 180 r / min for 14 days to obtain Agaricus fowleri culture solution.

[0055] Flatland mushroom seed liquid culture medium: soluble starch 10.0g / L, glucose 15.0g / L, yeast powder 10.0g / L, potassium dihydrogen phosphate 0.1g / L, magnesium sulfate 0.1g / L.

[0056] (2) The culture fluid of Agaricus rufipogon was inoculated into the fermentation medium at a volume fraction of 40%, and cultured at 25° C. and 160 r / min for 10 days. The liquid volume of the fermentation medium was 60% of the total volume.

[0057] Fermentation medium: Polygonatum sibiricum enzymatic hydrolysate 50g / L, ammonium chloride 25g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 1.0g / L, vitamin B1 0.002g / L.

[0058] (3) After fermentation for 10 days, the mycelia are broken; after boiling for 20 minutes, the mixture is cooled and centrifuged to obtain a fermented thioneine solution (denoted as A-1).

[0059] Example 2

[0060] (1) Take 4cm 2 Two Agaricus fowleri blocks (with culture medium) were inoculated into the seed liquid culture medium and cultured at 28°C and 180 r / min for 14 days to obtain Agaricus fowleri culture solution.

[0061] Flatland mushroom seed liquid culture medium: soluble starch 20.0g / L, glucose 30.0g / L, yeast powder 10.0g / L, potassium dihydrogen phosphate 0.2g / L, magnesium sulfate 0.2g / L.

[0062] (2) The culture fluid of Agaricus rufipogon was inoculated into the fermentation medium at a volume fraction of 20%, and cultured at 25° C. and 150 r / min for 10 days. The volume of the fermentation medium was 50% of the total volume.

[0063] Fermentation medium: Polygonatum sibiricum enzymatic hydrolysate 200 g / L, ammonium nitrate 55 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, vitamin B1 0.002 g / L.

[0064] (3) After fermentation for 7d, mycelia was broken; after boiling for 20min, cooling was performed, and centrifugation was performed to obtain thioneine solution fermentation liquid (denoted as A-2).

[0065] Example 3

[0066] (1) Take 6cm 2 Four Agaricus fowleri blocks (with culture medium) were inoculated into the seed liquid culture medium and cultured at 25°C and 150 r / min for 9 days to obtain Agaricus fowleri culture solution.

[0067] Flatland mushroom seed liquid culture medium: soluble starch 15.0g / L, glucose 20.0g / L, yeast powder 20.0g / L, potassium dihydrogen phosphate 0.2g / L, magnesium sulfate 0.2g / L.

[0068] (2) The culture liquid of Agaricus rufipogon was inoculated into the fermentation medium at a volume fraction of 40%, and cultured at 25°C and 180 r / min for 8 days. The volume of the fermentation medium was 25% of the total volume.

[0069] Fermentation medium: Polygonatum sibiricum enzymatic hydrolysate 100g / L, ammonium sulfate 25g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 1.5g / L, vitamin B1 0.005g / L.

[0070] (3) After fermentation for 14 days, the mycelia are crushed; after boiling for 20 minutes, the mixture is cooled and centrifuged to obtain a fermented thioneine solution (denoted as A-3).

[0071] Comparative Example 1

[0072] Compared with Example 3, the fermentation medium does not contain Polygonatum sibiricum enzymatic hydrolysate, but is replaced by glucose.

[0073] (1) Take 6cm 2 Four Agaricus fowleri blocks (with culture medium) were inoculated into the seed liquid culture medium and cultured at 25°C and 150 r / min for 9 days to obtain Agaricus fowleri culture solution.

[0074] Flatland mushroom seed liquid culture medium: soluble starch 15.0g / L, glucose 20.0g / L, yeast powder 20.0g / L, potassium dihydrogen phosphate 0.2g / L, magnesium sulfate 0.2g / L.

[0075] (2) The culture liquid of Agaricus rufipogon was inoculated into the fermentation medium at a volume fraction of 40%, and cultured at 25°C and 180 r / min for 8 days. The volume of the fermentation medium was 25% of the total volume.

[0076] Fermentation medium: glucose 100g / L, ammonium sulfate 25g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 1.5g / L, vitamin B1 0.005g / L.

[0077] (3) After fermentation for 14 days, the mycelia were crushed; after boiling for 20 minutes, the mixture was cooled and centrifuged to obtain a fermented thioneine solution (denoted as A-4).

[0078] Comparative Example 2

[0079] The only difference from Example 3 is that in step (3), no bacteria are introduced.

[0080] Preparation of fermentation broth for blank control group:

[0081] The fermentation medium was added with 100 g / L of Polygonatum sibiricum hydrolysate, 25 g / L of ammonium sulfate, 1.0 g / L of potassium dihydrogen phosphate, 1.5 g / L of magnesium sulfate, and 0.005 g / L of vitamin B1, and fermented for 14 days without inoculating bacteria. After boiling for 20 minutes and cooling, centrifugation was performed to obtain the blank control group fermentation liquid (denoted as A-5).

[0082] The products obtained in the above embodiments and comparative examples were subjected to the following inspections or tests:

[0083] Detection of thioneine (EGT): Under the same chromatographic conditions, thioneine standard substances and sample to be tested are subjected to HPLC determination, and the sample chromatogram is compared with the thioneine standard solution chromatogram, and the thioneine peak in the sample is determined according to the retention time. With the concentration of standard substance thioneine and the corresponding peak area, a standard curve is drawn, and when the standard substance is identical with the sample injection amount, the content of thioneine in the sample is calculated by external standard method quantitatively.

[0084] HPLC detection conditions: Agilent 1260 high performance liquid chromatography, XDB-C18 chromatographic column (250 mm × 4.6 mm, particle size 5 μm); mobile phase V (methanol): V (0.1% formic acid aqueous solution) = 1:9; flow rate 1.0 mL / min, column temperature 30°C, injection volume 10 μL, UV detection wavelength 254 nm. EGT detection results are shown in Table 2.

[0085] Determination of polysaccharide content: Determined in accordance with QB / T 2489-2018.

[0086] Determination of total sugar content: Determined in accordance with SN / T 4260-2015.

[0087] Determination of free amino acid content: refer to GB 5009.124-2016 Determination of amino acids in food.

[0088] The results of the polysaccharide, oligosaccharide and free amino acid detection of Polygonatum sibiricum hydrolysate are shown in Table 6.

[0089] Both DPPH clearance rate determination and ABTS clearance rate determination are determined in accordance with GB / T 39100-2020.

[0090] The DPPH removal rate and ABTS removal rate of the fermentation broth prepared in each example were close to 100%, so the test was performed after diluting 10 times. The test results are shown in Table 3.

[0091] Fibroblast proliferation experiment: Resuscitate cells, pass fibroblasts to the third generation (or wait for the cells to stabilize), and then inoculate 100,000 to 200,000 cells / mL into a 96-well plate (200 μL per well) after the cells cover the culture flask with one layer (70%-80%), and culture in a CO2 incubator (37°C, 5% CO2) for 24 hours;

[0092] (1) Preparation of test solution: Dilute the A-2 fermentation broth according to Table 1 to prepare different concentrations of the test substance.

[0093] Table 1 Test concentration setting table

[0094]

[0095] (2) Cell administration: After 24 h of culture, the density of fibroblasts can reach 50%-60%. At this time, administration can be performed. 200 μL of complete culture medium containing different concentration gradients is added to each well. Each group has 6 replicate wells. 200 μL of complete culture medium without cells is added to the blank group. After administration, the cells are placed in a CO2 incubator (37°C, 5% CO2) and cultured for 24 h.

[0096] (3) Detection: After 24 hours of cell culture, remove the cells, discard the supernatant, add 10% CCK8 solution (1mL DMED + 100μL CCK8 solution) in a dark place, 100μL per well, and place in a CO2 incubator (37°C, 5% CO2) in a dark place for 0.5-1h (determine the specific reaction time according to the color of the reaction), and detect the absorbance at 450nm with an enzyme reader after the reaction is completed. The test results are shown in Table 4. Proliferation ratio (%) = number of cells after proliferation / number of cells before proliferation*100%.

[0097] Collagen content detection: This experiment uses the human type I collagen ELISA detection kit. The experimental reagents are A-2 fermentation diluent and EGT standard solution. 1. Reagent preparation: prepare various test reagents, diluted standards and samples to be tested. 2. Determination of enzyme labeling strips: calculate the enzyme labeling strips required for the samples and standards to be tested, take the enzyme labeling strips out of the aluminum foil bag, put the remaining enzyme labeling strips back into the aluminum foil bag and seal the bag, and store at low temperature. 3. Soak the enzyme labeling plate: add 1× washing solution (350μL / well) to soak the enzyme labeling plate, let it stand for 30 seconds, then discard the liquid in the well and pat the enzyme labeling plate dry. 4. Sample addition and incubation: add each gradient standard and diluted sample to be tested, 100μL / well, and incubate at room temperature for 2 hours. 5. Wash the enzyme labeling plate: discard the liquid in the well, add 1× washing solution (350μL / well) to wash the plate 3 times, and pat the enzyme labeling plate dry. 6. Detection antibody incubation: Add the detection antibody prepared to the working concentration to the ELISA plate, 100μL / well, and incubate at room temperature for 2 hours. 7. Wash the ELISA plate: Discard the liquid in the well, add 1× washing solution (350μL / well) to wash the plate 3 times, and pat the ELISA plate dry. 8. Enzyme conjugate incubation: Add the enzyme conjugate prepared to the working concentration to the ELISA plate, 100μL / well, and incubate at room temperature for 20 minutes. 9. Wash the ELISA plate: Discard the liquid in the well, add 1× washing solution (350μL / well) to wash the plate 3 times, and pat the ELISA plate dry. 10. Color development: Return the substrate solution to room temperature 10 minutes before use, add the substrate solution to the ELISA plate, 100μL / well, and incubate at room temperature in the dark for 20 minutes. 11. Stop: Add 50μL / well stop solution to the ELISA plate, at this time the color turns from blue to yellow, and gently shake the ELISA plate until the color is uniform. 12. Reading: Read the light absorbance at 450nm within 10 minutes.

[0098] The experimental results are shown in Table 5:

[0099] Table 2 EGT content test results

[0100] name EGT content (ppm) A-1 539 A-2 555 A-3 526 A-4 245 A-5 Not detected

[0101] Table 3 DPPH and ABTS removal rates after the fermentation broth was diluted 10 times

[0102] name DPPH clearance rate (%) ABTS clearance rate (%) A-1 86.6 80.6 A-2 88.3 83.6 A-3 79.9 77.6 A-4 56.9 48.1 A-5 8.3 5.7

[0103] As can be seen from Table 2 and Table 3, the content of thioneine in the fermentation liquid after the combined fermentation of embodiment 1-embodiment 3 is more than 526ppm, and comparative example 1 adopts flat ground mushroom to ferment and produce thioneine without adding polygonatum sibiricum hydrolysate, and the output of thioneine in the fermentation liquid is 245ppm, which has improved by more than 115%. Comparative example 2 (A-5) is the blank control composed of fermentation medium, which hardly contains EGT and the antioxidant property is also very low. Due to the addition of polygonatum sibiricum hydrolysate, the output of thioneine is obviously improved, which causes the clearance rate of DPPH and ABTS of the fermentation liquid stock solution to be still more than 79.9% and 73.6% even if it is diluted 10 times. The DPPH and ABTS clearance rates of its 10vol% liquid (i.e., diluted 10 times) of Chinese patent application CN114214387A are all about 67%, indicating that the fermentation liquid prepared in the present invention has stronger antioxidant property.

[0104] Table 4 Fibroblast proliferation assay

[0105]

[0106]

[0107] Different concentrations of EGT and Agaricus rufipogon extract were added to the fibroblast culture medium, and it was found that both could promote cell proliferation. EGT had the best effect when added at a concentration of about 300ppm, with an increase in value of 117.6%. The Agaricus rufipogon extract of the present invention still had a high effect when diluted to an EGT concentration of 500ppm, and the cell count reached 131.5%. It can be seen that the Agaricus rufipogon extract prepared by this method can effectively improve the proliferation effect of fibroblasts to achieve the purpose of anti-aging. The fermented liquids of A-4 and A-5 were not diluted, and the stock solution was directly added, resulting in a cell count of only less than 113.3%.

[0108] Table 5 Determination of collagen content

[0109]

[0110]

[0111] After culturing fibroblasts with different concentrations of EGT and Agaricus sutchuenensis extract, a kit was used to detect the content of collagen.

[0112] By comparison, it was found that EGT can increase the collagen content in fibroblasts, but its effect is far weaker than that of the Agrocybe aegerita extract of the present invention. When the A-2 Agrocybe aegerita extract of the present invention was diluted to an EGT concentration of 500ppm, the collagen content of fibroblasts was still increased by 103.7%, indicating that it has a significant anti-aging effect. The A-4 and A-5 fermentation broths were not diluted, and the stock solution was directly added, and the collagen content was only increased by less than 32.9%.

[0113] Table 6 Determination of the content of main components of Polygonatum sibiricum enzymatic hydrolysate

[0114] name content(%) Polygonatum sibiricum polysaccharide 20±5 Polygonatum oligosaccharide 27±10 Free amino acids 13±5

[0115] Polygonatum sibiricum hydrolysate contains a large amount of carbon source and amino acids, which can be added to the culture medium as the main nutrient component. The large amount of free amino acids contained therein can not only provide the needs of mycelium growth and development, but also can serve as a precursor of EGT to promote the production of EGT.

[0116] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for preparing an extract rich in ergothioneine, characterized in that, The method comprises the following steps: inoculating a flat mushroom seed liquid into a fermentation medium containing a polygonatum hydrolysate for fermentation and culturing; after the fermentation and culturing is completed, the fermentation liquid is crushed, boiled and filtered in sequence to obtain an extract rich in ergothioneine; the fermentation medium comprises the following components: 50-200 g / L of polygonatum hydrolysate, 20-60 g / L of an inorganic nitrogen source, 0.1-5.0 g / L of an inorganic salt and 0.001-0.005 g / L of vitamins; The specific preparation process of the polygonatum hydrolysate is as follows: taking the polygonatum raw material according to 25% of the water mass, adding it into water, and then adding hydrolase according to 0.25% of the water volume for enzymolysis, the conditions of enzymolysis are: keeping warm at 40°C for 5h, the hydrolase is a mixed enzyme of α-amylase, pectinase, cellulase, acid protease, neutral protease, alkaline protease and papain, wherein cellulase accounts for 40%, α-amylase, pectinase, acid protease, neutral protease, alkaline protease and papain each account for 10%, after the enzymolysis is completed, boiling and filtering, the obtained filtrate is the polygonatum hydrolysate.

2. The preparation method according to claim 1, characterized in that: The inorganic nitrogen source in the fermentation medium is one or more of ammonium nitrate, ammonium chloride, and ammonium sulfate; the inorganic salt is one or more of sodium nitrate, sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, zinc sulfate, zinc chloride, and manganese chloride; and the vitamin is one or more of VB1, VB3, VB6, and VB12.

3. The preparation method according to claim 1, characterized in that: The raw material of polygonatum sibiricum includes polygonatum yunnanensis ( Polygonatum kingianum Coll.et Hemsl. )、Potamogeton Polygonatum sibiricum Red. ) and Polygonatum cyrtonema ( Polygonatum cyrtonema Hua ) 4. The preparation method according to claim 1, characterized in that: The ratio of polygonatum polysaccharide and polygonatum oligosaccharide in the polygonatum hydrolysate accounts for 30-60% of the total mass of the polygonatum hydrolysate; the inoculation amount of the Agaricus rutaecarpa seed liquid is 5-40% by volume; the fermentation temperature is 20-30°C and the fermentation time is 7-21d; stirring is performed during the fermentation process, and the stirring speed is 120-220r / min.

5. The preparation method according to claim 1, characterized in that: The preparation of the flatland mushroom seed liquid includes two processes: S1 strain culture and S2 seed liquid culture.

6. The preparation method according to claim 5, characterized in that: Step S1 specifically includes: inoculating the mycelium of the flatland mushroom on a PDA solid culture medium for cultivation to obtain a flatland mushroom strain containing the culture medium; step S2 specifically includes: inoculating the flatland mushroom strain containing the culture medium into a seed liquid culture medium for seed liquid cultivation to obtain a flatland mushroom seed liquid.

7. The preparation method according to claim 6, characterized in that: The culture temperature in step S1 is 20-30°C for 7-14 days; the seed solution culture temperature in step S2 is 20-30°C for 7-21 days; Stirring is performed during the culture process, and the stirring speed is 50-180r / min; the composition of the seed liquid culture medium includes the following components: 20.0-50.0g / L of carbon source, 10.0-30.0g / L of nitrogen source and 0.1-5.0g / L of inorganic salt.

8. The preparation method according to claim 6, characterized in that: The composition of the seed liquid culture medium in step S2 includes the following components: The invention discloses a fermentation medium comprising 10.0-20.0 g / L soluble starch, 10.0-30.0 g / L glucose, 10.0-30.0 g / L yeast powder, 0.1-1.0 g / L potassium dihydrogen phosphate and 0.1-1.0 g / L magnesium sulfate; the fermentation medium comprises the following components: 50-200 g / L polygonatum sibiricum hydrolysate, 20-60 g / L ammonium chloride, 0.1-3.0 g / L potassium dihydrogen phosphate and 0.1-1.5 g / L magnesium sulfate, and 0.001-0.005 g / L vitamin B12.

9. the extract rich in thioneine prepared according to the arbitrary described preparation method of claim 1-8.

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