A preparation method of flat mushroom extract

Abstract

The invention discloses a method for preparing a flat mushroom extract. The invention uses flat mushroom and Candida lipolytica to prepare an extract rich in polypeptides and EGT, and the extract has significant antioxidant and free radical scavenging abilities, and can be used as a cosmetic raw material. The method comprises 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 culture medium for fermentation; after the fermentation is completed, the fermentation liquid can be obtained after separation treatment. In the invention, the two strains are fermented together, which can exert a synergistic effect, not only making the EGT content reach 355.9 mg / L, but also significantly increasing the polypeptide yield to 4.93 g / L, and the DPPH and ABTS scavenging rates of the obtained flat mushroom extract after diluting 5 times are 93.7% and 80.4% respectively. It has significant antioxidant and free radical scavenging abilities.

Description

Technical Field

[0001] The invention relates to a method for preparing a flat mushroom extract, belonging to the technical field of microbial fermentation. Background Art

[0002] Ergothioneine (EGT), whose scientific name is 2-mercapto-L-histidine trimethylolate, has an imidazole-2-thione group in its molecular structure, which has been proven to be an excellent antioxidant and metal ion chelator.

[0003] Mushrooms are the main type of artificially cultivated edible fungi. They are not only delicious and tender, but also have extremely high nutritional value. They are ideal high-protein, low-fat, low-calorie health foods for humans. Basidiomycetes have high protein content, with a fresh weight content of up to 3.5-4.5%, which is several to dozens of times that of ordinary vegetables. They also contain vitamin B1, riboflavin, niacin, vitamin H, vitamin C and a variety of minerals.

[0004] Candida lipolytica is an important industrial fungus in my country and is widely used in many fields. Ceramide is a lipid substance present in the skin and plays an important role in the formation of the epidermal stratum corneum. Since there is free ceramide in Candida lipolytica, it is used to produce ceramide by taking advantage of its short growth cycle, low cultivation cost and mature related technology. It is also often used to produce active substances such as enzyme preparations, citric acid, vitamins, glutamic acid and fatty acids.

[0005] At present, a variety of edible fungi deep fermentation technology has been used for the production of ergothioneine, but the research depth of flat mushroom peptides in China is not enough. For example, the master's thesis "Preparation of Lentinus edodes stem peptides and study of their antioxidant activity" uses substrate concentration, enzyme dosage, pH and enzymatic hydrolysis temperature as influencing factors, and DPPH clearance rate as the response value to conduct a four-factor three-level response surface experiment. The results show that the DPPH free radical scavenging rate of the preparation of Lentinus edodes stem antioxidant peptides is only 63.20%. The master's thesis "Study on the preparation process of Agaricus bisporus antioxidant peptides" uses enzymatic hydrolysis to extract peptides from Agaricus bisporus. The extraction rate is only 27.42% and the DPPH clearance rate of the extract is only 68.72%. Patent CN 110922498 A uses defective shiitake mushrooms and shiitake stems to prepare shiitake polysaccharides and shiitake peptides. It uses 3090L of shiitake extract to obtain 8.67kg of polypeptide powder, and the content of polypeptide in the extract is only 2.69g / L. Patent CN104256044A uses 1% alkaline protease to hydrolyze flat mushrooms for 8 hours to obtain polypeptides, which is too time-consuming and laborious.

[0006] In summary, the current development and utilization of Agaricus bisporus mycelium fermentation mainly have the following problems: (1) The current method of extracting active substances is mainly enzymatic method and the cost is relatively high; (2) The current extraction rate of peptides and other active substances produced by mycelium fermentation is not high; (3) The activity of the extracted active substances is not high. Summary of the invention

[0007] The purpose of the present invention is to provide a flat mushroom extract rich in polypeptides and ergothioneine prepared by double-strain co-fermentation and a preparation method thereof in view of the problems that the extraction rate of active substances extracted by edible fungi in the prior art is low, the activity is insufficient, etc., wherein the flat mushroom extract and the preparation method thereof are provided, wherein the flat mushroom and the Candida lipolytica are co-fermented, the synergistic effect between the hyphae is improved through fermentation regulation, the yield of the polypeptide and the ergothioneine can be significantly increased, and the obtained combined fermentation liquid has significant antioxidant and free radical scavenging abilities.

[0008] In order to achieve the above object, the present invention provides a method for preparing a Pleurotus eryngii extract, comprising the following steps:

[0009] Step 1: inoculating the hyphae of Candida lipolytica and Agaricus rutaecarpa on cornmeal agar medium and PDA solid medium for cultivation, respectively, to obtain Candida lipolytica strains and Agaricus rutaecarpa strains containing culture medium, respectively;

[0010] Step 2: inoculating the Agaricus spp. containing the culture medium into the seed liquid culture medium I for seed liquid culture to obtain Agaricus spp. seed liquid, and inoculating the Candida lipolytica containing the culture medium into the seed liquid culture medium II for seed liquid culture to obtain Candida lipolytica seed liquid;

[0011] Step 3: inoculating the flat mushroom seed liquid into the fermentation medium at a volume fraction of 20-40%, adding the precursor for fermentation culture, and then inoculating the Candida lipolytica seed liquid at a volume fraction of 5-20%, and adding the precursor to continue fermentation culture, wherein the precursor is selected from at least one of cysteine, histidine and pyridoxal phosphate;

[0012] Step 4: After the fermentation is completed, the fermentation liquid containing ergothioneine and polypeptide is obtained by ultrasonication, boiling and centrifugal filtration in sequence, which is the Agaricus rutaecarpa extract.

[0013] Among them, the advantage of fermenting the flat ground mushroom first and then inoculating the lipolytica in step 3 is that the flat ground mushroom will produce unadded nutrients in the fermentation medium, for example, the glucose in the medium can produce a variety of organic acids that promote growth and development, and the starch can produce a variety of monosaccharides and oligosaccharides. Fermenting the flat ground mushroom first can enrich the nutrients in the medium. The inoculation of the lipolytica with the fermentation will produce a large amount of amino acids such as glutamic acid and aspartic acid, which promotes the process of flat ground mushroom fermentation to produce EGT, and the protease and lipase produced during the fermentation process can hydrolyze the flat ground mushroom mycelium to dissolve EGT and increase the polypeptide content, thereby eliminating the need for exogenous addition of hydrolases for the hydrolysis process.

[0014] Under the process conditions of the preparation method, Agaricus rufipogon and Candida lipolytica are co-fermented. The co-fermentation of the two strains not only does not cause competition, but also helps to increase the content of polypeptides and EGT in the fermentation product.

[0015] Preferably, the culture temperature in step 1 is 25-35° C. and the culture time is 3-7 days.

[0016] Preferably, the seed liquid culture medium I in step 2 comprises the following components: 20.0-200.0 g / L of carbon source, 10.0-50.0 g / L of nitrogen source and 0.1-5.0 g / L of inorganic salt; the seed liquid culture medium II comprises the following components: 10.0-30.0 g / L of carbon source, 5.0-30.0 g / L of nitrogen source and 0.1-3.0 g / L of inorganic salt.

[0017] Preferably, the seed liquid culture medium I comprises the following components: 100.0-200.0 g / L of potato, 10.0-30.0 g / L of glucose, 10.0-30.0 g / L of yeast powder, 0.1-1.0 g / L of potassium dihydrogen phosphate and 0.1-1.0 g / L of magnesium sulfate; the seed liquid culture medium II comprises the following components: 10-20 g / L of glucose, 5-20 g / L of peptone, 1.0-10 g / L of yeast extract, 0.1-1.0 g / L of potassium dihydrogen phosphate and 0.1-1.0 g / L of magnesium sulfate.

[0018] Preferably, in step 2, the seed solution is cultured at a temperature of 25 to 35° C., and stirring is performed during the culture process at a speed of 120 to 180 r / min.

[0019] Preferably, the fermentation medium in step 3 comprises the following components: 45.0-80.0 g / L of carbon source, 30.0-50.0 g / L of nitrogen source, 0.1-5.0 g / L of inorganic salt and 0.001-0.005 g / L of vitamin.

[0020] Preferably, the fermentation medium comprises the following components: 10.0-20.0 g / L of glucose, 35.0-50.0 g / L of soluble starch, 30.0-40.0 g / L of peptone, 0.1-8.0 g / L of inorganic salts, 0.001-0.005 g / L of nicotinic acid and 0.001-0.005 g / L of vitamin B1.

[0021] Preferably, the fermentation temperature in step 3 is 22-30° C.; stirring is performed during the fermentation process at a speed of 220-300 r / min, wherein after the Candida lipolytica seed solution is added, the stirring speed is reduced by 25-75 r / min every day to continue the fermentation.

[0022] Preferably, the initial addition amount of the precursor in step 3 is 1-5 g / L, and the additional addition amount of the precursor is 0.1-3.0 g / L.

[0023] The present invention also provides the Agaricus rufipogon extract prepared by the method for preparing the Agaricus rufipogon extract.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention selects Agaricus rutaegi and Candida lipolytica as the production strains of thioneine. Under the preparation process conditions of the present invention, the two strains are fermented together not only without competition, but also contribute to the promotion of polypeptide and EGT content in the fermentation product. Therefore, the output of polypeptide and thioneine is greatly improved.

[0026] (2) The co-fermentation of the two strains in the present invention can exert a synergistic effect, not only making the EGT content reach 355.9 mg / L, but also significantly increasing the polypeptide yield to 4.93 g / L. After the obtained Agaricus rufipogon extract was diluted 5 times, the DPPH and ABTS scavenging rates were 93.7% and 80.4%, respectively. It has significant antioxidant and free radical scavenging capabilities, can be used as a raw material for functional skin care products, and has good application prospects. DETAILED DESCRIPTION

[0027] In order to make the present invention more clearly understood, preferred embodiments are described in detail as follows.

[0028] In the following examples, the reagents and equipment used are conventional commercial products unless otherwise specified. The histidine, cysteine, and pyridoxal phosphate used in the following examples were purchased from Beijing Solarbio Technology Co., Ltd.; the ferrous sulfate, ferrous chloride, potassium dihydrogen phosphate, sodium chloride, magnesium sulfate, ammonium chloride, and ammonium citrate used in the following examples were purchased from Shanghai Titan Technology Co., Ltd.

[0029] In the following examples, the Candida lipolytica block and the Agaricus rutaecarpa block are prepared by inoculating the hyphae of Candida lipolytica and Agaricus rutaecarpa on corn flour agar medium and PDA solid medium, respectively, to obtain Candida lipolytica strains and Agaricus rutaecarpa strains containing culture medium, respectively.

[0030] Example 1

[0031] A method for preparing a flat mushroom extract comprises the following steps:

[0032] (1) Take 4cm 2 4 flat mushroom spawn (with culture medium) and 1cm 2 One Candida lipolytica block (with culture medium) was inoculated into the seed culture medium and cultured at 29°C and 180 rpm for 4 and 2 days.

[0033] Flatland mushroom seed liquid culture medium: potato 100.0g / L, glucose 10.0g / L, yeast powder 10.0g / L, potassium dihydrogen phosphate 0.1g / L, magnesium sulfate 0.1g / L.

[0034] Candida lipolytica seed liquid culture medium: glucose 10.0 g / L, peptone 5.0 g / L, yeast extract 1.0 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.1 g / L.

[0035] (2) 40% of Agaricus oleraceus culture fluid was added to new seed fluid culture medium, and cultured at 25°C and 260 r / min for 3 days. Subsequently, Candida lipolytica seed fluid was inoculated into the fermentation medium at a 15% inoculation rate. The speed was then reduced by 25 r / min every day, and fermented for 2 days. The volume of the fermentation medium was 40% of the total volume.

[0036] Fermentation medium: glucose 10.0g / L, soluble starch 50.0g / L, peptone 30.0g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 1.0g / L, ferrous sulfate 0.1g / L, niacin 0.005g / L, vitamin B1 0.002g / L.

[0037] (3) After inoculating Agaricus rufa into the fermentation medium, 3.0 g / L histidine was added, and after inoculating Candida lipolytica, 0.5 g / L histidine was added.

[0038] (4) after fermentation is completed, add a small amount of fermented liquid and cross 0.22 micron filter membrane and then cryopreserve (denoted as A-1). The remaining fermented liquid is then ultrasonically treated for 45min under the condition of 1500W and temperature of 40°C; after boiling for 20min, cool down, centrifuge at 5000r / min to obtain thioneine solution (denoted as A-2). Take a small amount of fermented liquid and use 500 Dalton filter membrane 12000r / min to recover volume after repeated centrifugation (denoted as A-3).

[0039] Example 2

[0040] A method for preparing a flat mushroom extract comprises the following steps:

[0041] (1) Take 6cm 2 Four Agaricus spp. blocks (with culture medium) and four Candida lipolytica blocks (with culture medium) of 1 cm2 were inoculated into the seed culture medium and cultured at 25°C and 180 r / min for 4 and 2 days.

[0042] Flatland mushroom seed liquid culture medium: potato 150.0g / L, glucose 15.0g / L, yeast powder 10.0g / L, potassium dihydrogen phosphate 0.1g / L, magnesium sulfate 0.1 / L.

[0043] Candida lipolytica seed liquid culture medium: glucose 14.0 g / L, peptone 10.0 g / L, yeast extract 1.0 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.5 g / L.

[0044] (2) 20% of the Agaricus rufipogon culture solution was inoculated into the new seed solution culture medium, and the culture was carried out at 28°C and 220 r / min for 5 days. The Candida lipolytica seed solution was inoculated into the fermentation medium at a 20% inoculation rate. The speed was then reduced by 40 r / min every day, and the fermentation was carried out for 4 days. The liquid volume of the fermentation medium was 30% of the total volume.

[0045] Fermentation medium: glucose 20.0g / L, soluble starch 35.0g / L, peptone 20.0g / L, potassium dihydrogen phosphate 0.3g / L, magnesium sulfate 0.1g / L, ferrous sulfate 0.05g / L, niacin 0.002g / L, vitamin B1 0.002g / L.

[0046] (3) After inoculation into the fermentation medium, 3.0 g / L histidine and 1.5 g / L cysteine ​​were added, and after inoculation with Candida lipolytica, 1.0 g / L histidine was added.

[0047] (4) after fermentation is completed, add a small amount of fermented liquid and cross 0.22 micron filter membrane and then cryopreserve (denoted as B-1). The remaining fermented liquid is then ultrasonically treated for 60min under the condition of 2800W and temperature of 50 DEG C; after boiling for 15min, cool down, centrifuge at 8000r / min to obtain thioneine solution (B-2). Take a small amount of fermented liquid and use 500 Dalton filter membrane 12000r / min to recover volume (B-3) after repeated centrifugation.

[0048] Example 3

[0049] A method for preparing a flat mushroom extract comprises the following steps:

[0050] (1) Take 8cm 2 6 flat mushroom spawn blocks (with culture medium) and 4cm 2 Four Candida lipolytica blocks (with culture medium) were inoculated into the seed culture medium and cultured at 27°C and 125 rpm for 7 and 3 days.

[0051] Flatland mushroom seed liquid culture medium: potato 150.0g / L, glucose 15.0g / L, yeast powder 5.0g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 1.0 / L.

[0052] Candida lipolytica seed liquid culture medium: glucose 15.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L.

[0053] (2) 30% of the Agaricus rufipogon culture fluid was inoculated into the new seed liquid culture medium, and cultured at 22°C and 255 r / min for 4 days. Subsequently, the Candida lipolytica culture fluid was inoculated into the fermentation medium at a 5% inoculation rate. The speed was then reduced by 35 r / min every day, and the fermentation was continued for 5 days. The liquid volume of the fermentation medium was 20% of the total volume.

[0054] Fermentation medium: glucose 15.0g / L, soluble starch 45.0g / L, peptone 20.0g / L, potassium dihydrogen phosphate 0.6g / L, magnesium sulfate 0.8g / L, ferrous sulfate 0.1g / L, niacin 0.002g / L, vitamin B1 0.001g / L

[0055] (3) After inoculation into the fermentation medium, 1.0 g / L histidine was added, and after inoculation with the Candida lipolytica strain, 0.5 g / L histidine, 0.5 g / L pyridoxal phosphate, and 1.5 g / L cysteine ​​were added.

[0056] (4) after fermentation is completed, add and take a small amount of fermented liquid and cross 0.22 micron filter membrane and then cryopreserve (denoted as C-1). The remaining fermented liquid is then ultrasonically treated for 30min under the condition of 2050W and temperature of 45 DEG C; After boiling for 15min, cool down, 6500r / min centrifuge filtration, obtain thioneine solution (denoted as C-2). Take a small amount of fermented liquid and use 500 Dalton filter membrane 12000r / min to recover volume after repeated centrifugation (denoted as C-3).

[0057] Example 4

[0058] A preparation method of a flat mushroom extract, the difference from Example 3 is that: the precursor added when accessing the flat mushroom seed liquid to the fermentation medium is 1.5g / L histidine and 1.5g / L cysteine, the precursor added when accessing the Candida lipolytica seed liquid to the fermentation medium is 0.5g / L histidine and 0.5g / L pyridoxal phosphate, and after fermentation is completed, a small amount of fermented liquid is added and stored at low temperature after passing through a 0.22 micron filter membrane (denoted as D-1). The remaining fermented liquid is then ultrasonically treated for 30min under the conditions of 3000W and a temperature of 45°C; After boiling for 20min, it is cooled and centrifuged at 7000r / min to obtain an ergothioneine solution (denoted as D-2). A small amount of fermented liquid is taken and the volume is restored after multiple centrifugation using a 500 Dalton filter membrane 12000r / min (denoted as D-3).

[0059] Example 5

[0060] A method for preparing a flat mushroom extract, which is different from Example 4 in that the precursor added when the flat mushroom seed liquid is accessed to the fermentation medium is 2.0g / L histidine, and the precursor added when the Candida lipolytica seed liquid is accessed to the fermentation medium is 0.5g / L histidine and 0.5g / L pyridoxal phosphate. The inorganic salts added to the fermentation broth are 0.3g / L ferrous sulfate, 0.5g / L magnesium sulfate, 1.5g / L potassium dihydrogen phosphate, 0.5g / L sodium chloride, and 0.5g / L ammonium citrate. After the fermentation is completed, a small amount of fermentation broth is added and passed through a 0.22 micron filter membrane and then stored at low temperature (denoted as E-1). The remaining fermentation broth is then ultrasonically treated for 60min at 2500W and a temperature of 45°C; after boiling for 15min, cooled, and centrifuged at 7500r / min to obtain an ergothioneine solution (denoted as E-2). Take a small amount of fermentation broth and centrifuge it several times at 12000 r / min using a 500 Dalton filter to restore the volume (denoted as E-3).

[0061] Comparative Example 1

[0062] A method for preparing a flat mushroom extract comprises the following steps:

[0063] (1) Take 1cm 2 Four pieces of Agaricus spp. mycelium (with culture medium) were inoculated into seed liquid culture medium and cultured for 7 days.

[0064] (2) Then, the fermentation medium was inoculated with 10% inoculum, and fermented at 25° C. and 255 r / min for 5 days, and then the speed was reduced by 35 r / min every day and the fermentation was continued for 5 days. The liquid volume of the fermentation medium was 20% of the total volume.

[0065] (3) After the Pingdu mushroom seed liquid was inoculated into the fermentation medium, 1.0 g / L histidine and 0.5 g / L cysteine ​​were added. After culturing for 5 days, 0.5 g / L histidine and 1.5 g / L cysteine ​​were added again.

[0066] (4) after fermentation is completed, add a small amount of fermented liquid and cross 0.22 micron filter membrane and then cryopreserve (denoted as F-1). The remaining fermented liquid is then subjected to ultrasonic treatment for 40min under the condition of 2450W and 45°C for fermented liquid; after boiling for 10min and cooling, 5500r / min centrifugal filtration is performed to obtain thioneine solution (denoted as F-2). Take a small amount of fermented liquid and use 500 Dalton filter membrane 12000r / min to recover volume after repeated centrifugation (denoted as F-3).

[0067] Comparative Example 2

[0068] A method for preparing a Candida lipolytica fermentation extract comprises the following steps:

[0069] (1) 4cm 2 Four Candida lipolytica blocks (with culture medium) were inoculated into the seed culture medium and cultured at 27°C and 125 r / min for 3 days.

[0070] (2) Subsequently, the fermentation medium was inoculated with 10% inoculum, and fermented at 25°C, 255 r / min for 5 days, then the speed was reduced by 35 r / min every day and continued to ferment for 5 days. The liquid volume of the fermentation medium was 20% of the total volume.

[0071] (3) After inoculating Candida lipolytica for 5 days, 0.5 g / L histidine, 0.5 g / L pyridoxal phosphate, and 1.5 g / L cysteine ​​were added and the fermentation was continued for 5 days.

[0072] (4) After fermentation, a small amount of the fermentation liquid was filtered through a 0.22 μm filter membrane and stored at low temperature (denoted as G-1). The remaining fermentation liquid was then ultrasonicated at 1500 W for 30 min, boiled for 20 min, cooled, filtered through a 0.22 μm filter membrane, and stored at low temperature (denoted as G-2).

[0073] Comparative Example 3

[0074] Preparation of fermentation broth for blank control group:

[0075] The fermentation medium was supplemented with 1.0 g / L histidine and 0.5 g / L cysteine, and cultured at 25°C and 250 r / min for 4 days, and then 0.5 g / L histidine, 1.5 g / L cysteine, and 0.5 g / L pyridoxal phosphate were added again and cultured for 3 more days.

[0076] Ultrasonication was performed at 3000W for 30 min, boiling was performed for 10 min, and then centrifugation was performed after cooling to obtain the fermentation broth of the blank control group (denoted as H-1).

[0077] The products obtained in the above embodiments and comparative examples were subjected to the following inspections or tests:

[0078] Detection of thioneine: Under the same chromatographic conditions, thioneine standard substances and the sample to be tested are subjected to HPLC determination, 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. A standard curve is drawn with the concentration of the standard substance thioneine and the corresponding peak area, and when the standard substance is the same as the sample injection amount, the content of thioneine in the sample is calculated by external standard method quantitatively.

[0079] 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 0.7 mL / min, column temperature 25°C, injection volume 10 μL, UV detection wavelength 254 nm.

[0080] Determination of polypeptide content: The polypeptide content in the fermentation broth was determined using the Folin phenol method.

[0081] The DPPH clearance rate determination and ABTS clearance rate determination are both conventional methods.

[0082] The activity of protease was determined by spectrophotometry according to GB / T28715-2012.

[0083] The experimental results are shown in Tables 1 to 4:

[0084] Table 1 EGT and polypeptide content

[0085] EGT content in fermentation broth (mg / L) Peptide content in fermentation broth (g / L) A-2 338.6 4.79 B-2 349.4 4.55 C-2 355.9 4.93 D-2 343.5 4.89 E-2 346.6 4.71 F-2 133.5 1.36 G-1 Not detected 0.37 H-1 Not detected 0.23

[0086] Table 2 DPPH and ABTS removal rates

[0087]

[0088]

[0089] Among them, the data in Table 2 were obtained by diluting the sample solution 5 times during the determination process.

[0090] As can be seen from Table 1, the content of thioneine in the fermented liquid after embodiment 1~5 combined fermentation has reached up to 355.9mg / L, and the content of polypeptide has reached up to 4.93g / L, and comparative example 1 adopts flat ground mushroom to ferment and produce polypeptide and thioneine separately, and the output of thioneine in fermented liquid is 133.5mg / L, and the content of polypeptide is 1.36g / L, and comparative example 3 is the blank control of fermentation medium and amino acid composition, wherein hardly containing EGT also has almost no antioxidant property. Due to the synergistic effect of two kinds of bacteria, the output of thioneine is obviously improved, and the content of polypeptide has also had obvious rising, which causes the clearance rate of 5 times of DPPH and ABTS of common fermented liquid to still reach 93.7% and 80.4% even if diluting.

[0091] Table 3 Antioxidant activity of peptides

[0092] DPPH clearance rate (%) ABTS clearance rate (%) A-3 87.9 75.7 B-3 89.6 70.4 C-3 88.5 72.3 D-3 86.3 73.9 E-3 89.1 76.6 F-3 34.6 29.5

[0093] After using a 500Da microfiltration membrane to remove substances with smaller molecular weight such as EGT, it was found that the DPPH clearance rate and ABTS clearance rate of the fermentation broth reached 89.6% and 76.6%, which were at a relatively high level.

[0094] Table 4 Protease activity detection

[0095]

[0096]

[0097] The activity of the protease in the fermentation broth of Examples 1 to 5 and Comparative Example 1 was tested, and it was found that the activity of the protease in Examples 1 to 5 with the addition of Candida lipolytica reached 1499.6 to 1831.3 U / mL, while the activity of the protease in Comparative Example 1 was only 22.5 U / mL. When Candida lipolytica was fermented alone, it was found that 1399.6 U / mL of protease could be produced, and the protease produced during mixed fermentation mainly came from Candida lipolytica.

[0098] The above embodiments are only preferred embodiments of the present invention and are not any formal or substantial limitations of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a flat mushroom extract, characterized in that: The following steps are involved: Step 1: inoculating the hyphae of Candida lipolytica and Agaricus rutaecarpa on cornmeal agar medium and PDA solid medium for cultivation, respectively, to obtain Candida lipolytica strains and Agaricus rutaecarpa strains containing culture medium, respectively; Step 2: inoculating the Agaricus spp. containing the culture medium into the seed liquid culture medium I for seed liquid culture to obtain Agaricus spp. seed liquid, and inoculating the Candida lipolytica containing the culture medium into the seed liquid culture medium II for seed liquid culture to obtain Candida lipolytica seed liquid; Step 3: inoculating the flat mushroom seed liquid into the fermentation medium at a volume fraction of 20-40%, adding the precursor for fermentation culture, and then inoculating the Candida lipolytica seed liquid at a volume fraction of 5-20%, and adding the precursor to continue fermentation culture, wherein the precursor is selected from at least one of cysteine, histidine and pyridoxal phosphate; Step 4: After the fermentation is completed, the fermentation liquid containing ergothioneine and polypeptide is obtained by ultrasonication, boiling and centrifugal filtration in sequence, which is the Agaricus rufipogon extract.

2. The method for preparing the flat mushroom extract according to claim 1, characterized in that: The culture temperature in step 1 is 25-35° C. and the culture time is 3-7 days.

3. The method for preparing the flat mushroom extract according to claim 1, characterized in that: The seed liquid culture medium I in step 2 includes the following components: 20.0-200.0 g / L of carbon source, 10.0-50.0 g / L of nitrogen source and 0.1-5.0 g / L of inorganic salt; the seed liquid culture medium II includes the following components: 10.0-30.0 g / L of carbon source, 5.0-30.0 g / L of nitrogen source and 0.1-3.0 g / L of inorganic salt.

4. The method for preparing the flat mushroom extract according to claim 3, characterized in that: The seed liquid culture medium I comprises the following components: 100.0-200.0 g / L of potato, 10.0-30.0 g / L of glucose, 10.0-30.0 g / L of yeast powder, 0.1-1.0 g / L of potassium dihydrogen phosphate and 0.1-1.0 g / L of magnesium sulfate; the seed liquid culture medium II comprises the following components: 10-20 g / L of glucose, 5-20 g / L of peptone, 1.0-10 g / L of yeast extract, 0.1-1.0 g / L of potassium dihydrogen phosphate and 0.1-1.0 g / L of magnesium sulfate.

5. The method for preparing the flat mushroom extract according to claim 1, characterized in that: In the step 2, the seed solution is cultured at a temperature of 25 to 35° C., and stirring is performed during the culture process at a speed of 120 to 180 r / min.

6. The method for preparing the flat mushroom extract according to claim 1, characterized in that: The fermentation medium in step 3 includes the following components: 45.0-80.0 g / L of carbon source, 30.0-50.0 g / L of nitrogen source, 0.1-5.0 g / L of inorganic salt and 0.001-0.005 g / L of vitamin.

7. The method for preparing the flat mushroom extract according to claim 5, characterized in that: The fermentation medium comprises the following components: 10.0-20.0 g / L of glucose, 35.0-50.0 g / L of soluble starch, 30.0-40.0 g / L of peptone, 0.1-8.0 g / L of inorganic salt, 0.001-0.005 g / L of nicotinic acid and 0.001-0.005 g / L of vitamin B1.

8. The method for preparing the flat mushroom extract according to claim 1, characterized in that: The fermentation temperature in step 3 is 22-30° C. The fermentation is stirred at a speed of 220-300 r / min. After the Candida lipolytica seed solution is added, the stirring speed is reduced by 25-75 r / min every day to continue the fermentation.

9. The method for preparing the flat mushroom extract according to claim 1, characterized in that: In step 3, the initial addition amount of the precursor is 1-5 g / L, and the additional addition amount of the precursor is 0.1-3.0 g / L.

10. The Agaricus rufipogon extract prepared by the method for preparing the Agaricus rufipogon extract according to any one of claims 1 to 9.

Images