1. A method for producing selenium-rich Lyophyllum nuciferum mycelium in small scale is characterized by comprising the following steps:

s1, preparing a lyophyllum decastes seed culture solution: adding 100mL of enriched PDA culture medium into a 250mL triangular flask, inoculating activated Lyophyllum decastes strain, performing shake culture for 4-8 days to obtain Lyophyllum decastes seed culture solution, and placing in a refrigerator at 4 deg.C for use;

s2, preparing a fermentation medium, wherein the fermentation medium is prepared by sterilizing protein hydrolysate, corn starch hydrolysate and glucose;

s3, inoculating Lyophyllum decastes seeds: inoculating the lyophyllum decastes seed culture solution obtained in the step S1 in the fermentation culture medium, wherein the inoculation amount is 5.5-10%; introducing sterile air, and performing sterile culture under the condition that the pressure in the tank is 0.02 MPa;

s4, sterile culturing 3d, adding sterilized Na into the fermentation medium₂SeO₃Solution containing 4. mu.g of Na per 1mL of fermentation medium₂SeO₃Continuing fermenting for 5-10 days;

and S5, after the fermentation is finished, filtering the fermentation liquor by a 300-mesh filter screen, repeatedly washing the bacterial balls by distilled water, drying, and weighing to obtain the selenium-rich mycelium of the Lyophyllum nuciferum mycelium.

2. The method for small-scale production of selenium-enriched Lyophyllum decastes mycelia according to claim 1, wherein the shaking culture time of step S1 is 5 days.

3. The method for small-scale production of selenium-enriched Lyophyllum decastes mycelia according to claim 1, wherein the preparation method of the protein hydrolysate is as follows: weighing 100g of soybeans, soaking overnight, pulping, boiling, cooling, adding 5 wt% of protease, and carrying out water bath in a constant-temperature water bath kettle at 40 ℃ for 5 hours to obtain protein hydrolysate.

4. The method for small-scale production of selenium-rich Lyophyllum decastes mycelia according to claim 3, wherein the protease is added after the boiled soybean milk is cooled to 40 ℃.

5. The method for small-scale production of selenium-rich Lyophyllum decastes mycelia according to claim 1, wherein the preparation method of the corn starch hydrolysate is as follows: 600g of corn flour is weighed, 6000g of hot water at 65 ℃ is added, 5 wt% of amylase is added, and the mixture is placed in a constant-temperature water bath kettle at 65 ℃ and mixed until the iodine solution is colorless, so that corn starch hydrolysate is obtained.

6. The small-scale production method of selenium-rich Lyophyllum decastes mycelia according to claim 1, wherein the sterilization temperature in step S2 is 121 ℃, and the sterilization time is 40 min.

7. The method for small-scale production of selenium-rich Lyophyllum decastes mycelia according to claim 1, wherein the drying temperature in step S5 is-25 deg.C-15 deg.C.

8. Use of selenium-enriched Lyophyllum Inophyllum mycelium produced by the method according to any one of claims 1-7 in preparation of health food, medicine or cosmetic for reducing blood lipid and blood glucose and resisting oxidation.

Background

Selenium is used as essential trace element, and has effects of stimulating production of human immunoglobulin and antibody, and resisting aging. Lyophyllum decastes is fungus belonging to Lyophyllum, and has high nutritive value. The lyophyllum decastes has strong selenium enrichment capability, so that inorganic selenium is safely and effectively converted into organic selenium polysaccharide and selenoprotein, and the nutritional value and the application range of the edible fungi are greatly improved.

At present, the large-scale technology for artificially culturing the selenium-rich Lyophyllum decastes is not mature, and how to realize the method for producing the selenium-rich Lyophyllum decastes mycelium in a large scale is an important subject continuously explored by the industry at present.

Disclosure of Invention

In order to overcome the problems, the invention provides a method for producing selenium-rich Lyophyllum nuciferum mycelium in a small scale, the method can produce the selenium-rich Lyophyllum nuciferum mycelium in a 20L fermentation tank, the preparation process is simple and convenient to operate, and the obtained mycelium has high batch stability and higher biological activity.

The invention relates to a method for producing selenium-rich Lyophyllum decastes mycelia in small scale, which comprises the following steps:

s1, preparing a lyophyllum decastes seed culture solution: adding 100mL of enriched PDA culture medium into a 250mL triangular flask, inoculating activated Lyophyllum decastes strain, performing shake culture for 4-8 days to obtain Lyophyllum decastes seed culture solution, and placing in a refrigerator at 4 deg.C for use;

s2, preparing a fermentation medium, wherein the fermentation medium is prepared by sterilizing protein hydrolysate, corn starch hydrolysate and glucose;

s3, inoculating Lyophyllum decastes seeds: inoculating the lyophyllum decastes seed culture solution obtained in the step S1 in the fermentation culture medium, wherein the inoculation amount is 5.5-10%; introducing sterile air, and performing sterile culture under the condition that the pressure in the tank is 0.02 MPa;

s4, sterile culturing 3d, adding sterilized Na into the fermentation medium₂SeO₃Solution containing 4. mu.g of Na per 1mL of fermentation medium₂SeO₃Continuing fermenting for 5-10 days;

and S5, after the fermentation is finished, filtering the fermentation liquor by a 300-mesh filter screen, repeatedly washing the bacterial balls by distilled water, freeze-drying, and weighing to obtain the selenium-rich mycelium of the Lyophyllum nuciferum mycelium.

Preferably, the shake cultivation time in step S1 of the present invention is 5 days.

Preferably, the preparation method of the protein hydrolysate is as follows: weighing 100g of soybeans, soaking overnight, pulping, boiling, cooling, adding 5 wt% of protease, and carrying out water bath in a constant-temperature water bath kettle at 40 ℃ for 5 hours to obtain protein hydrolysate.

More preferably, the temperature of the boiled soybean milk is reduced to 40 ℃, and then the protease is added.

Preferably, the preparation method of the corn starch hydrolysate is as follows: 600g of corn flour is weighed, 6000g of hot water at 65 ℃ is added, 5 wt% of amylase is added, and the mixture is placed in a constant-temperature water bath kettle at 65 ℃ and mixed until the iodine solution is colorless, so that corn starch hydrolysate is obtained.

Preferably, the sterilization temperature in step S2 is 121 ℃ and the sterilization time is 40 min.

Preferably, the drying temperature in step S5 of the present invention is-25 ℃ to 15 ℃.

The invention also aims to provide selenium-rich Lyophyllum decastes mycelium with strong oxidation resistance and the functions of reducing blood fat and blood sugar, and the selenium-rich Lyophyllum decastes mycelium is used for preparing functional food, medicine or cosmetics with the functions of reducing blood fat and blood sugar and resisting oxidation.

Compared with the prior art, the invention has the following beneficial effects:

1. according to the invention, the biological activity of hyphae is improved and the growth rate is improved by adjusting the composition of the fermentation medium and the culture solution of the lyophyllum decastes seeds;

2. inorganic selenium is added in the fermentation step 3d, so that the time of mycelium entering the decline period is delayed, and the yield is obviously improved;

3. according to the invention, the lyophyllum decastes mycelium is utilized to convert inorganic selenium into organic selenium, so that the safety of people for selenium intake is improved, the utilization added value of the lyophyllum decastes can be improved, meanwhile, the production mode can realize small-scale mass production, and the adopted fermentation device is a 20L fermentation tank, so that the lyophyllum decastes has a wide application prospect.

Drawings

FIG. 1 is a graph of the effect of a suspension of Lyophyllum nuciferum mycelium on alpha-glucosidase activity;

FIG. 2 is a graph of the effect of Lyophyllum nuciferum mycelium polysaccharides on alpha-glucosidase activity;

FIG. 3 is a graph showing the effect of Lyophyllum decastes mycelium protein on alpha-glucosidase activity;

FIG. 4 shows the scavenging ability of hydroxyl radical by soluble polysaccharide in Lyophyllum decastes mycelium;

FIG. 5 shows DPPH radical scavenging ability of soluble polysaccharides in Lyophyllum decastes mycelia;

FIG. 6 shows the ABTS free radical scavenging ability of soluble polysaccharides in Lyophyllum decastes mycelia;

FIG. 7 shows the reducing power of soluble polysaccharides in Lyophyllum decastes mycelia.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1

A method for producing selenium-rich Lyophyllum decastes mycelia in small scale comprises the following steps:

s1, preparing a lyophyllum decastes seed culture solution: adding 100mL of enriched PDA culture medium into a 250mL triangular flask, inoculating activated Lyophyllum decastes strain, performing shake culture for 5d to obtain Lyophyllum decastes seed culture solution, and placing in a refrigerator at 4 deg.C for use;

s2, preparing protein hydrolysate: weighing 100g of soybeans, soaking overnight, pulping, boiling, cooling to 40 ℃, adding 5% of protease, and carrying out water bath in a constant-temperature water bath kettle at 40 ℃ for 5 hours to obtain protein hydrolysate;

s3, preparing corn starch hydrolysate: weighing 600g of corn flour, adding 6000g of 65 ℃ hot water, adding 5% of amylase, placing in a 65 ℃ constant-temperature water bath kettle, and mixing until iodine solution is colorless to obtain corn starch hydrolysate;

s4, mixing to prepare a fermentation medium, filtering the protein hydrolysate obtained in the step S2 and the corn starch hydrolysate obtained in the step S3 by using a 300-mesh sieve, mixing, adding 200g of glucose, sterilizing at 121 ℃ for 40min, and cooling to obtain the fermentation medium;

s5, inoculating Lyophyllum decastes seeds: inoculating the lyophyllum decastes seed culture solution obtained in the step S1 in the fermentation culture medium, wherein the inoculation amount is 8.5%; introducing sterile air, and performing sterile culture under the condition that the pressure in the tank is 0.02 MPa;

s6, sterile culturing 3d, adding sterile Na into the fermentation medium₂SeO₃The solution was prepared by adding 4. mu.g of Na to 1mL of the fermentation medium₂SeO₃Continuing to ferment for 5 d;

s7, after fermentation, filtering the fermentation liquor by a 300-mesh filter screen, repeatedly washing the bacterial balls with distilled water, drying in a freeze dryer at-25 ℃, and weighing to obtain the selenium-rich mycelium of the Lyophyllum decastes.

Detection shows that after fermentation, the dry weight of the mycelium and the selenium content of the mycelium reach the maximum values of 8.154g/L and 234.785 mug/100 mL respectively, and a blank control group (without Na addition)₂SeO₃) The dry weight of the mycelium was only 4.758 g/L. The content of polysaccharide in the mycelium is 11.6mg/g, and the extraction amount of protein is 13.5 mg.

Performance test (the following test is only to adjust the selenium addition time in example 1, and no other parameters are adjusted)

1. In the invention, mycelium obtained at different selenium adding time in the fermentation process is subjected to in vitro cholate adsorption test, and the specific experimental result is shown in table 1.

TABLE 1

Note: comparing with blank group, it represents P < 0.05, and it represents P < 0.01

As can be seen from Table 1, the mycelium obtained by adding selenium-enriched mycelium on the third day has the best capacity of adsorbing cholate in vitro and has better cholate-reducing capacity.

2. The mycelium obtained in different selenium adding time in the fermentation process of the invention is subjected to in vitro cholesterol adsorption test, and the experimental results are shown in table 2.

TABLE 2

Note: comparing with blank group, it represents P < 0.05, and it represents P < 0.01

As can be seen from Table 2, the mycelium obtained by adding selenium at 3d has the highest cholesterol adsorption amount and the highest cholesterol-lowering ability.

3. Study on inhibitory activity of mycelium suspension on alpha-glucosidase

The mycelium suspensions obtained in different selenium adding time in the fermentation process are utilized to measure the inhibition activity of the mycelium suspensions on alpha-glucosidase, and the inhibition activity is shown in figure 1.

As can be seen from FIG. 1, the inhibition rate of alpha-glucosidase by the suspension of selenium-enriched Lyophyllum decastes on the third day is 65.03% at the highest, and the standard deviation is the smallest. The results show that the bacterial suspension of the selenium-enriched Lyophyllum decastes on the third day has better blood sugar reducing effect compared with the bacterial suspension of the selenium-enriched Lyophyllum decastes on other days. Selenium is added after the third day of fermentation, and the inhibitory activity of the mycelium to alpha-glucosidase is in a descending trend.

4. Study on inhibitory Activity of mycelia polysaccharide on alpha-glucosidase

The mycelium is obtained at different selenium adding time in the fermentation process, the polysaccharide is extracted from the mycelium, and the inhibitory activity of the mycelium polysaccharide (the concentration is 0.5mg/mL) to alpha-glucosidase is determined, as shown in figure 2.

As can be seen from FIG. 2, the highest inhibition rate of polysaccharide extracted from the selenium-added Lyophyllum decastes thallus on alpha-glucosidase on the third day is 69.97%. According to the comparison of the green brick tea crude polysaccharide on the inhibition of the activity of the alpha-glucosidase (shuting 2019), the inhibition rate of the green brick tea crude polysaccharide on the alpha-glucosidase is (57.59 +/-0.54)%, when the concentration of the green brick tea crude polysaccharide is 3.20 mg/mL. Compared with the polysaccharide extracted from selenium-rich Lyophyllum nuciferum mycelium, the polysaccharide has better alpha-glucosidase inhibitory activity. And the polysaccharide extracted from the selenium-rich Lyophyllum decastes mycelium has higher inhibition rate on alpha-glucosidase than the polysaccharide extracted from the selenium-free Lyophyllum decastes mycelium, which shows that the selenium polysaccharide obtained from the selenium-rich mycelium has better hypoglycemic effect. However, selenium is added after the third day of fermentation, and the polysaccharide of the mycelium has a descending trend on the inhibitory activity of alpha-glucosidase.

5. Study on inhibitory Activity of mycelial protein on alpha-glucosidase

Mycelium proteins are extracted from mycelium obtained at different selenium adding time in the fermentation process, and the inhibitory activity of the mycelium proteins (with the concentration of 1mg/mL) on alpha-glucosidase is determined, as shown in figure 3.

As can be seen from FIG. 3, the maximum inhibition rate of the protein extracted from the selenium-added mycelium on the third day of fermentation to alpha-glucosidase is 65.79%.

6. Research on hydroxyl radical scavenging ability of soluble polysaccharide in mycelium

Soluble polysaccharides are extracted from mycelia obtained at different selenium adding time in the fermentation process, and hydroxyl radical scavenging capacity is detected by using the soluble polysaccharides with different concentrations, as shown in figure 4 (sample 1 in figure 4 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 2 nd stage, sample 2 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 3 rd stage, sample 3 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 4 th stage, and sample 4 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 5 th stage).

As can be seen from FIG. 4, after selenium is added at different fermentation times, the soluble polysaccharide in the fermented mycelium has certain scavenging capacity for hydroxyl free radicals, and the scavenging rate gradually increases with the increasing concentration, thus showing an obvious dose-effect relationship.

Under the condition that the concentration of the soluble polysaccharide is 32 mug/mL, the removal rate of hydroxyl radicals of the selenium-rich soluble polysaccharide in the sample 1 is the maximum and reaches 35.37%; sample 4, minimum, 26.11%, indicates that the time of selenium addition during fermentation is related to its ability to scavenge hydroxyl radicals, and that the scavenging ability diminishes with time. The clearance rate of soluble polysaccharide in the blank mycelia without selenium enrichment is 19.28%, and the clearance is weaker than that of the selenium enrichment group. This is similar to the research of qiruiting on the antioxidant activity of selenium-rich lactococcus lactis and its selenium-rich polysaccharide, where mentioned: the scavenging ability of selenium-rich polysaccharide to hydroxyl radical increases with the increase of concentration, and is generally higher than that of selenium-free polysaccharide.

7. Research on DPPH free radical scavenging capacity of soluble polysaccharide in mycelium

Soluble polysaccharides are extracted from mycelia obtained at different selenium adding time in the fermentation process, and the DPPH free radical scavenging capacity is detected by using the soluble polysaccharides with different concentrations, as shown in figure 5 (sample 1 in figure 5 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 2 nd time, sample 2 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 3 rd time, sample 3 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 4 th time, and sample 4 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 5 th time).

As can be seen from FIG. 5, after selenium is added at different fermentation times, the soluble polysaccharides in the fermented mycelia have certain scavenging capacity for DPPH free radicals, and the scavenging rate gradually increases with the increasing concentration, thus showing an obvious dose-effect relationship.

Under the condition that the concentration of the soluble polysaccharide is 32 mug/mL, the clearance rate of the selenium-rich soluble polysaccharide in the sample 1 to DPPH free radicals is the maximum and reaches 52.29%; sample 4 minimum 33.95%; it is shown that the time of selenium addition during fermentation is related to its ability to scavenge DPPH free radicals, and that the scavenging ability diminishes with the delay of the time of addition. The clearance rate of soluble polysaccharide in the blank mycelia without selenium enrichment is 23.73%, and the clearance is weaker than that of the selenium enrichment group. Sample 2 has a greater clearance than sample 1 at a soluble polysaccharide concentration of 25.6 μ g/mL, consistent with the study of the antioxidant activity of Enterobacter Cloacae Z0206 bacteria extracellular selenium-rich polysaccharide by Xun Chun orchid et al, which indicates that the Enterobacter Cloacae Z0206 bacteria extracellular selenium-rich polysaccharide has a certain scavenging activity on DPPH free radicals in a certain concentration range.

8. Research on ABTS free radical scavenging capacity of soluble polysaccharide in mycelium

Soluble polysaccharides are extracted from mycelia obtained in different selenium adding time in the fermentation process, and ABTS free radical scavenging capacity is detected by using the soluble polysaccharides with different concentrations, as shown in figure 6 (sample 1 in figure 6 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation in the 2 nd time, sample 2 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation in the 3 rd time, sample 3 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation in the 4 th time, and sample 4 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation in the 5 th time).

As can be seen from FIG. 6, after selenium is added at different fermentation times, soluble polysaccharide in the fermented mycelia has a certain scavenging capacity for ABTS free radicals, and the scavenging rate gradually increases with the increasing concentration, thus showing an obvious dose-effect relationship.

Under the condition that the concentration of the soluble polysaccharide is 32 mug/mL, the clearance rate of the selenium-rich soluble polysaccharide in the sample 1 to ABTS free radicals is the maximum and reaches 44.88 percent; sample 4 was a minimum of 26.62%, indicating that the time of selenium addition during fermentation is related to its ability to scavenge ABTS free radicals, and that clearance diminishes with time. The clearance rate of soluble polysaccharide in the blank mycelium without selenium enrichment is 16.1%, and the clearance capability is weaker than that of the selenium enrichment group.

9. Research on reducing power of soluble polysaccharide in mycelium

Soluble polysaccharides are extracted from mycelia obtained at different selenium adding time in the fermentation process, and the reduction capability detection is carried out by using the soluble polysaccharides with different concentrations as shown in figure 7 (in figure 7, sample 1 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 2 nd stage, sample 2 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 3 rd stage, sample 3 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 4 th stage, and sample 4 is the soluble polysaccharides of mycelia obtained by selenium adding fermentation at the 5 th stage).

As can be seen from FIG. 7, after adding selenium for different fermentation times, the soluble polysaccharides in the fermented mycelia have a certain reducing power, and the clearance rate gradually increases with the increasing concentration, showing an obvious dose-effect relationship.

Under the condition that the concentration of the soluble polysaccharide is 32 mug/mL, the reducing power of the selenium-rich soluble polysaccharide in the sample 1 is the maximum, the absorbance value reaches 0.080 at the wavelength of 700nm, and the samples 2, 3 and 4 are 0065, 0.047 and 0.038 respectively; the time of adding selenium in the fermentation process is related to the reduction power, and the removal capacity is weakened along with the delay of the adding time. The absorbance value of soluble polysaccharide in the blank mycelia without selenium enrichment is 0.032 under the wavelength of 700nm, and the reduction capability is weaker than that of the blank mycelia without selenium enrichment.

Through research on the linear relation between the soluble polysaccharide and the antioxidant capacity in the early selenium-rich Lyophyllum decastes mycelium, the higher the content of the soluble polysaccharide is, the stronger the antioxidant capacity is, and the clearance rate is higher than that of selenium-rich mycelia.

The above embodiments are merely examples of the present invention, and it is obvious that the present invention is not limited to the above embodiments, but may be modified. All modifications directly or indirectly derivable by a person skilled in the art from the present disclosure are to be considered within the scope of the present invention.