WO2020098397A1

WO2020098397A1 - Fermented mycoplasm prepared from cordyceps militaris fermented tenebrio molitor and application thereof in preparation of agent for improving immunity

Info

Publication number: WO2020098397A1
Application number: PCT/CN2019/108391
Authority: WO
Inventors: 朴美子
Original assignee: 青岛海思达生物科技有限公司
Priority date: 2018-11-16
Filing date: 2019-09-27
Publication date: 2020-05-22
Also published as: CN109221927A

Abstract

Provided are a fermented mycoplasm prepared from cordyceps militaris fermented tenebrio molitor and an application thereof in the preparation of an agent for improving immunity. The influences of each single factor of culture medium ratio, material-water ratio, inoculation amount and fermentation time on the content of cordycepin in the cordyceps militaris solid fermentation tenebrio molitor mycoplasm are studied; and then on the basis of single-factor experiment results, an orthogonal optimization experiment using four factors and three levels is performed to obtain the optimum fermentation process, in which the mass ratio of tenebrio molitor degreased protein powder to rice flour is 6:4, the material-water ratio is 10:8, the inoculation amount is 25%, the fermentation time is 11 days in total, and the content of cordycepin in yeast is 8.63 mg/g. Novel application of the tenebrio molitor is exploited, the added value of the tenebrio molitor is improved, and meanwhile development of new cordyceps militaris products is also promoted.

Description

Fermented bacterium prepared by fermentation of Tenebrio molitor with Cordyceps militaris and its application in preparation for improving immunity

Technical field

The invention belongs to the technical field of food, and particularly relates to a fermented bacterium prepared by Cordyceps militaris fermentation of Tenebrio molitor and its application in the preparation of a preparation for improving immunity.

Background technique

Mealworms are rich in nutrients such as protein, unsaturated fatty acids, trace elements and amino acids. Currently, they are mainly used for making simple dishes and adding them to economic animal feed. Mealworms high-quality protein resources need to be further developed. Tenebrio molitor was originally regarded as a storage pest. With the increase of storage conditions and pest control, the number of tenebrio molitor in the storage environment has decreased, and it has entered the breeding stage. In recent years, the production of yellow mealworms in Shandong Province has increased rapidly, causing slow sales in many areas of Shandong. . At present, the development of mealworms is only limited to traditional methods, which can only be processed into feed, canned food, biscuits, etc., which greatly reduces the value of mealworms themselves, and due to the processing methods and sales difficulties, a large number of mealworm resources Was wasted.

Cordyceps militaris and Cordyceps sinensis belong to the same species and contain biologically active ingredients such as cordycepin, cordycepic acid, cordyceps polysaccharides, etc. It has antibacterial, anti-aging, anti-tumor, and immunity enhancement effects, but the current market products are expensive and have poor market promotion. People's economic level and health awareness continue to increase, and they pay more and more attention to the nutrition and health of food. Cordyceps militaris is comprehensive and can enhance the body's immunity. Wild Cordyceps militaris has problems such as scarcity of resources, long production cycle, degradation of strains and high price.

Summary of the invention

The purpose of the invention of the present invention is to provide a fermented bacterium prepared from Cordyceps militaris fermented by Tenebrio molitor and its application in preparing a preparation for improving immunity. The invention combines edible fungi and edible insects, utilizes the fermentation ability of Cordyceps militaris, and Tenebrio molitor provides the nutrients needed for its fermentation, and adopts Cordyceps militaris solid fermentation technology to increase the content of cordycepin in the fermentation to about 8mg / g; The use of mealworm larvae as the main fermentation substrate has opened up new applications of mealworms as an insect resource, which has increased the added value of mealworms, and also promoted the development of new products of Cordyceps militaris.

In order to achieve the above-mentioned object of the invention, the present invention is implemented using the following technical solutions:

The invention provides a fermented bacterium prepared by fermentation of Tenebrio molitor with Cordyceps militaris. The preparation method of the fermented bacterium includes the following steps:

(1) Inoculate Cordyceps militaris into the culture medium to prepare the seed culture solution;

(2) Weigh the mealworm powder and rice as a fermentation substrate, add water, and insert the seed solution of Cordyceps militaris for dark culture;

(3) After the dark culture is finished, it is cultured under light; the prepared culture medium is fermented fungus.

Further: the mass ratio of mealworm powder to rice in the step (2) is 5: 5-9: 1.

Further: the material-water ratio of the fermentation substrate and water in the step (2) is 10: 6 to 10:14.

Further: the inoculation amount in the step (2) is 10% -30%.

Further: in the step (2), the dark culture is 5 days.

Further: light culture in step (3) is 6 days.

Further: the best conditions are the ratio of 6: 4 of the powdered mealworm powder and rice flour, the ratio of feed water to 10: 8, the inoculation volume is 25%, and the fermentation time is 11 days.

The invention also provides the application of the fermented bacterium prepared by fermenting yellow mealworm by the Cordyceps militaris to prepare the preparation for improving immunity.

Further: when the concentration of the fermentation bacterium is gradually increased from 2 to 10 mg / mL, the scavenging rate of DPPH radicals, the scavenging ability and the reducing ability of hydroxyl radicals gradually increase.

Further: the fermentation bacterium is in the range of 20-100 mg / mL, and as the concentration increases, the clearance rate of superoxide anion free radicals increases.

Compared with the prior art, the advantages and technical effects of the present invention are: the present invention utilizes the high-quality animal protein resource of mealworm, and uses Cordyceps militaris to ferment the mealworm, which eases the protein resource crisis that China is about to face. The development of functional food provides a scientific basis. Combining Cordyceps militaris resources with high protein resources of Tenebrio molitor, through solid-state fermentation to obtain bacteria rich in higher cordycepin. It not only makes full use of insect protein resources, enhances the value of insect products, but also shortens the long production cycle, improves production efficiency, and reduces costs. The rigorous mouse experiments to study the antioxidant activity and immune activity of the fermented materials provide data references for the research and development of new insect functionalities.

BRIEF DESCRIPTION

Figure 1 is a graph of the experimental results of Cordycepin standard curve;

Fig. 2 is a graph showing the experimental results of the content of cordycepin in Tenebrio molitor mycoplasma with different ratios of mealworm powder and rice flour;

Figure 3 is a graph showing the effect of different feed-water ratios on the content of Cordycepin in the fermentation bacterium of Tenebrio molitor;

Figure 4 is a graph showing the effect of different inoculation doses on the content of cordycepin in the powdery mildew fungus;

Fig. 5 is an experimental result diagram of the influence of different fermentation time on the content of cordycepin in the fermentation bacterium of Tenebrio molitor;

6 is a graph of the experimental results of the scavenging ability of DPPH free radicals with different concentrations of fermenting fungi;

7 is a graph of the experimental results of the scavenging ability of different concentrations of fermenting bacterium on superoxide anion free radicals;

8 is a graph of the experimental results of the scavenging ability of different concentrations of fermenting bacterium on hydroxyl radicals;

Fig. 9 is a graph showing the experimental results of the reducing ability of different concentrations of fermented bacterium.

detailed description

The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

Example 1: Method for preparing fermentation bacterium by solid-state fermentation of Tenebrio molitor using Cordyceps militaris

1. Activation of Cordyceps militaris strains

Cordyceps militaris strains were inoculated on PDA solid plate medium, and cultured at 25 ° C for 6 days in the dark, until the mycelium was covered with slant medium and was white, dense and free of bacteria. Store in a refrigerator at 4 ° C, protected from light for use.

2. Preparation of liquid seeds of Cordyceps militaris

Fill 500mL Erlenmeyer flask with 200mL medium, sterilize at 121 ℃ for 30min, select the activated mycelium white and dense Cordyceps militaris slant medium, use the inoculation spatula to take 3 pieces of hyphae about 5mm ² and transfer to the liquid medium . Incubate at 130r / min and 23 ° C for 3 days under constant temperature and dark, so that the mycelium is evenly covered with the liquid medium for the inoculation of the solid medium of the mealworm powder.

3. Solid-state fermentation of Tenebrio molitor by Cordyceps militaris

Accurately weigh 20g of fermentation substrate (mixture of mealworm powder and rice) and measure 16mL of distilled water. Sterilize in a 500mL Erlenmeyer flask at 121 ° C for 30min. After the sterilization, put it in an ultra-clean workbench to cool, each bottle is connected with 25% The seed liquid of Cordyceps militaris and the glass rod were stirred evenly during the aseptic operation. Incubate in a 25 ° C constant temperature incubator in the dark for 6 days, shaking every 8 hours to make the solid medium evenly covered with small white particles. After the dark culture was completed, it was cultured under 300lx light for 4 days, shaking every 8 hours to prevent clumping, and the color of the medium changed from white to orange. Take out the solid medium overgrown with mycelium and freeze-dry it. The dried medium is the prepared fermented fungus, which is crushed by a grinder at 4 ° C and stored in the refrigerator.

Example 2: Determination of cordycepin content

The content of Cordycepin in the fermentation of Cordyceps militaris and Tenebrio molitor was determined by using Agilent 1260 high-performance liquid chromatography analyzer, C ₁₈ column (4.6mm × 150mm) mobile phase was acetonitrile: water = 1: 9, flow rate was 0.8mL / min, injection volume 5μL, column temperature 25 ℃, detection wavelength 260nm. The cordycepin standard was dissolved in 20% ethanol to prepare a 1 mg / mL solution, which was then diluted into 6.25, 12.5, 25, 50, and 100 μg / mL standard solutions, and each was prepared with a 0.22 μm microporous filter membrane. After sampling and measuring separately, a standard curve was prepared with cordycepin as the abscissa and the corresponding peak area as the ordinate, as shown in Figure 1. The standard curve equation of cordycepin is y = 22.384x-48.15, the correlation coefficient R ² = 0.9997, and the linear range is 6.25-100 μg / mL.

Weigh 5g of the mealworm fermented bacterium powder, add 125mL of 20% ethanol, weigh it, place it in an ultrasonic cleaner at 40 ° C, extract for 30min, cool to room temperature, and make up for the lost with 20% ethanol after weighing Weight, mix and filter to collect the extraction solution, filter with a membrane with a pore size of 0.22 μm, and store at 4 ° C for a short time to be tested.

Ferment three bottles for each experimental factor to reduce experimental errors. SPSS statistical software was used to analyze the results significantly, and the experimental data was used

To represent. According to the standard curve of cordycepin. Calculate the content of fermented cordycepin.

Example 3: Single factor test of solid-state fermentation of Tenebrio molitor by Cordyceps militaris

1. The effect of different ratios of mealworm meal powder and rice meal meal on the content of cordycepin in mealworm fungus

Mix the dried mealworm powder and rice flour in the ratio of 5: 5, 6: 4, 7: 3, 9: 1, add water according to the feed water ratio of 10: 8 (g / mL), and kill at 121 ℃ Bacteria for 30min, inoculation volume 15%, fermentation temperature 25 ℃, culture in dark environment for 5 days, light culture for 4 days, shaking every 8 hours to prevent agglomeration. The experimental results are shown in Figure 2.

As can be seen from FIG. 2, the content of cordycepin in the fermentation showed a trend of increasing first and then decreasing as the content of yellow mealworm increased. When the ratio of mealworm powder to rice powder reached 6: 4, the cordycepin content of Cordyceps militaris fermented mealworm fermentation reached a maximum of 8.56 mg / g; thereafter, with the increase of the mealworm content, the content of cordycepin in the fermentation was Decreasing state. The growth of Cordyceps militaris requires a nitrogen source and a carbon source. Mealworm meal and rice powder provide the corresponding nutritional factors. When the proper ratio is appropriate, it is just suitable for the growth of Cordyceps militaris mycelia and the production of Cordycepin.

2. The effect of feed-water ratio on the content of cordycepin in Tenebrio molitor

The powder medium of the mealworm defatted protein powder and the rice powder is prepared at 5: 5, mixed well, and the distilled water is added according to the feed liquid ratio of 10: 6, 10: 8, 10:10, 10:12, 10:14 (g / mL), Sterilize at 121 ℃ for 30min, inoculation volume 15%, fermentation temperature 25 ℃, first culture in dark environment for 5 days, light culture for 4 days, shake every 8 hours to prevent agglomeration The experimental results are shown in Figure 3.

It can be seen from Figure 3 that as the amount of water added during fermentation increases, the content of cordycepin in the fermentation of Tenebrio molitor Cordyceps militaris first increases and then gradually decreases. When the feed-liquid ratio is 10: 8, the content of cordycepin in the fermentation reaches the highest 8.32mg / g. When the feed water ratio is too large, the culture medium of mealworm powder and rice flour is dry, which is not suitable for fermentation of Cordyceps militaris; when the feed liquid ratio is too small, the water content of the culture medium becomes higher, the viscosity increases, it is easy to agglomerate, and the permeability becomes Poor, the oxygen supply of the mycelium is reduced, the mycelium only grows on the surface of the substrate, and cannot penetrate into the substrate, which eventually makes the culture medium fermentation incomplete, and even susceptible to mixed bacteria.

3. The effect of inoculum on the content of Cordycepin in Tenebrio molitor

The mealworm powder and rice flour were prepared with 5: 5 medium, mixed well, and the amount of water added was 10: 8 (g / mL) according to the feed water ratio, sterilized at 121 ° C for 30min, and the inoculation amount was 10%, 15%, 20%, 25%, 30%, fermentation temperature 25 ℃, first culture in dark environment for 5 days, light culture for 4 days, shaking every 8 hours to prevent agglomeration. The results are shown in Figure 4.

As can be seen from Fig. 4, the content of cordycepin in the fermentation of Cordyceps militaris, Tenebrio molitor, with the increase of the inoculation amount, showed a trend of gradually increasing first and then decreasing. When the inoculation amount was 20%, the content of cordycepin in the fermentation reached a maximum of 8.46 mg / g. In the experiment, the amount of inoculum directly affected the growth rate of Cordyceps militaris in the fermentation substrate. When the inoculation amount is too small, the substrate inoculation area is small, the mycelium grows slowly, and the substrate fermentation is not complete, which reduces the production efficiency. The formation of a bacterial film on the surface of the substrate limits the growth of the internal mycelium, and the increase in metabolic waste also affects the production of beneficial substances such as cordycepin.

4. The effect of fermentation time on the content of cordycepin in Tenebrio molitor

The mealworm powder and rice flour are prepared with a 5: 5 medium and mixed well. The amount of water added is 10: 8 (g / mL) according to the feed water ratio, sterilized at 121 ° C for 30 min, the inoculation amount is 15%, and the fermentation temperature is 25 ° C. Incubate for 5 days in dark environment, see light for 3, 4, 5, 6 and 7 days, shaking every 8 hours to prevent clumping.

It can be seen from Fig. 5 that as the fermentation time increases, the visible light fermentation time increases, and the content of cordycepin in the fermentation of Tenebrio molitor fermented by Cordyceps militaris first increases and then decreases. When the fermentation time is 9-10d (see light time 4-5d), the content of cordycepin in the fermentation reaches the highest 8.53mg / g. In the early stage of fermentation, with the increase of fermentation time, fermentation products such as cordycepin continue to accumulate; when the fermentation time is too long, the mycelium of Cordyceps militaris aging phenomenon, the accumulation rate of cordycepin decreases, and may also be accompanied by the decomposition of cordycepin, Therefore, the fermentation time should be strictly controlled.

Example 4: Orthogonal optimization experiment of solid-state fermentation of Tenebrio molitor by Cordyceps militaris

Based on the above single-factor experimental results, L ₉ (3 ⁴ ) orthogonal optimization experimental design was used, with the medium ratio, feed-water ratio, inoculation amount and fermentation time as influencing factors, and the content of cordycepin in the fungus as Indicators, study the relationship between these four factors, determine the best combination between them, determine the best fermentation conditions for Cordyceps militaris fermentation of Tenebrio molitor, the factor levels are shown in Table 1.

Table 1. Orthogonal experimental factors and levels

Based on the four factors of medium ratio, feed-water ratio, inoculation volume and fermentation time, based on single-factor experiment results, three levels adjacent to the optimal conditions were selected to determine the content of cordycepin after fermentation to further study Cordyceps militaris The best process for bacterial fermentation of Tenebrio molitor, the experimental results are shown in Table 2.

Table 2. Orthogonal optimization experiment results and statistical analysis

It can be seen from Table 2 that the range analysis of the content of cordycepin in the bacterium shows that R _A ＞ R _B ＞ R _D ＞ R _C , and it can be seen that the influence of different factors on the content of cordycepin in the fermentation of Tenebrio militaris by Cordyceps militaris The main order is: medium ratio> feed water ratio> fermentation time> inoculation amount. That is, the ratio of culture medium has the greatest effect on the content of cordycepin in the fermentation, followed by the feed-water ratio and fermentation time, and the inoculation amount has the smallest effect. And determine the best fermentation process combination is A ₂ B ₂ C ₃ D ₃ , that is, the best conditions for Cordyceps militaris fermentation of Tenebrio molitor are the ratio of the powdered meal powder and rice powder of Tenebrio molitor to 6: 4, the feed water ratio to 10: 8, the inoculation amount 25%, fermentation time is 11 days.

According to the best fermentation plan of the orthogonal experiment results, the ratio of the powdered mealworm powder to rice powder is 6: 4, the feed water ratio is 10: 8, the inoculation amount is 25%, and the fermentation time is 11 days. The content of cordycepin in the mass is 8.63mg / g, which is close to the theoretical expected result and has good repeatability, thus verifying the reliability of the orthogonal optimization experiment results.

Analysis of variance and significance test of orthogonal experiment results. The results are shown in Table 3.

Table 3. Analysis of variance of orthogonal optimization experiment results

When the experiment level is α = 0.10, the significance of each factor in the experiment is analyzed. It can be seen from Table 3 that the ratio of F medium> F critical value, that is, the ratio of factor A medium has a significant effect on the content of cordyceps in fungi; factor B feed water ratio, factor C inoculation amount and factor D fermentation time The effect of cordycepin content is not significant.

Example 5: Determination method of antioxidant capacity of Tenebrio molitor in vitro

1. Determination of DPPH free radical scavenging ability of Tenebrio molitor

With reference to the DPPH method revised by Brand-Williams et al., DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) is a stable organic radical, soluble in ethanol, and its solution is deep purple-red at 517nm. Characteristic absorption peak, when DPPH free radicals contact antioxidants, they combine with lone pairs of electrons, so that the deep purple red DPPH free radical solution is reduced to yellow, and the absorbance value at 517nm is reduced. The degree of reduction is quantitatively related to the number of electrons received. Therefore, DPPH free radical scavenging rate can be quantitatively analyzed with the change of absorbance value.

Accurately draw 0.5mL sample solutions of different concentrations (2, 4, 6, 8, 10mg / mL) into the test tube, add 2.4mL DPPH solution (prepared with absolute ethanol, concentration is 0.2mmol / L), and then add the extraction reagent to make Make up 4mL of each test tube, mix well, let stand for 30min in the dark, measure the absorbance at 517nm, use the leaching reagent instead of the sample solution as a control experiment, and use absolute ethanol instead of the DPPH solution as the sample reference experiment. Three parallel. The calculation formula of DPPH free radical scavenging ability is:

In the formula: A ₁ is the absorbance of the sample tube; A ₂ is the absorbance of the sample tube; A ₀ is the absorbance of the control tube.

The experimental results are shown in Figure 6. As the concentration of Tenebrio molitor fermented by Cordyceps militaris increased, its scavenging rate of DPPH free radicals also increased. The ability of 2 ～ 4mg / mL to scavenge DPPH free radicals is larger than that of 4 ～ 10mg / mL. It has been verified that after the concentration is greater than 10 mg / mL, the scavenging ability of the bacterium to DPPH free radicals gradually stabilizes and does not continue to increase. When the concentration of Cordyceps militaris fermented mealworm ferment was 10 mg / mL, its scavenging rate of DPPH free radicals reached 80.34%, and its IC ₅₀ was 5.08 mg / mL.

2. Determination of the ability of Tenebrio molitor to remove superoxide anion

The pyrogallol autoxidation method is used for the determination. The principle is that pyrogallol oxidizes rapidly under alkaline conditions to produce O ₂ ^- and dark intermediate products. The accumulation of colored intermediate products within 4 minutes has a linear relationship with the reaction time. At the same time, it has a maximum absorption peak at 316nm. The degree of autoxidation of pyrogallol and O ₂ ^- concentration in a positive correlation, an antioxidant and O ₂ ^- binding, inhibit the accumulation of the colored intermediate product, therefore, a measurement of antioxidant substances super quantified by UV spectrophotometer Scavenging effect of oxygen anion free radicals.

Set blank tube, empty loss tube, sample damaged tube and sample undamaged tube in the experiment. Pipette 5mL Tris-HCl buffer (pH = 8.2) of 1mmol / mL into each tube, and then add each in blank tube and empty loss tube. 50μL of leaching reagent, the sample damaged tube and the sample non-damaged tube were added with equal volumes of different concentrations (2, 4, 6, 8, 10mg / mL) of sample solution. After fully mixed, the sample was allowed to stand in a water bath at 25 ℃ for 20min. In a 25 ° C water bath, add 40 μL of 10 mmol / L hydrochloric acid to the blank tube and the sample tube, mix well, add 40 μL of 25 mmol / L pyrogallol to the empty tube, and count the sample for 30 seconds. Add an equal volume of pyrogallol to the tube. After 3 min, add 50 μL of 50 mg / mL DTT to the empty loss tube, add equal volume of DTT to the sample tube after 3 min and 30 s, and finally add 50 μLDTT to the blank tube and sample damage tube. Let stand at room temperature for 15min, and measure the absorbance at 316nm. Make three parallels per tube. The calculation formula for the superoxide anion radical scavenging rate is:

In the formula: A ₁ is the absorbance value of the empty loss tube; A ₂ is the absorbance value of the sample loss tube; A ₃ is the absorbance value of the sample undamaged tube.

The experimental results are shown in Figure 7. The strain of Tenebrio molitor fermented by Cordyceps militaris is in the range of 20-100 mg / mL, and as the concentration increases, the scavenging rate of superoxide anion radicals increases. It has been verified that when the concentration is greater than 100 mg / mL, the scavenging rate of superoxide anion free radicals by the mealy mildew fermented by Cordyceps militaris gradually stabilizes and does not continue to increase. When the concentration is 100 mg / mL, the scavenging rate of superoxide anion radical is 66.30%, and the IC ₅₀ is 86.73 mg / mL.

3. Determination of the ability of Tenebrio molitor to scavenge hydroxyl free radicals

Hydroxyl radical scavenging ability measurement principle is based in fenton ^{_{_{reaction: Fe 2+ + H 2 O 2}}} → Fe 3+ + · OH + OH -. In the system, Fe ²⁺ and o-phenanthroline form a red complex, which has a characteristic absorption peak at 536 nm. As the reaction progresses, the absorbance at the maximum absorption wavelength gradually decreases. The combination of antioxidant components and hydroxyl radicals can weaken the oxidation, so The corresponding change in absorbance decreases.

In the experiment, a blank tube, an undamaged tube, a damaged tube, a sample tube and a sample tube are set. 3.25 mL of a phosphate buffer solution with a pH of 7.4 is added to each tube, and 0.75 and 0.25 are added to the blank tube, the undamaged tube and the sample tube respectively. And 0.75mL of distilled water, then add 1mL of leaching reagent to the blank tube, undamaged tube, and damaged tube, add 1mL of sample solution to the sample reference tube and sample tube, mix well, and then undamaged tube, damaged tube, and sample tube Add 0.25mL 7.5mmol / L o-phenanthroline, mix well, then add 0.25mL 7.5mmol / L ferrous sulfate to the test tube with o-phenanthroline, mix quickly, and finally, add to the damaged tube and sample Add 0.25 mL of 1% H ₂ O ₂ to the tube, and measure the absorbance at 536 nm after a 90 ° C water bath for 90 min. Make three parallels per tube. The calculation formula of hydroxyl radical scavenging ability is:

In the formula: A ₁ is the absorbance of the undamaged tube; A ₂ is the absorbance of the damaged tube; A ₃ is the absorbance of the sample tube; A ₄ is the absorbance of the sample tube. The experimental results are shown in Figure 8. When the concentration of Tenebrio molitor fermented with Cordyceps militaris gradually increased from 2 to 10 mg / mL, its scavenging capacity for free hydroxyl groups would also increase. It can also be found that the concentration of bacteria in the concentration of 2 ~ 4mg / mL is larger than that of 4 ~ 10mg / mL. It has been verified that after the concentration is greater than 10 mg / mL, the scavenging ability of the bacterium to DPPH free radicals gradually stabilizes and does not continue to increase. When the concentration is 10 mg / mL, the clearance rate reaches 77.28%, and the IC ₅₀ is 6.61 mg / mL.

4. Determination of the reduction ability of the mealworm bacteria

Antioxidants, while resisting oxidation, release electrons to free radicals, thereby eliminating free radicals. There is a relationship between the reducing power of antioxidants and their antioxidant activity. The stronger the reducing power, the stronger the antioxidant activity. The antioxidant components in this experimental sample can reduce the ferric iron of potassium ferricyanide to ferric iron (potassium ferrocyanide). There is Prussian blue (Fe ₄ [Fe (CN) ₆ ] ₃ ) with maximum absorbance at 700 nm. Therefore, the absorbance value at 700 nm indirectly reflects the reduction capacity of antioxidants. The greater the absorbance, the stronger the reduction capacity.

Refer to Ding Yanru's method to improve the experiment, add diluted sample 1mL, 0.2mol / L (pH = 6.6) phosphate buffer solution 0.2mL, 1% potassium ferricyanide solution 0.5mL in a centrifuge tube, mix well, at 50 ℃ After 20 minutes of reaction, add 1mL of 10% trichloroacetic acid solution after rapid cooling, mix well, centrifuge at 5000r / min for 10min, and take out the supernatant; add 1.5mL of supernatant, 3mL of distilled water, and 1% ferric chloride solution to the test tube 0.2mL, mix well, let stand for 10min, use distilled water as a blank control, and measure the absorbance at 700nm.

The experimental results are shown in Figure 9. When the concentration is in the range of 2 ～ 10mg / mL, as the concentration of Cordyceps militaris fermented by Tenebrio molitor increases, its reducing ability also increases. The concentration of 2 ~ 6mg / mL changed significantly compared with 6 ~ 10mg / mL. When the concentration is 10mg / mL, the reducing ability reaches 0.967.

Example 6: Antioxidant activity of the fermentation bacterium of Tenebrio molitor fermented with Cordyceps militaris

1. Effect of Cordyceps militaris fermentation of Tenebrio molitor on the weight gain of mice

In the experiment, 8 healthy male Kunming SPF mice weighing 8 to 22g were selected and randomly divided into a blank group, a D-galactose-induced aging model group, a positive control group, a low-dose group, and a medium-dose group. There are 6 groups in the high-dose group, 10 in each group. The room temperature was controlled at 22-25 ° C, ventilation was good, and the mice were free to eat and drink, and were kept adaptively for one week. After adapting to the environment, the mice were tested according to Table 4, and food and water were administered every 12 hours. Among them, vitamin C solution is given by intragastric administration at 100 mg / (kg bw · d), and D-galactose solution is injected subcutaneously at 200 mg / (kg bw · d). Both intragastric and subcutaneous injections follow the principle of equal volume to ensure that each mouse Deal with the same damage. Mice can eat and drink freely. After 42 days of feeding, observe the physical signs and mental state of the mice every day, and weigh them once a week.

Table 4. Different treatments for each group of mice

Table 5. Effect of Tenebrio molitor on the body weight of mice

Note: The same superscript in the same column means that the difference between the values is not significant, and the different superscript means that the difference between the values is significant (p <0.05).

It can be seen from Table 5 that during the 42-day feeding period, the weight of the mice has increased. The average weight gain of the model group mice is the smallest, and the positive control group mice have the largest average weight gain. The body weight gain of the model group was significantly lower than that of the blank group. There was no significant difference in body weight gain between the model group and the low-dose and middle-dose groups, which was significantly higher than that of the high-dose group. Data analysis shows that the weight gain of mice is proportional to the amount of Tenebrio molitor fermentation bacteria added in the feed. There is no significant difference in the weight gain of mice in the high-dose group compared with the normal group and the positive control group, indicating that the fermentation bacteria of Tenebrio molitor can be improved The phenomenon of weight loss in aging mice.

Example 7: Determination of the antioxidant activity index of mouse plasma and liver

1. Mouse sacrifice and pretreatment of plasma and liver tissue samples

After 42 days of modeling and administration, the body weight was weighed, and blood was collected from the eyeballs in order and the cervical spine was dislocated and sacrificed. The complete liver tissue of the mice was dissected and removed. The whole blood of mice was centrifuged at 3000r / min for 10min, and the upper serum was collected and stored in a refrigerator at -80 ℃; the liver tissue was washed in physiological saline at 4 ℃, the excess water was absorbed by absorbent paper, and part of the liver tip tissue was cut out Add 9 times pre-chilled physiological saline to make 10% tissue homogenate, centrifuge at 3000r / min for 10min at low temperature, draw the supernatant, store it in a refrigerator at -80 ℃ after dispensing.

2. Determination of protein content in mouse liver tissue

The Coomassie Brilliant Blue method was used to determine the protein content of mouse liver tissues. The protein quantitative test kit was used. In accordance with the kit instructions, distilled water was used instead of the sample as a blank, and the standard solution instead of the sample as a control. The absorbance was measured at 595 nm, based on the absorbance. Calculate the protein content in the liver of mice.

3. Determination of malondialdehyde (MDA) in mouse plasma and liver

Malondialdehyde (MDA) is a degradation product of cell membranes attacked by oxygen free radicals. The polyunsaturated fatty acids in the membranes are lipid peroxidation reactions. The level of MDA content can reflect the degree of cell damage caused by oxygen radicals attack. MDA can react with thiobarbituric acid (TBA) to form a red substance with a characteristic absorption peak at 532nm. According to the instructions of the malondialdehyde (MDA) test box, the blood and liver tissue homogenates of the mice were taken to determine the MDA content.

4. Effect of fermentation of Tenebrio molitor by Cordyceps militaris on the content of malondialdehyde in the plasma and liver of mice

Table 6. Effect of fermented bacterial biomass on the content of malondialdehyde in the plasma and liver of mice

Malondialdehyde (MDA) is an important indicator of aging evaluation. The higher the MDA content, the greater the degree of oxidative damage to the body. It can be seen from Table 6 that compared with the blank group, the MDA content in the plasma and liver of the model group is higher than that in the blank group and the difference is significant, indicating that the experiment successfully established a model of sub-acute aging caused by D-galactose. With the increase of the dosage of Tenebrio molitor fermentation bacteria, the content of MDA in plasma and liver gradually decreased. Plasma and liver MDA in the middle-dose group, there is no significant difference from the blank group, indicating that the normal group level. At the same time, compared with the positive control group, the MDA content of the medium and high dose groups was not significantly different, indicating that the medium- and high-dose groups of mealworm fermented fungi have good antioxidant capacity, so that the aging model mice basically returned to normal levels.

5. Determination of hydroxyl radical scavenging ability in mouse plasma and liver

As a part of oxygen free radicals, hydroxyl free radicals are the most reactive oxygen free radicals, second only to F strong oxidants, and are the intermediate medium for the body's oxidation reaction. When the body's aging antioxidant capacity is reduced or the cells are damaged, the number of hydroxyl radicals in the body will increase relatively. In the Fenton reaction system, the amount of H ₂ O ₂ is proportional to the amount of · OH. When an electron acceptor is given, the color is developed with the griess solution to produce a red substance. The color is proportional to the amount of · OH. Using 37 ° C reaction for 1 min, the concentration of H ₂ O _{2 in the} system is reduced by 1 mmol / L as a unit for scavenging hydroxyl radicals. According to the hydroxyl radical measurement kit, the serum and liver tissues in mice are tested and calculated to eliminate hydroxyl radicals ability.

Table 7. Effect of Tenebrio molitor fermentation bacterium on the ability of mouse plasma and liver to scavenge hydroxyl radicals

It can be seen from Table 7 that compared with the blank group, the model group and the liver have low hydroxyl radical scavenging ability and significant difference, indicating that the D-galactose-induced aging model was successfully modeled. With the increase of the dosage of Tenebrio molitor fermentation bacteria, the plasma and liver scavenging capacity of hydroxyl radicals also gradually increased. There was no significant difference between the low-dose group and the model group, but the medium-dose group and the high-dose group were significantly different from the model group, and there was no significant difference compared with the blank group, indicating that the mice in the middle-dose and high-dose groups have recovered their ability to freely remove hydroxyl groups. Level.

6. Determination of catalase (CAT) activity in mouse plasma and liver

Peroxidase (CAT) is an important antioxidant enzyme of the body, it can decompose the hydrogen peroxide produced by the body's metabolism into water and oxygen, protect the body from the damage of organic hydrogen peroxide, delay the aging of cells, its activity Can reflect the body's antioxidant capacity. Ammonium molybdate can quickly terminate the reaction of catalase to decompose hydrogen peroxide, and react with the remaining hydrogen peroxide to produce a pale yellow complex. Measure its absorbance at 405nm, according to the peroxidase (CAT) test box Instructions, and calculate the activity of CAT in mouse serum and liver tissue.

Table 8. Effect of Tenebrio molitor fermentation bacterium on catalase activity in mouse plasma and liver

As can be seen from Table 8, compared with the blank group, the activity of catalase in the plasma and liver of the D-galactose-induced aging model group was significantly lower, indicating that the D-galactose-induced aging model has been established. In the low-, medium-, and high-dose groups, as the dose increased, the activity of catalase in the plasma and liver of mice gradually increased. There was no significant difference between the CAT in the low-dose group and the model group, but the difference was significant in the middle and high-dose groups, and the CAT content in the liver tissue was low, the medium- and high-dose groups were significantly different from the model group, and the mouse plasma in the high-dose group Compared with the CAT activity in the liver and the blank group, there was no significant difference, indicating that when the dose reached the level of the high-dose group, the CAT activity in the plasma and liver of the aging mice had returned to normal levels.

7. Determination of total superoxide dismutase (T-SOD) activity in mouse plasma and liver

Superoxide dismutase (SOD) has a special inhibitory effect on superoxide anion radicals (O _2- ·), causing it to disproportionate and generate O ₂ and H ₂ O ₂ . The level of superoxide dismutase in the body is negatively correlated with the content of free radicals, which can reflect the body's antioxidant capacity. In this experiment, the reaction of xanthine and xanthine oxidase was used to produce superoxide anion free radicals, and then hydroxylamine was oxidized to generate nitrite. A color reagent was added to show a magenta color, and the absorbance at 550 nm was measured. If the system contains SOD, it will have a scavenging effect on superoxide anion free radicals, so that the content of nitrite is reduced and the absorbance value becomes smaller. According to the total superoxide dismutase (T-SOD) test kit instructions to measure and calculate the activity of T-SOD in mouse serum and liver tissue.

Table 9. Effect of Tenebrio molitor fermentation bacterium on the activity of total superoxide dismutase in mouse plasma and liver

Superoxide dismutase (SOD) is an important antioxidant enzyme in the body. It has the ability to specifically inhibit superoxide anion free radicals. It can be seen from Table 9 that the T-SOD activity of the model group is lower than that of the blank group and the difference is significant, indicating that the aging model induced by D-galactose is established. The activity of T-SOD in the low-, medium-, and high-dose groups increased with the increase of the dose of Tenebrio molitor fermentation bacterium. The difference between the low-dose group and the blank group was significant, but at the medium- and high-dose groups, the T-SOD activity and the blank group had no effect. The obvious difference indicates that the T-SOD activity of mice in the middle-dose group has recovered to the level of healthy mice.

8. Determination of the activity of glutathione peroxidase (GSH-Px) in mouse plasma and liver

Glutathione peroxidase (GSH-Px) is also an important endogenous antioxidant enzyme in the body, it can catalyze the reaction of reduced GSH and H ₂ O ₂ to produce water and oxidized glutathione (GSSG ), Play the role of scavenging free radical protection film structure. The rate of enzymatic reaction reflects the activity of GSH-Px. The activity of GSH-Px is calculated by measuring the consumption of GSH. Glutathione reacts with dithiodinitrobenzoic acid to form 5-thiodinitrobenzoic acid anion The product was yellow, and the absorbance at 412nm was measured. The GSH-Px test kit was used to determine the activity of GSH-Px in the plasma and liver of mice.

Table 10. Effect of Tenebrio molitor fermentation bacterium on the activity of total glutathione peroxidase in mouse plasma and liver

Glutathione peroxidase (GSH-Px) is also an important antioxidant enzyme in the body, and its vitality can be used to measure the body's antioxidant capacity. From Table 10, the GSH-Px activity of the model group was significantly lower than that of the blank group, indicating that the D-galactose-induced aging model was successfully modeled. The activity of GSH-Px in the plasma of low, medium and high dose mice was significantly higher than that of the model group, and the activity of GSH-Px in the middle dose group was not significantly different from that of the blank group, indicating that the activity of GSH-Px in the plasma of the middle dose group had Normal mice levels; while the activity of GSH-Px in the liver, the mice in the middle and high doses were significantly higher than the model group, and the activity of GSH-Px in the high dose group was not significantly different from that in the blank group, indicating that the mice in the liver of the high dose group GSH-Px vitality has returned to normal mouse levels.

Example 8: Study on immune activity of Tenebrio molitor fermentation by Cordyceps militaris

In this experiment, the D-galactose-induced aging model was used to study the spleen index, thymus index, serum IgG, IgM, interleukin-4 and interferon-γ content as indicators to study -Impact of immune function of galactose-induced aging mice, explore its mechanism of delaying immune aging.

1. Mice grouping and feeding

Sixty eight-week-old 18-22g healthy male Kunming SPF grade mice were adaptively reared for one week and randomly divided into a blank group, a model group, a vitamin C positive control group, a low-dose group, a medium-dose group, and a high-dose group. 6 groups with 10 animals in each group. The room temperature is stable at 22 ~ 25 ℃, with proper ventilation, the mice can freely eat and drink water, and keep them adaptively for one week. After adapting to the environment, the mice were tested according to Table 11, and food and water were administered every 12 hours. Among them, vitamin C solution is given by intragastric administration at 100mg / (kg bw · d), and D-galactose solution is injected subcutaneously at 200mg / (kg bw · d). Both intragastric and subcutaneous injections follow the principle of equal volume to ensure that each mouse Deal with the same damage. Mice can eat and drink freely. After 42 days of feeding, the signs and mental state of the mice were observed and recorded every day.

Table 11 Different treatments for each group of mice

2. Mice blood collection and dissection

After 12 hours of the last meal, blood was taken from the eyeballs, and then the cervical spine was dislocated. The state of each organ was dissected and separated into complete liver, thymus, and spleen, rinsed in sterile saline, and the excess water was absorbed by absorbent paper. , Accurately weighed, stored in refrigerator at -80 ℃. The whole blood of mice was centrifuged at 3000r / min for 10min, and the upper serum was collected and stored in a refrigerator at -80 ℃ after aliquoting.

3. Determination of the index of the effect of the fermentation of Tenebrio molitor by Cordyceps militaris on the immune function of mice

The spleen is the body's largest peripheral immune organ, and the thymus is an important central immune organ. The immune organ index reflects the development and immune function of the immune organ.

The thymus and spleen are stripped off, washed with sterile saline, and the excess water is absorbed with absorbent paper. The electronic analysis balance accurately weighs the weight. The ratio of organ weight (mg) to mouse body weight (g) represents the corresponding organ index.

The effect of the fermentation of Tenebrio molitor by Cordyceps militaris on the immune organ index of mice is shown in Table 12.

Table 12. Effect of Tenebrio molitor fermentation bacterium on the immune organ index of mice

It can be seen from Table 12 that the immune organ index of the model group is significantly lower than that of the blank group, indicating that the D-galactose-induced aging model is successfully modeled. Compared with the model group, the spleen index and thymus index of the low, medium and high dose groups were significantly increased. The thymus index and spleen index of the low-dose group were not significantly different from the blank group, indicating that the weight of the spleen and thymus reached the level of normal mice in the low-dose group. With the increase in the dosage of the mealworm ferment, there was no significant difference between the high-dose group and the positive group, indicating that the high-dose group of the mealworm ferment has reached the level of mice in the positive group, but the thymus index between the low, medium and high dose groups The difference was significant, but there was no significant difference in spleen index.

4. Determination of the effect of the fermentation of Tenebrio molitor by Cordyceps militaris on the content of immunoglobulin G (IgG) in mice plasma

Immunoglobulin G (IgG) accounts for 75% of the total plasma immunoglobulin, mainly synthesized by plasma cells, can specifically bind to antigens, and is the main antibody produced in the immune response again. The kit adopts the double antibody one-step sandwich enzyme-linked immunosorbent assay. To the coated microwells pre-coated with immunoglobulin G (IgG) antibodies, samples, standards, and labeled detection antibodies are added in sequence. After 37 ° C Incubate at a constant temperature and wash thoroughly. A color reaction occurs with the substrate TMB, the substrate turns blue under the action of peroxidase, then reacts with an acid and eventually turns yellow. The color depth is positively correlated with the IgG content in the sample. Measure the OD value at 450nm with a microplate reader to calculate the sample concentration.

As can be seen from Table 13, compared with the blank group, the IgG content in the plasma of the model group mice was significantly lower, indicating that the D-galactose-induced aging model was successfully modeled. Compared with the model group, the plasma levels of IgG in the low, medium and high dose groups were significantly increased. Compared with the blank group, the fermented bacteria of Tenebrio molitor has a significant difference compared with the blank group, and no significant difference compared with the positive group. The high dose group has a significant difference compared with the positive control group, indicating that the low and medium dose groups have reached the level of mice in the positive group The effect of the high-dose group was higher than that of the positive control group, and the difference between the low-, medium-, and high-dose groups was significant.

Table 13. Effect of Tenebrio molitor fermentation bacterium on IgG content in mouse plasma

5. Determination of the effect of fermentation of Tenebrio molitor by Cordyceps militaris on the content of immunoglobulin M (IgM) in mice plasma

Immunoglobulin M is the body's first antibody against antigens, and it is also the immunoglobulin with the largest molecular weight. The level of IgM in serum can reflect the body's immune function. The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay. To the coated microwells pre-coated with immunoglobulin M (IgM) antibodies, samples, standards, and labeled detection antibodies are added in sequence. After 37 ° C Incubate at a constant temperature and wash thoroughly. A color reaction occurs with the substrate TMB, the substrate turns blue under the action of peroxidase, then reacts with an acid and eventually turns yellow. The color depth is positively correlated with the IgM content in the sample. Measure the OD value at 450nm with a microplate reader to calculate the sample concentration. The results are shown in Table 14.

Table 14. Effect of fermented bacterium on IgM content in mouse plasma

It can be seen from Table 14 that the model group and the blank group have significant differences in immunoglobulin content, indicating that the D-galactose-induced aging model was successfully modeled. With the increase in the dosage of Tenebrio molitor fermentation bacterium, the content of immunoglobulin M in the low, medium and high dose groups increased significantly. There was no significant difference between the positive control group and the low and medium dose groups, and there was a significant difference from the high dose group, indicating that the low and medium dose groups had reached the level of the positive control group. The effect of the high dose group exceeded the level of the positive control group, but the low, There was no significant difference between the middle doses, and the difference between the low and middle dose groups and the high dose group was significant. The experimental results show that the content of immunoglobulin M in the serum of mice increases with the increase of dose, indicating that the fermentation of Tenebrio molitor with Cordyceps militaris can increase the content of IgM in the serum of aging model mice.

6. Determination of the effect of the fermentation of Tenebrio molitor by Cordyceps militaris on the content of interleukin 4 (IL-4) in the plasma of mice

Interleukin 4 (IL-4) is the main cytokine secreted by type II helper T cells (Th2) and plays an important role in regulating humoral immunity and adaptive immunity. The kit adopts double antibody one-step sandwich enzyme-linked immunosorbent assay. To the pre-coated microwells coated with IL-4 antibody, samples, standards, and labeled detection antibodies are added in sequence, followed by constant temperature incubation at 37 ° C. Wash thoroughly. A color reaction occurs with the substrate TMB, the substrate turns blue under the action of peroxidase, then reacts with an acid and eventually turns yellow. The color depth is positively correlated with the IL-4 content in the sample. Measure the OD value at 450nm with a microplate reader to calculate the sample concentration.

Table 15. Effect of fermented bacteria on interleukin 4 (IL-4) content in mouse plasma

As can be seen from Table 15, compared with the blank group, the content of interleukin 4 (IL-4) in the plasma of the model group mice was significantly lower, and the difference was significant, indicating that the D-galactose-induced aging model was successfully modeled. Compared with the model group, the levels of interleukin 4 (IL-4) in the plasma of mice in the low, medium and high dose groups increased significantly. There was no significant difference between the low-dose group and the positive control group, the difference between the medium- and high-dose groups and the positive control group was significant, and the difference between the low-, medium- and high-dose groups was significant, indicating that with the increase in the dose of Tenebrio molitor fermentation bacteria, The content of IL-4 in rat plasma increased significantly. The content of IL-4 in the plasma of the low-dose group returned to the level of the control group, and the content of IL-4 in the plasma of the middle- and high-dose group exceeded the level of the positive control group.

7. Determination of the effect of the fermentation of Tenebrio molitor by Cordyceps militaris on the content of IFN-γ in the plasma of mice

Interferon is divided into three types of α, β and γ, of which interferon-γ is mainly involved in immune regulation and antiviral and antitumor effects. The mouse IFN-γ ELISA detection kit was selected. The kit uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay. To the coated microwells pre-coated with IFN-γ antibody, samples, standards, and labeled detection antibodies are added in sequence, followed by constant temperature incubation at 37 ° C. Wash thoroughly. A color reaction occurs with the substrate TMB, the substrate turns blue under the action of peroxidase, then reacts with an acid and eventually turns yellow. The color depth is positively correlated with the IFN-γ content in the sample. Measure the OD value at 450nm with a microplate reader to calculate the sample concentration.

Table 16. Effect of fermented bacterium on IFN-γ content in mouse plasma

It can be seen from Table 16 that the content of interferon-γ in the serum of the model group was lower than that of the blank group, and the difference was obvious, indicating that the D-galactose-induced aging model was successfully modeled. Compared with the model group, the levels of interferon-γ in the serum of mice in the low, medium and high dose groups were obviously on the rise, indicating that the content of interferon-γ in the serum of the aging model mice was different from the dose of Cordyceps militaris. Increase and increase. There was no significant difference between the low- and medium-dose fermentative bacteria of the mealworm mealworm and the positive control group. The difference was significant between the high-dose group and the low-, medium-, and high-dose groups. Experiments have shown that the fermented bacteria of Tenebrio molitor can increase the content of interferon-γ in the serum of aging model mice, and it is directly proportional to the increase of the dosage of fermented bacteria of Tenebrio molitor.

The experimental results of this example show that compared with the aging model group, the low, medium, and high dose groups of Tenebrio molitor can significantly increase the spleen index and thymus index of aging mice (p <0.05), and significantly increase the serum of mice The content of IgG and IgM and the content of IL-4 and IFN-γ (p <0.05), and there is a dose dependence, the low-dose group and the high-dose group have a significant difference (p <0.05), the high-dose group of mice The spleen index, thymus index, serum IgG and IgM content and IL-4 and IFN-γ content were significantly greater than the positive control group (p <0.05). Therefore, fermentation of Tenebrio molitor fermented with Cordyceps militaris can improve the immune function of aging model mice, regulate the content of cytokines in serum, and maintain the immune balance of the body.

The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still The recorded technical solutions are modified, or some of the technical features are equivalently replaced; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed by the present invention.

Claims

A fermented bacterium prepared by fermentation of Tenebrio molitor with Cordyceps militaris, characterized in that the preparation method of the fermented bacterium includes the following steps:

(1) Inoculate Cordyceps militaris into the culture medium to prepare the seed culture solution;

(2) Weigh the mealworm powder and rice as a fermentation substrate, add water, and insert the seed solution of Cordyceps militaris for dark culture;

(3) After the dark culture is finished, it is cultured under light; the prepared culture medium is fermented fungus.
The fermentation of Tenebrio molitor mycelia of Cordyceps militaris according to claim 1, characterized in that: the mass ratio of Tenebrio molitor powder to rice in the step (2) is 5: 5-9: 1.
The fermentation of Tenebrio molitor mycelia of Cordyceps militaris according to claim 1, characterized in that: the material-to-water ratio of the fermentation substrate and water in the step (2) is 10: 6-10: 14.
The fermentation of Tenebrio molitor fungus by Cordyceps militaris according to claim 1, characterized in that the inoculation amount in the step (2) is 10% to 30%.
The fermentation of Tenebrio molitor mycoplasma by Cordyceps militaris according to claim 1, characterized in that: in the step (2), the dark culture is 5 days.
The fermentation of Tenebrio molitor mycelia of Cordyceps militaris according to claim 1, characterized in that in step (3), the light culture is 6 days.
The fermentation of Tenebrio molitor fungus by Cordyceps militaris according to claim 1, characterized in that: the optimal conditions are the ratio of 6: 4 of mealworm powder and rice flour, the ratio of feed water to 10: 8, the inoculation amount is 25% The fermentation time is 11 days.
Use of the fermented bacterium prepared by fermentation of Tenebrio molitor Cordyceps militaris according to claim 1 in the preparation of a preparation for improving immunity.
The application according to claim 8, characterized in that: when the concentration of the fermentation bacterium is gradually increased from 2 to 10 mg / mL, the scavenging rate to DPPH radicals, the scavenging ability to hydroxyl radicals and the reducing ability to gradual increase.
The use according to claim 8, characterized in that the fermentation bacterium is in the range of 20-100 mg / mL, and as the concentration increases, the scavenging rate of superoxide anion radicals increases.