Liquid fermentation process of armillaria mellea

Abstract

The present application relates to a kind of Armillaria mellea liquid fermentation process, comprising the following steps: step one: Armillaria mellea pure living mycelium is cultivated by PDA culture medium;Step six: when fermentation to stationary phase, fermentation broth is divided into 3 identical volume fermentation sub-tank, different fresh culture medium is supplemented, and continue fermentation culture;Step seven: Armillaria mellea liquid in different fermentation sub-tank is collected to total fermentation tank and fresh culture medium is supplemented to carry out slow stirring depth fermentation;1.the present application describes the best culture medium component of Armillaria mellea liquid fermentation process and the best culture condition, greatly improve the biomass of Armillaria mellea liquid fermentation;2.the present application is combined with segmentation fermentation process, and fermentation culture time is shortened, and efficiency is high, and process is simple and stable, suitable for batch production, and cost is low;3.newly added fusion step, form multistage fermentation system, improve strain amplification efficiency;4.Armillaria mellea liquid fermentation, mycelium can be made into Armillaria mellea tablet and related product, with broad market prospect and good economic value.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a liquid fermentation process for Armillaria mellea. Background Technology

[0002] Armillaria mellea, belonging to the order Agaricales and genus Armillaria, is also commonly known as hazel mushroom, honey mushroom, and oak mushroom. It is an aerobic facultative parasitic fungus and an important associated fungus of Gastrodia elata. Depending on its growth and development stages, Armillaria mellea can be divided into mycelium and fruiting body. The mycelium, as the vegetative organ of Armillaria mellea, exists in two forms: hyphae or mycelial cords. The fruiting body is the mature mushroom. Liquid submerged fermentation technology for Armillaria mellea can obtain large quantities of mycelium and metabolites. Liquid submerged fermentation technology is the main way to achieve the industrial production of medicinal fungi. Compared with traditional solid-state fermentation, liquid submerged fermentation technology has advantages such as continuous production, shorter fermentation cycle, and the production of abundant physiologically active substances.

[0003] Traditional Armillaria mellea fermentation processes generally suffer from low raw material utilization and low production capacity, which significantly increases production costs. Summary of the Invention

[0004] (a) Technical problems to be solved To address the aforementioned problems in the prior art, this invention provides a liquid fermentation process for Armillaria mellea, which can improve the low utilization rate of raw materials, increase production capacity, and reduce production costs.

[0005] (II) Technical Solution To achieve the above objectives, the main technical solutions adopted by the present invention include: A liquid fermentation process for Armillaria mellea includes the following steps: Step 1: Cultivate pure live mycelia of Armillaria mellea using PDA medium; Step 2: Primary shake flask seed culture: Take mycelia and inoculate them into a shake flask containing liquid culture medium for shaking culture; Step 3: Secondary shake-flask seed culture: Transfer to fresh liquid culture medium at an inoculum rate of 10–18% and culture with shaking. Step 4: Three-stage shake flask seed culture: Take the seeds from the two-stage shake flasks and stir them for culture to establish a stable seed system; Step 5: Transfer the mature seeds into a fermentation tank and add fermentation medium for initial fermentation culture; Step 6: When fermentation reaches the stable phase, divide the fermentation broth into 3 fermentation tanks of equal volume, add different fresh culture media, and continue fermentation. Step 7: Collect the Armillaria mellea liquid from the different fermentation tanks into the main fermentation tank and add fresh culture medium for slow, deep fermentation with stirring. Step 8: Concentrate, dry, and pulverize the Armillaria mellea liquid after deep fermentation in Step 7.

[0006] Furthermore, the liquid culture medium in steps two, three, and four is: 5% wheat bran juice, 2% glucose, 0.5% peptone, 0.2% yeast powder, 0.05% magnesium sulfate, 0.001% vitamin B1, with the remainder being drinking water, pH 5.5–6.0, 24–26℃, and culture time of 36h–48h.

[0007] Furthermore, the fermentation culture medium in step five is: 5-8% corn flour, 2-4% soybean meal, 3% silkworm pupa powder, 0.003% VB1, 0.15-0.2% potassium dihydrogen phosphate, 0.075-0.1% magnesium sulfate heptahydrate, 0.05-0.08% zinc sulfate, with the remainder being drinking water; the pH is 5.5-6.0, the fermentation conditions are 25-28℃, the fermentation time is 36-50 h, 200 r / min, and the aeration ratio is 1:(0.5-1.0)V / V.min.

[0008] Furthermore, 0.05% saccharifying enzyme and 0.02% acidic protease were added during the fermentation process.

[0009] Furthermore, the three fermentation tanks of equal volume are designated as the first fermentation tank, the second fermentation tank, and the third fermentation tank.

[0010] Furthermore, the culture medium in the first fermentation tank consists of: 5% soybean meal powder treatment liquid, 2% glucose, 4% sucrose, 0.05% defoamer, and the remainder is drinking water; the fermentation temperature is 24~28℃, the fermentation time is 68~82h, the fermentation tank pressure is 0.04~0.06MPa, and the aeration ratio is 1:(0.6-1.0)V / V.min.

[0011] Furthermore, the culture medium in the second fermentation tank consists of: 5% corn flour, 2% glucose, 4% sucrose, 0.05% defoamer, and the remainder is drinking water; the fermentation temperature is 24~28℃, the fermentation time is 68~82h, the fermentation tank pressure is 0.04~0.06MPa, and the aeration ratio is 1:(0.6-1.0)V / V.min.

[0012] Furthermore, the culture medium in the third fermentation tank consists of: 5% wheat bran, 2% glucose, 4% sucrose, 0.05% defoamer, and the remainder is drinking water; the fermentation temperature is 24~28℃, the fermentation time is 68~82h, the fermentation tank pressure is 0.04~0.06MPa, and the aeration ratio is 1:(0.6-1.0)V / V.min.

[0013] Furthermore, the culture medium in the total fermentation tank in step seven is: 8% potato, 2-4% soybean meal, 3% silkworm pupa powder, 0.003% VB1, 0.15-0.2% potassium dihydrogen phosphate, 0.075-0.1% magnesium sulfate heptahydrate, 0.05-0.08% zinc sulfate, and the remainder is drinking water; the pH is 5.5-6.0, the fermentation conditions are 25-28℃, the fermentation time is 32-46 h, 200 r / min, and the aeration ratio is 1:(0.5-1.0)V / V.min.

[0014] (III) Beneficial Effects

[0015] The beneficial effects of this invention are:

[0016] 1. This invention describes the optimal culture medium composition and optimal culture conditions for the liquid fermentation process of Armillaria mellea, which greatly improves the biomass of Armillaria mellea during liquid fermentation;

[0017] 2. This invention combines a segmented fermentation process, which shortens the fermentation time, is highly efficient, has a simple and stable process, is suitable for mass production, and has low cost;

[0018] 3. A new fusion step is added to form a multi-stage fermentation system, improving the efficiency of strain amplification;

[0019] 4. The liquid fermentation of Armillaria mellea mycelium can be made into Armillaria mellea tablets and related products, which have broad market prospects and good economic value. Attached Figure Description

[0020] Figure 1 This is a flow chart of the liquid fermentation process of Armillaria mellea according to an embodiment of the present invention; Detailed Implementation

[0021] Example 1

[0022] Please refer to Figure 1 A liquid fermentation process for Armillaria mellea includes the following steps: Step 1: Cultivate pure live mycelia of Armillaria mellea using PDA medium; Step 2: Primary shake flask seed culture: Take mycelia and inoculate them into a shake flask containing liquid culture medium for shaking culture; Step 3: Secondary shake-flask seed culture: Transfer to fresh liquid culture medium at an inoculum rate of 10–18% and culture with shaking. Step 4: Three-stage shake flask seed culture: Take the seeds from the two-stage shake flasks and stir them for culture to establish a stable seed system; Step 5: Transfer the mature seeds into a fermentation tank and add fermentation medium for initial fermentation culture; Step 6: When fermentation reaches the stable phase, divide the fermentation broth into 3 fermentation tanks of equal volume, add different fresh culture media, and continue fermentation. Step 7: Collect the Armillaria mellea liquid from the different fermentation tanks into the main fermentation tank and add fresh culture medium for slow, deep fermentation with stirring. Step 8: Concentrate, dry, and pulverize the Armillaria mellea liquid after deep fermentation in Step 7; The liquid culture medium in steps two, three, and four is: 5% wheat bran juice, 2% glucose, 0.5% peptone, 0.2% yeast powder, 0.05% magnesium sulfate, 0.001% vitamin B1, with the remainder being drinking water, pH 5.5–6.0, 24–26℃, and incubation time of 36h–48h. The fermentation medium in step five is as follows: corn flour 5-8%, soybean meal 2-4%, silkworm pupa powder 3%, VB1 0.003%, potassium dihydrogen phosphate 0.15-0.2%, magnesium sulfate heptahydrate 0.075-0.1%, zinc sulfate 0.05-0.08%, with the remainder being drinking water; pH 5.5-6.0; fermentation conditions 25-28℃; fermentation time 36-50 h; 200 r / min; aeration ratio 1:(0.5-1.0) V / V.min. During fermentation, 0.05% saccharifying enzyme and 0.02% acidic protease were added. The three fermentation tanks of the same volume are designated as the first fermentation tank, the second fermentation tank, and the third fermentation tank. The culture medium in the first fermentation tank consists of: 5% soybean meal powder treatment liquid, 2% glucose, 4% sucrose, 0.05% defoamer, and the remainder is drinking water; the fermentation temperature is 24~28℃, the fermentation time is 68~82h, the fermentation tank pressure is 0.04~0.06MPa, and the aeration ratio is 1:(0.6-1.0)V / V.min; The culture medium in the second fermentation tank consists of: 5% corn flour, 2% glucose, 4% sucrose, 0.05% defoamer, and the remainder is drinking water; the fermentation temperature is 24~28℃, the fermentation time is 68~82h, the fermentation tank pressure is 0.04~0.06MPa, and the aeration ratio is 1:(0.6-1.0)V / V.min. The culture medium in the third fermentation tank is: 5% wheat bran, 2% glucose, 4% sucrose, 0.05% defoamer, and the remainder is drinking water; the fermentation temperature is 24~28℃, the fermentation time is 68~82h, the fermentation tank pressure is 0.04~0.06MPa, and the aeration ratio is 1:(0.6-1.0)V / V.min. The culture medium in the total fermentation tank in step seven is as follows: 8% potato, 2-4% soybean meal, 3% silkworm pupa powder, 0.003% VB1, 0.15-0.2% potassium dihydrogen phosphate, 0.075-0.1% magnesium sulfate heptahydrate, 0.05-0.08% zinc sulfate, and the remainder is drinking water; pH is 5.5-6.0, fermentation conditions are 25-28℃, fermentation time is 32-46 h, 200 r / min, and aeration ratio is 1:(0.5-1.0)V / V.min.

[0023] Example 2

[0024] The liquid culture medium in steps two, three, and four is: 7% potato, 2% glucose, 0.5% peptone, 0.2% yeast powder, 0.05% magnesium sulfate, 0.001% vitamin B1, with the remainder being drinking water, pH 5.5–6.0, 24–26℃, and incubation time of 36–48 hours.

[0025] Example 3

[0026] The culture medium in the total fermentation tank in step seven is: dextrin 5%, soybean meal 2-4%, silkworm pupa powder 3%, VB1 0.003%, potassium dihydrogen phosphate 0.15-0.2%, magnesium sulfate heptahydrate 0.075-0.1%, zinc sulfate 0.05-0.08%, and the remainder is drinking water; pH is 5.5-6.0, fermentation conditions are 25-28℃, fermentation time is 32h~46h, 200r / min, and aeration ratio is 1:(0.5-1.0)V / V.min.

[0027] The above description shows and illustrates the basic principles, main features, and advantages of the present invention. Standard parts used in the present invention can be purchased from the market, and irregular parts can be customized according to the description and drawings. The specific connection methods of each part adopt conventional methods such as bolts, rivets, and welding that are mature in the prior art. The machinery, parts, and equipment adopt conventional models in the prior art, and the circuit connection adopts conventional connection methods in the prior art, which will not be described in detail here.

[0028] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A liquid fermentation process for Armillaria mellea, characterized in that: The steps include the following: Step 1: Cultivate pure live mycelia of Armillaria mellea using PDA medium; Step 2: Primary shake flask seed culture: Take mycelia and inoculate them into a shake flask containing liquid culture medium for shaking culture; Step 3: Secondary shake-flask seed culture: Transfer to fresh liquid culture medium at an inoculum rate of 10–18% and culture with shaking. Step 4: Three-stage shake flask seed culture: Take the seeds from the two-stage shake flasks and stir them for culture to establish a stable seed system; Step 5: Transfer the mature seeds into a fermentation tank and add fermentation medium for initial fermentation culture; Step 6: When fermentation reaches the stable phase, divide the fermentation broth into 3 fermentation tanks of equal volume, add different fresh culture media, and continue fermentation. Step 7: Collect the Armillaria mellea liquid from the different fermentation tanks into the main fermentation tank and add fresh culture medium for slow, deep fermentation with stirring. Step 8: Concentrate, dry, and pulverize the Armillaria mellea liquid after deep fermentation in Step 7.

2. The liquid fermentation process of Armillaria mellea as described in claim 1, characterized in that: The liquid culture medium in steps two, three, and four is: 5% wheat bran juice, 2% glucose, 0.5% peptone, 0.2% yeast powder, 0.05% magnesium sulfate, 0.001% vitamin B1, with the remainder being drinking water, pH 5.5–6.0, 24–26℃, and incubation time of 36–48 hours.

3. The liquid fermentation process of Armillaria mellea as described in claim 1, characterized in that: The fermentation medium in step five is: 5-8% corn flour, 2-4% soybean meal, 3% silkworm pupa powder, 0.003% VB1, 0.15-0.2% potassium dihydrogen phosphate, 0.075-0.1% magnesium sulfate heptahydrate, 0.05-0.08% zinc sulfate, and the remainder is drinking water; the pH is 5.5-6.0, the fermentation conditions are 25-28℃, the fermentation time is 36-50 h, 200 r / min, and the aeration ratio is 1:(0.5-1.0)V / V.min.

4. The liquid fermentation process of Armillaria mellea as described in claim 3, characterized in that: During fermentation, 0.05% saccharifying enzyme and 0.02% acidic protease were added.

5. The liquid fermentation process of Armillaria mellea as described in claim 1, characterized in that: The three fermentation tanks of the same volume are designated as the first fermentation tank, the second fermentation tank, and the third fermentation tank.

6. The liquid fermentation process of Armillaria mellea as described in claim 5, characterized in that: The culture medium in the first fermentation tank consists of: 5% soybean meal powder treatment liquid, 2% glucose, 4% sucrose, 0.05% defoamer, and the remainder is drinking water; the fermentation temperature is 24~28℃, the fermentation time is 68~82h, the fermentation tank pressure is 0.04~0.06MPa, and the aeration ratio is 1:(0.6-1.0)V / V.min.

7. The liquid fermentation process of Armillaria mellea as described in claim 6, characterized in that: The culture medium in the second fermentation tank consists of: 5% corn flour, 2% glucose, 4% sucrose, 0.05% defoamer, and the remainder is drinking water; the fermentation temperature is 24~28℃, the fermentation time is 68~82h, the fermentation tank pressure is 0.04~0.06MPa, and the aeration ratio is 1:(0.6-1.0)V / V.min.

8. The liquid fermentation process of Armillaria mellea as described in claim 7, characterized in that: The culture medium in the third fermentation tank is: 5% wheat bran, 2% glucose, 4% sucrose, 0.05% defoamer, and the remainder is drinking water; the fermentation temperature is 24~28℃, the fermentation time is 68~82h, the fermentation tank pressure is 0.04~0.06MPa, and the aeration ratio is 1:(0.6-1.0)V / V.min.

9. The liquid fermentation process of Armillaria mellea as described in claim 8, characterized in that: The culture medium in the total fermentation tank in step seven is as follows: 8% potato, 2-4% soybean meal, 3% silkworm pupa powder, 0.003% VB1, 0.15-0.2% potassium dihydrogen phosphate, 0.075-0.1% magnesium sulfate heptahydrate, 0.05-0.08% zinc sulfate, and the remainder is drinking water; pH is 5.5-6.0, fermentation conditions are 25-28℃, fermentation time is 32-46 h, 200 r / min, and aeration ratio is 1:(0.5-1.0)V / V.min.

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