CN111903428A - Novel mushroom liquid strain and preparation method and use method thereof - Google Patents

Abstract

The invention provides a novel mushroom liquid strain and a preparation method and a use method thereof. The preparation steps of the liquid strain are as follows: 1) preparing a plate to obtain strong hyphae; 2) performing shake flask culture to obtain a culture solution of Lentinus Edodes strain seed; 3) amplifying, culturing and fermenting to obtain liquid strains of the mushroom strains. Wherein, during liquid culture, the formula of the used liquid culture medium is as follows: 2.0g of glucose, 2.0g of corn flour, 1.5g of fine wood chips, 1.2g of soybean meal and MgSO₄ 0.05g、KH₂PO₄ 0.1g、H₂O100 mL. The C/N ratio in the medium was 23.6: 1. the preparation method of the mushroom liquid strain has the characteristics of low cost, short period, strong impurity resistance, high purity and strong activity of the obtained strain and the like, and is suitable for commercial application.

Description

A new type of mushroom liquid strain and its preparation method and use method

technical field

The invention belongs to the technical field of edible and medicinal fungal liquid strains, and in particular relates to a novel Lentinus edodes liquid strain and a preparation method and use method thereof.

Background technique

Shiitake mushroom (Lentinula edodes) is a well-known edible and medicinal fungus, known as "the king of mushrooms". The shiitake mushroom industry plays an important role in my country's edible mushroom industry, with a wide range of production areas, and it is produced in all parts of the north and south of my country. Mushrooms are rich in essential amino acids. The umami component of shiitake mushroom is a class of water-soluble substances, the main components of which are glutamic acid and guanylic acid. The medicinal functions of shiitake mushrooms have been recorded by famous Chinese medicine practitioners in all dynasties. Some studies have reported that the lentinan contained in mushrooms can regulate the activity of T cells with immune function in the human body and play a role in resisting cancer; adenine and choline can prevent liver cirrhosis and arteriosclerosis; tyrosine oxidase can effectively Lower blood pressure; double-stranded ribonucleic acid can induce the production of interferon, which has antiviral effect.

With the large-scale expansion of the mushroom industry, the traditional solid strain production mode is difficult to meet the requirements of large-scale and annual production of modern edible fungi due to the long production cycle, while the liquid strain has a short production cycle, high strain purity, The high degree of standardization can better meet the needs of factory production. However, because of its rich nutrition, liquid strains are easily contaminated by miscellaneous bacteria in the actual production process, resulting in impure strains and huge economic losses in severe cases.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned defects in the prior art, the purpose of the present invention is to provide a new type of Lentinus edodes liquid strain and its preparation method and use method. The preparation method of the mushroom liquid strain has the characteristics of low cost, short cycle, strong impurity resistance, high purity and strong vitality of the obtained strain, and is suitable for commercial application.

To achieve the above object, the present invention adopts the following technical solutions:

A kind of preparation method of novel Lentinus edodes liquid strain, is characterized in that: comprises the following steps:

1) Plate preparation: Pick the tip of the mycelium of the Lentinus edodes strain in a test tube and inoculate it in the center of the PDA plate, culture at 25°C for 10 days in the dark, repeat 3-5 times until robust mycelium is obtained;

2) shake flask culture: under aseptic conditions, punch holes at the edge of the mycelium obtained in the above-mentioned step 1) to obtain a mycelium block with a diameter of 1 cm; insert three pieces of the bacteria described in the triangular flask of the liquid culture medium. The silk block was incubated at a constant temperature of 25°C for 10 d on a shaking table with a rotational speed of 150 r/min, and then the culture was terminated to obtain a Lentinus edodes strain seed culture solution;

3) Enlarged cultivation: by inoculation amount 0.5%, the Lentinus edodes strain seed culture liquid obtained in step 2) is inoculated into the fermentor for enlarged cultivation, and adjusting the ventilation rate and the volume ratio of the fermented liquid in the tank is 0.5vvm, and the maintenance tank pressure is 0.02- 0.04MPa, the fermentation temperature is 25°C, and the fermentation time is 6-8d to obtain the liquid strain of the Lentinus edodes strain.

According to the above scheme, preferably, the formula of the liquid medium in step 2) is: glucose 2.0g, corn flour 2.0g, fine sawdust 1.5g, soybean meal powder 1.2g, MgSO ₄ 0.05g, KH ₂ PO ₄ 0.1g, H ₂ O 100 mL. The C/N ratio in the medium was 23.6:1.

The present invention also provides a novel liquid strain of Lentinus edodes, which is characterized in that the liquid strain is prepared by the above method.

The present invention also provides a method for preserving the above-mentioned novel liquid Lentinus edodes, which is characterized by storing at 5° C. for 3 days.

The present invention also provides a method for using the above-mentioned novel Lentinus edodes liquid strain, characterized in that, when using the Lentinus edodes liquid strain to inoculate the cultivation material, the adopted solid-liquid ratio (W/V) is 37.5-40:1.

Compared with the prior art, the present invention has the following positive effects:

1. The preparation method of the Lentinus edodes liquid strain provided by the present invention has the advantages of low cost, short fermentation period, consistent bacterial age and the like.

2. The time of culturing the liquid strain of Lentinus edodes prepared by the present invention in the cultivation bag is shortened, and when the inoculation amount is 7mL, 12mL, and 17mL, the time for the mycelium to fill the bag is 6.9d, 5.1d, and 4.6d earlier than the solid strain, respectively. reached a significant level of difference.

3. The C/N ratio in the liquid medium used in the present invention is 23.6:1 when preparing the liquid strain of Lentinus edodes, and the dry weight of the mycelium of the obtained liquid strain after culture is increased by 307% compared with that before optimization.

4. The liquid strain of Lentinus edodes of the present invention is used in the cultivation of Lentinus edodes bag material, which reduces the pollution rate. When the inoculum volume of liquid strains is 12mL and 17mL, the contamination rate is only 2%, which is lower than the contamination rate of solid strains by 10%.

Detailed ways

Example 1

A new type of mushroom liquid strain is characterized in that: comprising the following steps:

1. Production of culture medium

1) Preparation of PDA medium (potato dextrose agar medium)

Peel and slice the potatoes, weigh 200g and place them in a pot, add 1L of water, and cook until the potato slices can be gently pierced with chopsticks. Filter the filtrate with several layers of gauze, add 20 g of agar powder to the filtrate, cook until the agar powder is completely melted, add 20 g of glucose and make up to 1 L of water. Dispense into 250mL conical flasks, 200mL per bottle. Seal with parafilm and sterilize in a high pressure steam sterilizer at 121°C for 30min. After the sterilization, the plate was poured in time to obtain PDA medium.

2) Liquid culture medium preparation

Select fresh and mildewed fine wood chips and soybean meal and place them in an electric heating constant temperature oven for drying. Use a pulverizer to pulverize the materials and pass them through a 120-mesh sieve (pore size: 0.125mm) to ensure the particle size of the materials used. Put in a sealed bag and store in a cool, dry place for later use.

Add 2.0g glucose, 2.0g corn flour, 1.5g fine sawdust, 1.2g soybean meal powder, 0.05g _MgSO4 , 0.1g _KH2PO4 , and 100mL water to each 250mL conical flask _. Seal with parafilm and sterilize in a high pressure steam sterilizer at 121°C for 30min.

2. Preparation of liquid strains

1) Plate preparation: pick the tip of the mycelium of the test tube Lentinus edodes strain L808 (provided by the Bacteria Test Center of Huazhong Agricultural University) and inoculate it in the center of the PDA plate, place it at 25°C for 10 days in the dark, and repeat 3 times until a robust mycelium is obtained. . The mycelium after purification and rejuvenation is ready for use.

2) Shake flask culture: under aseptic conditions, punch holes on the edge of the mycelium obtained in the above step 1), with a hole diameter of 1 cm. Three pieces of Lentinus edodes strain L808 mycelium were inserted into each Erlenmeyer flask containing 100 mL of liquid medium, and after culturing at 25°C for 10 d on a shaking table with a rotating speed of 150 r/min, the culture was terminated, and the seed culture of Lentinus edodes strain L808 was obtained. liquid for inoculation.

3) Amplified culture: inoculate 2.5L of the L808 seed culture liquid of step 2) into a 500L fermentor for enlarged culture, adjust ventilation, and the volume ratio of ventilation to the fermented liquid in the tank is 0.5vvm, and the maintenance tank pressure is 0.03MPa, and the fermentation The temperature is 25°C, and the fermentation time is 156h, to obtain the liquid strain of Lentinus edodes strain L808, which is stored at 5°C for 3 days for use.

The liquid bacterial classification of Lentinus edodes strain L808 obtained from the formula of the liquid culture medium provided in the present embodiment is carried out to measure the dry weight of mycelium, and the specific steps are as follows:

Shake the liquid strain (fermentation liquid) of the Lentinus edodes strain L808 obtained by fermentation, filter the mycelium with an 80-mesh sieve (0.180mm), and rinse repeatedly until there is no visible solid residue in the rinsed water. In the finished plate, dry at a constant temperature of 60°C until the weight of the plate does not change within 12 hours, take out the plate, and place it in an analytical balance to weigh the mass to obtain the mycelial biomass (g/100mL). The formula for calculating the dry weight of mycelium is as follows:

Dry weight of mycelium = mass of petri dish after drying - actual mass of petri dish.

The change of the dry weight of the obtained mycelium according to the culture time is shown in Table 1.

Comparative Example 1

A kind of preparation of Lentinus edodes solid bacterial classification, comprises the following steps:

1) Preparation of PDA medium

The specific steps are the same as those in Example 1.

2) Bacterial activation: Pick the tip of the Lentinus edodes strain L808 (provided by the Bacteria Test Center of Huazhong Agricultural University) in a test tube and inoculate it in the center of the PDA plate, place it at 25°C for 10 days in the dark, repeat 3 times until a robust mycelium is obtained . The mycelium after purification and rejuvenation is ready for use.

3) Preparation of cultivars

Select dry, fresh, mildew-free fine sawdust and add accessories according to the mass ratio of 80% sawdust, 19% bran, and 1% gypsum, mix well, adjust the humidity, and put it into a 15cm×30cm polypropylene bacteria bag. The bag fits snugly. Sterilize in an autoclave at 121°C for 2 hours, and after cooling, connect the activated first-class species in step 2) in strict accordance with the aseptic operation on the ultra-clean workbench. Inoculation should be divided into three sections, so that the upper, middle and lower parts of the bacterial bag have bacterial blocks. Place in a 25°C culture room to avoid light, pay attention to ventilation, and remove contaminated bacteria bags in time. After a month or so, put it in a refrigerator at about 4°C for later use.

Comparative Example 2

A kind of Lentinus edodes liquid bacterial classification, is characterized in that: comprises the following steps:

1. Production of culture medium: The specific steps are the same as in Example 1, except that the formula of the liquid culture medium is changed from "glucose 2.0g, corn flour 2.0g, fine sawdust 1.5g, soybean meal powder 1.2g, MgSO ₄ 0.05g, KH ₂ PO ₄ 0.1 g, water 100 mL" was adjusted to "corn flour 2%, glucose 2%, soybean meal 1.2%, MgSO4 0.05 g, KH2PO4 0.1 g, water 100 mL".

2. Preparation of liquid strains: the specific steps are the same as those in Example 1.

The liquid culture of the Lentinus edodes strain L808 obtained from the formula of the liquid medium provided in this example was carried out in shake flask liquid culture, and the change of the dry weight of the obtained mycelium according to the culture time is shown in Table 1.

Table 1

Note: "**" represents significant difference at P<0.05 level.

It can be seen from Table 1 that from the 6th day to the 15th day of culturing the liquid Lentinus edodes, the mycelial biomass of the two formulations provided in Example 1 and Comparative Example 1 both showed an upward trend. Wherein, the dry weight of the mycelium in Example 1 reached a maximum of 1.8788g/100mL on the 15th day, and the dry weight of the mycelium in Comparative Example 2 (the medium was composed of 2% corn meal, 2% glucose, and 1.2% soybean meal) was A maximum of 0.6326 g/100 mL was reached on day 13. According to the independent sample T test analysis, the difference between the two formulas on the dry weight of mycelium reached a significant difference, indicating that the formula provided in Example 1 can promote the growth of mycelium and increase the biomass of the liquid bacteria obtained by culture.

Application Example 1

The liquid strain of the Lentinus edodes strain L808 prepared in Example 1 was used for cultivation in the cultivation bag, and the specific steps were as follows:

1) Preparation and sterilization of cultivation bags

According to the mass ratio of 80% sawdust, 19% bran, and 1% gypsum, mix well, adjust the humidity, and put it into a polypropylene bacteria bag with a size of 15cm×55cm. Sterilize for 2h.

2) Cultivation of cultivation bags

After cooling, 12 mL of the liquid strain obtained by the culture in Example 1 was inserted into the ultra-clean workbench in strict accordance with the aseptic operation, and each cultivation bag was inoculated with 4 holes. Place in a 25°C culture room to avoid light for cultivation, pay attention to ventilation, and remove contaminated bacteria bags in time. Among the 50 bags of culture bags, one bag of culture bags was contaminated by miscellaneous bacteria, and the contamination rate was 2%.

3) Put on the shelf, turn color, and fruit

After culturing for 50 days, the bacteria bags were overgrown, and then the bacteria bags were moved to the outdoor greenhouse shelf. In order to stimulate the fungus stick and speed up the color change, use bamboo chopsticks to puncture the fungus stick before putting it on the shelf. The shelf is divided into 6 layers, which are divided into 3 blocks according to the upper 2 layers, the middle 2 layers, and the lower 2 layers. Using the stimulation of the temperature difference between day and night in late autumn, the fungus sticks began to turn color and fruit.

4) Harvest

Harvest after the rupture of the biofilm of the fruiting bodies. The average yield of fruiting bodies per bag of fungus sticks was 104.9 g, and the biological efficiency was 11.7%.

Comparative Application Example 1

The solid bacterial classification of the Lentinus edodes strain L808 prepared in Comparative Example 1 is used for cultivation in the cultivation bag, and the concrete steps are as follows:

1) Preparation and sterilization of cultivation bags

The specific steps are the same as in Application Example 1.

2) Cultivation of cultivation bags

After cooling, the solid bacteria were inserted into the ultra-clean workbench in strict accordance with the aseptic operation. Each cultivation bag was inoculated with 4 holes, and each hole was connected with 6g of solid bacteria. Place in a 25 ℃ culture room to avoid light, pay attention to ventilation, and remove the contaminated bacteria bags in time. Among the 50 bags of cultivation bags, 5 bags were contaminated by miscellaneous bacteria, and the pollution rate was 10%.

3) Put on the shelf, turn color, and fruit

The specific steps are the same as in Application Example 1.

4) Harvest

Harvest after the rupture of the biofilm of the fruiting bodies. The average yield of neutrophils per bag was 102.2 g, and the biological efficiency was 11.3%.

Claims (6)

1. A preparation method of a novel mushroom liquid strain is characterized by comprising the following steps:

1) preparing a plate: selecting the tip of hypha of Lentinus Edodes in test tube, inoculating to the center of PDA plate, culturing at 25 deg.C in dark for 10 days, and repeating for 3-5 times until strong hypha is obtained;

2) and (3) shake flask culture: under aseptic conditions, perforating the edge of the hypha obtained in the step 1) to obtain a hypha block with the diameter of 1 cm; inoculating the mycelium block into a triangular flask filled with a liquid culture medium, culturing for 10 days at a constant temperature of 25 ℃ on a shaking table at the rotating speed of 150r/min, and terminating the culture to obtain a mushroom strain seed culture solution;

3) amplification culture: inoculating the culture solution of the lentinus edodes strain seeds obtained in the step 2) into a fermentation tank for amplification culture according to the inoculation amount of 0.5%, adjusting the volume ratio of the ventilation volume to the fermentation liquid in the tank to be 0.5vvm, maintaining the tank pressure to be 0.02-0.04MPa, and fermenting to obtain the liquid strain of the lentinus edodes strain.

2. The method for preparing a novel liquid spawn of shiitake mushroom according to claim 1, wherein the formula of the liquid culture medium in the step 2) is: 2.0g of glucose, 2.0g of corn flour, 1.5g of fine wood chips, 1.2g of soybean meal and MgSO₄ 0.05g、KH₂PO₄ 0.1g、H₂O 100mL。

3. The method for preparing the novel liquid spawn of shiitake mushroom according to claim 1, wherein the fermentation temperature in the step 3) is 25 ℃ and the fermentation time is 6-8 days.

4. A novel liquid strain of Lentinus edodes, which is obtained by the method of any one of claims 1 or 2.

5. A method for preserving a liquid spawn of Lentinus edodes according to claim 4, wherein the liquid spawn of Lentinus edodes is stored at 5 ℃ for 3 days.

6. The use method of the novel mushroom liquid spawn according to claim 4, wherein the solid-to-liquid ratio is 37.5-40: 1 when the mushroom liquid spawn is inoculated into a cultivation material.