SU719513A3 - Method of culturing basidiomycets - Google Patents

Description

This invention relates to a technique for cultivating basidiomycetes and can be used in the food, pharmaceutical industry and D medicine. Formed in the mycelium of basidiomycetes, the polysaccharide has pro-tumor properties and can also serve as a food additive.

There is a well-known method of cultivating basidio eggs, which envisages their cultivation under conditions of aeration and transfer on a liquid nutrient medium containing carbohydrates as a carbon source, a nitrogen source, a source of necessary mineral salts and organic additives, with a certain medium loading in the fermenter 1.

However, when basidiomycete fungi are cultivated in a liquid aqueous medium, abundant foam is formed during cultivation; as a result, the efficiency of cultivation decreases when a large amount of medium in the fermenter is used. Reducing the flow of the medium into the fermenter to 40-60% of its capacity during long-term cultivation leads to a S1shzhzheniyu productivity, which creates economic difficulties.

Diluting the medium to inhibit foaming and increase the loading of the fermenter leads to a reduction in the yield of the mycelium to the loading.

The aim of the invention is to intensify the cultivation process and increase the yield of the product.

This is achieved by the fact that in the described method of cultivating basidiomycetes, the latter are prepared when the medium is loaded at 40 to 70% of the volume of the fermenter, and as the foam is grown during the cultivation, the nutrient medium or solution containing one of its components, with a frequency of once every two days or daily up to 80-85% filling of the volume of the fermenter, and the total number of additives in the cultivation process is from 2 to 5.

The cultivation of basidiomycetes belonging to the family of Polyporaceae of the genus Coliolus is carried out.

37

Glucose is used as a carbon source, and yeast extract, peptone, casein hydrolytic or meat extract as an organic additive.

The method is carried out as follows. Basidiomycetes, which belong to the family of the Polyporaceae family of the genus Colioluc, are grown under conditions of aeration and replenishing on a liquid nutrient medium containing carbohydrates as a carbon source, the source of nitrogen, the source of the required mineral salts and organic additives from 40 to 70% of the volume of the fermenter.

In the process of growing basidiomycetes

An abundant foam forms on the surface of the liquid aqueous medium, which disappears after the initial cultivation phase, which is accompanied by the rapid growth of basidiomycetes. In this connection, as the foam disappears, an additional nutrient medium is added to the fermenter to ensure the growth of microorganisms once every two days or daily to 80-85% filling of the fermenter's volume, the total concentration of additives in the process of cultivation being from 2 up to 5

The aqueous liquid medium as a carbon source contains saccharides such as glucose, lactose, shsh starch.

Yeast extract, peptone, casein hydrolyzate or meat extract in an amount of more than 0.1 wt.% Are used as an organic additive.

As the main product in the basidiomycete gap and in the medium, polysz orvd is formed, which is separated and regenerated by extraction with an aqueous solvent,

PRI me R 1. 1000 l of an aqueous liquid medium containing ingredients, wt.%: Glucose - 10; OO5-burnt extract - 1.5; malt extract - 0,2; MgS04 THjO - 0.1; KI2P04 - 0,1, loaded into the fermenter in the form of a tank with a capacity of 2 m, having two-stage flat-bladed turbines. The medium is sterilized and inoculated with 20 l of Coiiolus versicolor (Fr) Quel mycelium sludge, which is preliminarily cultivated as seed culture in a 50 l canned fermentor, and then immediately start cultivation with an aeration rate of 0.5 l of air per 1 l of medium per minute at a rotation speed pipe 150 rpm at 26 ° C.

During the initial stage of cultivation, abundant foam is formed and its level reaches the top of the fermenter. After 1520 hours, the foam level is smoothed and, 24 hours after the start of cultivation, additional

200 liters of sterilized medium of the composition described above are added, and the addition is carried out for 1 hour and then the mixture is continued, while maintaining the aeration rate of 600 liters / min.

At 48 hours after the start of cultivation, another 200 liters of medium are added and the cultivation is continued, maintaining the aeration rate of 700 liters / min. After 72 hours from the start of cultivation, 200 liters of medium are added under the same conditions as for the first and second additions, and the cultivation is continued, delaying the aeration rate of 800 liters / min. These. the three nutrient supplements are 1600 l, or 80% of the capacity of the fermenter, which is the limiting value of the feed, therefore, further purification is continued under these conditions and is completed on the 7th day. Then the mycelium is separated from the medium by centrifugation and sushi. 15.6 g / l.

Example 2. 1000 l of aqueous liquid Creschl containing the ingredients, wt.%: Glucose - 15; yeast extract - 2.25; malt extract - 0.2; MgS04-7H2O - 0.1; KI2P04 - 0.1, loaded into a vertical fermenter with a capacity of 2 m in the form of a vat, having two-stage smooth-lobed turbines. 20 l of the Coliolus versicolor (Fr) Quel mycelium sludge is inoculated on the medium and is pre-cultured in a 50 l bottle fermentor, and immediately after this, the cultivation process at a speed of 0.5 l of air per liter of medium per minute is used. min at 26 ° C.

The foam level starts up to the top of the fermenter during the initial stage of curing and then after 30-40 hours the foam begins to fall, due to which 48 hours after the start of the cultivation, 200 l of sterilized medium of the composition described above was added. The addition was allowed to proceed for one hour, after which the cultivation was continued, maintaining the aeration rate of 600 l / min.

At 72 hours after the start of cooling, another 200 liters of medium are added under the same conditions as in the first addition, and the cultivation is continued, keeping the aeration rate of 700 liters / min. At 96 h after the start of cultivation, another 200 l of medium are added under the same conditions as with the first and second additions, maintaining the aeration rate of 800 l / min. A total of 1600 L of nutrient medium, or 80% of the capacity of the fermenter, was added in three times.

The cultivation is completed on the 7th day after the start of cultivation, after which the mycelium is separated from the nutrient medium by centrifugation and injected. Output 18.4 g / l. 5 EXAMPLE 3 A control comparative example was carried out, for which 1200 l of the medium with the same composition as in example 1 was immediately added to the fermenter, using the synthetic method in example 1. After sterilization, the medium is inoculated with 20 l of the mycelium Colioius versicolor sludge (Fr) Quel, which is preliminarily cultivated as a seed culture in a 50 l beam fermenter, after which they immediately begin cultivating {500 during aeration of 500 l / min, speed of rotation of the turbine is 150 rpm and at 26 ° C. 7 days. The yield of mycelium is 16.9 t / l. Example 4. Cultivation was carried out as in Example 3, loading 1000 into the fermenter.

Indicator, measurement unit 6 of the nutrient medium, the composition of which is described in Example 2. It is practically impossible to supply more than 1000 liters of medium to the fermenter due to its foaming. Froze Cultivation was carried out as in Example 3, except that 1600 were added. 1 liter of medium containing ingredients, wt.%: Glucose - 5; yeast extract - 0.7; malt extract - 0.2; MgS047H20 - 0.1; KHjTO4 - 0.1. The medium is added as described in examples 1 and 2. The degree of foaming is reduced by diluting the medium. Comparative data indicators for examples 1-5 are given in table. 1. Table 1

The composition of the medium, glucose

yeast extract

malt extract

MgSO4 / Ig O KHjPO.i

The final feed among ((%

Obiga load, l

Production by IU Eliro, kg / load

Example 6. For additional loading, instead of the whole environment, one of its components is used. 35 l of liquid medium containing glucose and yeast extract are loaded into a 50 l vertical fermenter in the form of a vat having planar turbines. The medium is sterilized and inoculated with mycelium slime (0.01% micelle) Colioius versicolor (Fr) Quel CM-101 under deposit number FERM-P No. 2412, which is obtained by shaking the culture. Immediately after this, the cultivators start15, 00

5.00

10.00 0.75 1.50

2.25

0.20

0.20

0.20 0.10 0.10 0.10 0.10 0.10 0.10

80 1600

50 1000

60,100

10,9017,76

10.28

aeration rate of 0.5 l / l / mic to the mixer rotation speed of 250 rpm at

25 ± 2 ° С.

During cultivation, the level of foam rises to the upper part of the fermenter, after 15-20 hours the foam falls off, and after 24 hours 2 liters of aqueous solution containing yeast extract are added. Cultivation is continued under the aeration rate of aeration 0.6 l / l / min. 48 hours after the start of cultivation, 2 aqueous solution of yeast extract was added again.

and the cultivation is continued, maintaining the aeration rate of 0.6 l / l / min. After 72 hours, another 2 L of an aqueous solution of the yeast extract is added and culture is continued. The total amount of medium with additives is

41 liters, or 82% of the capacity of the fermenter.

The cultivation is carried out for 7 days, then the midelium is separated from the medium by ceterification and dried. The yield and productivity are 19.8 g / l and 810 g, respectively.

Indicator, unit

The proposed method of measurement

Initial loading environment, l

Content in the environment, g: glucose

yeast extract

Additive solution of yeast extract, l;

rl24 h - 100 g; M-48 h - 100 g; 111-72 h - 100 g

Total amount of loaded medium, l

Cultivation time, Temperature, ° С

Productivity by

mycelium, g Note.

PRI me R 7. Mycelium. Coliolus versicolor (Fr) Quel CM-lOl is cultivated under the same conditions as in Example 1. 30 L of medium containing glucose and yeast extract are loaded into a 50 L fermentor and inoculated with mycelium slurry. 48 and 72 hours after the start of cultivation, an additive of 5 liters of yeast extract is added, and the cultivation is continued.

To compare the mycelium, Coliolus versicolor (Fr) Quel f is cultivated in the same fermenter without adding yeast extract to a liquid medium containing a relatively larger amount of yeast extract than glucose (see Table 2). In this case, no more than 25 liters of medium is added due to strong agitation. 7 days are cultivated, the yield of the mycelium is 22.0 g / l, and the productivity is 550 g.

The results of comparative studies are shown in Table. 2

table 2

Control

25

1875 375

25

2

25 ± 2

550

Mixed with a large amount of yeast extract without additional addition of yeast extract to the medium. In this case, abundant foaming is observed, which does not allow more than 20 liters to be loaded into the fermenter. The performance of the mycelium in this case is low. In both cases, the concentration of glucose in the medium is 7.5% by weight, and the concentration of yeast extract (the total amount in additives and initial load in a comparative example) is 1.5% by weight.