Nutrient medium for submerged culturing fungus blakeslea trispora vkpm f-117 biomass as producer of carotene

FIELD: microbiological industry.

SUBSTANCE: invention relates to nutrient media used for culturing producers of carotene. Invention proposes nutrient medium containing barley flour, soybean meal, potassium dihydrogen phosphate, sunflower oil, vitamin B1, β-ionone and tap water being components are taken in the following ratio, wt.-%: barley flour, 3.0-4.0; soybean meal, 4.0-4.7; sunflower oil, 3.8-4.0; potassium dihydrogen phosphate, 0.04-0.05; vitamin B1, 0.0002-0.0005; β-ionone, 0.098-0.099, and tap water, the balance. The proposed nutrient medium is low-priced and enhances the biosynthetic ability of producer of carotene.

EFFECT: valuable properties of nutrient medium.

3 tbl, 3 ex

 

The invention relates to the microbiological industry, and specifically to nutrient media for cultivation of producers of big quantity of carotene biomass.

Known nutrient medium Ziegler for cultivation producers carcinosarcoma biomass, including soy flour is 4.7%, corn flour and 2.3%, sunflower oil - 4%, potassium phosphate one-deputizing - 0,05%, vitamin B1- is 0.0002% water and the rest (Cigler, A., Arnold U., nlrson R.F. Microbiological production carotinoides. 1958, V7, R-98).

The disadvantages of the nutrient medium Ziegler are low biosynthetic ability of the producer and the use of food raw materials for its preparation.

Closest to the technical essence and the achieved technical result is a nutrient medium for submerged cultivation of the biomass of the fungus lakeslea trispora producer carotene containing soybean oil and 43%, crushed corn, potassium phosphate one-deputizing - 0,05%, vitamin b11 mg%, β-Ionen - 0,1% (see US 2890989, 16.06.1959). However, this medium does not provide a sufficiently high biosynthetic capacity of a producer carotene.

The technical result is to increase the biosynthetic ability of the producers of big quantity of carotene biomass and the reduction of nutrient medium.

The technical problem is solved by a nutrient medium DL is submerged cultivation of the biomass of the fungus lakeslea trispora VKPM F-117 producer carotene contains barley meal, soy flour, potassium phosphate one-deputizing, sunflower oil, vitamin b1that β-ionon, tap water in the following ratio of components, wt.%:

Barley meal 3,0-4,0

Soy flour 4,0-4,7

Sunflower oil 3,8-4,0

Potassium phosphate one-deputizing 0,04-0,05

Vitamin b1is 0.0002-0,0005

β-ionon 0,098-0,099

Tap water is the rest

The invention consists in that the increased biosynthetic capacity of a producer carotene provides a high concentration in barley meal isoprenoid hydrocarbons nature of carotenoids and terpenes. Use as a source of carbon isoprenoid hydrocarbons nature, in particular carotenoids, dramatically increases the content of carotene in biomass and microbial lipids. This is because probably the microorganisms include vegetable carotenoids in products of lipid synthesis, while the energy cost of synthesis of lipids in microbial cell is minimized. Increased biosynthetic capacity of producers carotene, may contribute to the presence of sterols in barley meal (1,5-4,5%).

It was established experimentally that the achievement of higher biosynthetic ability of producers carotene is possible using Acme the Noah husking bran, characterized by the following chemical composition (table 1).

Table 1

The chemical composition of barley husking bran
Humidity, %In % dry matter
ProteinFatStarchFiberAsh
12-1312-138-1050-608 - 108-9

The intensification of the process of biosynthesis of carotene may help the vitamins and carotenoids content in the meal varies within the following limits, mg%:

Vitamin b10,50-0,65

Vitamin b20,45-0,55

Vitamin PP 1,10-1,3

Vitamin E 4,8-4,95

Carotenoids 0,40-0,42

To increase the biosynthetic ability of producers carotene can influence and other groups of biologically active substances, in particular some fatty acids. The content of major fatty acids of lipids barley husking bran presents the following data (table 2).

Table 2

Fatty acid composition of lipids barley husking bran
Fatty acid% from amount
With16:06,9
With18:01,2
With18:125,4
With18:265,6
With18:30,9
The amount of saturated acids8,1
The amount of unsaturated acids91,9

The main representative of fatty acids of lipids barley husking bran is a dietary linoleic acid.

A nutrient medium is prepared as follows: barley meal, the resulting peeling barley, sieved through a sieve with a diameter of 1 mm. of Salt in a given amount dissolved in cold tap water. In salt solution add barley meal, soy flour, sunflower oil. The medium is sterilized at 121°C for 45-60 min, then cooled.

In fermentation flasks containing 50 ml of medium, seeded with a culture of a strain of microorganism in the amount of 5-10% of the volume of medium in the volume ratio 1:10. Conduct in-depth ferment for 4-6 days at 26-28°under aerobic conditions with continuous stirring and aeration in the ratio of the amount of air 1/2:1 to the volume of the nutrient medium.

After 36-48 h of fermentation download β-ionon in the number 0,099 weight. PM

Upon reaching the maximum concentration of carotene, the mycelium is separated from the liquid Phi is Tricia.

Example 1

Cultivation producer lakeslea Trispora PMBC-117 is carried out in laboratory flasks-the rocking V=730 mm Used nutrient medium of the following composition, wt.%: soy flour is 4.7%, barley meal 3%, sunflower oil 4%KN2P04- a 0.05%, vitamin B1- 0,0005%, β-Ionen - 0,099% and water up to 100%. In fermentation flasks containing 50 ml of medium, seeded two-day culture of the fungus l. Trispora in the amount of 10 percent of the total environment, volume ratio of 1:10. The cultivation is carried out at 26°and With stirring at 220 rpm After 48 hours from the beginning of the fermentation flasks download 0,099 parts by weight of β-ionone, volume environment.

When reaching the maximum concentration of carotenoid mass is separated by filtration. A portion of the biomass of 0.02 g placed in tube containing 0.5 ml of trichloroethylene and 7 ml of acetone. After transfer of the pigments in a solution of biomass additional pound in a mortar to a colorless state.

The solution of carotene in acetone transferred into a volumetric flask PA 200 ml. Volume was adjusted to the mark with acetone. The content of carotene determined photometrically according to the standard solution of the synthetic carotene. In addition, determine the content of carotene in the culture fluid (μg/100 ml of culture fluid) and the content is absolutely dry matter biomass (g/100 ml of kulturalny liquid). The carotene content in biomass 46,25 MHC, what 2.27 more than the known environment (43,98 g/kg). The content of carotene in the culture fluid 185315 µg/100 ml, which is 30026 µg/100 ml compared with the known environment (155289 µg/100 ml), biomass accumulation also is more intense and 3.72 g/kg against 3,63 g/kg on a known environment.

Example 2

A nutrient medium is prepared as in example 1, the difference is that the nutrient medium contains barley meal in the amount of 3.5%, vitamin b1- is 0.0002%, the number of remaining components analogously to example 1.

According to the results carotene content was 49,93 g/kg, which is 13.5% more in comparison with the known environment. The content of carotene in the culture fluid was 188654 µg/100 ml, which is 21% more than the results obtained in known environment. Biomass accumulation 3,83 g/100 ml, i.e. by 5.5% compared with the known environment.

Example 3.

A nutrient medium is prepared as example 1 and 2, the difference is that the nutrient medium contains barley meal in the amount of 4%, soy flour 4.3%), vitamin b1- is 0.0002%. A number of other components similarly indicated in examples 1 and 2.

When this ratio of components in the environment carotene content in biomass 44,23 g/kg, which is 0.5% more than in the known environment. The content of carotene in the culture fluid 165534 µg/100 ml, which is 6.5% more kemna known environment, the biomass accumulation of 3.75 g/100 ml, which is 3.3% higher compared with the results of the known environment.

When using heavy concentration values husking bran barley in a medium positive effect could not be reached. The results are shown in table 3.

Table 3
The concentration of components in a nutrient medium, %The carotene contentThe accumulation of biomass, g/100 ml culture fluid

Barley mealSoy flourSunflower oilVitamin b1β-ionong/kg of biomassµg/100 ml culture fluid
14,75the 3.80,00050,09931,011101003,71
1,54,75the 3.80,00050,09931,101101153,79
24,75the 3.8is 0.00020,09931,821352143,85
2,54,70the 3.8is 0.00020,098 38,841412763,79
4,54,00the 3.8is 0.00020,09840,771532143,60
5,04,00the 3.8is 0.00020,09835,141501143,57

The proposed nutrient medium can increase the yield of the target product in comparison with the prototype 0.85 kg in terms of crystalline β-carotene.

Nutrient medium for submerged cultivation of the biomass of the fungus can trispora VKPM F-117 - producer carotene containing barley meal, soy flour, potassium phosphate one-deputizing, sunflower oil, vitamin b1that β-ionon, tap water in the following ratio, wt.%:

Barley meal 3,0-4,0

Soy flour 4,0-4,7

Sunflower oil 3,8-4,0

Potassium phosphate one-deputizing 0,04-0,05

Vitamin b1is 0.0002-0,0005

β-Ionon 0,098-0,099

Tap water is the Rest



 

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