RU1768638C - Bacterial strain streptococcus thermophilus a producer of @@@-galactosidase - Google Patents

Abstract

Usage: biotechnology, food industry, dairy industry for the hydrolysis of lactose in milk and dairy products. SUMMARY OF THE INVENTION: An enzyme producing strain / 2 gzlactosidase strain is based on the Streptococcus thermophllus strain (VKPM B-2242), which is currently used as a component of starter cultures in the production of cottage cheese. The strain Streptococcus trermophilus (VKPM B-5392) (CM1351) was obtained as a result of multistage selection using nitrosguanidine. The proposed strain when grown on M21 medium with 5% lactose at a constant pH of 8.0 and a temperature of 37 ° C for 12 hours, the activity of the enzyme / 3-galactosidase is 34 u / ml, which corresponds to a specific activity of 33 u / mg protein.

Description

The invention relates to the microbiological industry, in particular to strains producing extracellular D-galactosidase. The enzyme / 5-galactosidase is used in a number of food industries, and the use of this enzyme in the dairy industry for the hydrolysis of lactose milk and dairy products is particularly promising. Known strain Penlcilllum canescens VKPM F-436. The enzyme activity on a medium containing 3% beet pulp, 5% BVK, and 2.B% monosubstituted sodium phosphate after 120 hours of cultivation is 106.4 u / ml. after 144 h - 133.7 units / ml.

Currently, there are no producers of ultrasonic galactosidase derived from lactic acid bacteria that could be used as components

starter cultures in the production of cottage cheese and other dairy products. The use of such strains of lactic streptococci will make it possible to obtain serum in which glucose and galactose will be present instead of lactose.

The purpose of the invention is the creation of a producer of ultrasound-galactosidase for the dairy industry, which has high productivity and is capable of secreting the enzyme into the culture fluid based on Str. thermophilus. The goal is achieved by obtaining a new highly active Str producer strain. thermophllus CM1351. This strain is based on the Str strain. thermophllus 6 kb, which we obtained from the VIIKMI collection (Str. thermophllus 6 kb strain was deposited in the All-Union Collection of Industrial Microorganisms of the Institute of VNIIgenetics under

Xi

Ltd

about

W 00

number B-2242). Currently, this strain is used as a component of the starter culture for the production of cottage cheese. Strain Str. thermophllus CM 1351 was obtained as a result of multistage selection using nitrosguanidine. Selective selection was performed according to the ability of cells of a growing culture to secrete the enzyme / 3-galactosidase into the culture fluid. The initial 6 kb Streptococcus thermophllus strain did not have the ability to isolate the enzyme / J-galactosidase into the culture fluid, the ft-galactosidase activity of Streptococcus thermophllus 6 kb is 20,000 units / ml, if the amount of enzyme that hydrolyzes 1, M is taken per unit of activity substrate sz 1 minute at 37 ° C, when converted to 1 µM substrate, the activity is 20 units / ml, which corresponds to a specific activity of 19.6 units / mg protein. The activity of the proposed strain Str. thermophllus CM1351 is 34,000 units / ml, if the amount of enzyme that hydrolyzes 1 M substrate in 1 min at 37 ° C is taken as a unit of activity, when converted to 1 μM substrate, the activity is 34 units / ml, which corresponds to the specific activity 33 units / ml without. Fermentation time 12 h. The enzyme is secreted into the culture fluid. The advantage of this strain over the known strain is the short fermentation time, as well as the ability of this strain to produce extracellular / 2-galactoidase. It should be noted that the new producer of / 3-galactosidase was obtained on the basis of the 6 kb Streptococcus thermophilus strain already used in the dairy industry. Thus, the use of the strain we created allows us to obtain serum with hydrolyzed lactose without naming the existing technology.

The strain Streptococcus thermophilus CM 1351 is deposited in the All-Union Collection of Industrial Microorganisms of the Institute of NI AND Genetics under number B - 5392.

The strain Streptococcus thermophllus CM 1351 has the following cultural and morphological characteristics:

Cell morphologists. Sedentary gram-positive cocci in pairs and chains do not form spores.

On agar medium M21 10 with 1% lactose at a temperature of 37 ° C, after 24 hours it forms round, whitish, slightly convex colonies with a diameter of 0.7-1.5 mm.

In milk with hydrolyzed protein (1.5% agar) at a temperature of 37 ° C, after 24 hours it forms round, whitish, slightly convex colonies with a diameter of 0.5-1 mm.

On lactose agar 11 at a temperature of 37 ° C after 24 hours it forms round, whitish, slightly convex colonies with a diameter of 0.3-0.6 mm.

In liquid medium M21 with 1% lactose and in

liquid hydrolyzed milk after 12 hours forms a strong uniform turbidity, with further cultivation, cell precipitation is observed.

Physiological signs. The strain grows well on synthetic media (M21), milk and whey.

Attitude to carbohydrate nutrition. Absorbs glucose, galactose, lactose, sucrose and fructose.

Attitude to oxygen. Optional aerob.

Attitude to nitrogen sources. Well absorbed by peptone yeast extract. Attitude to temperature. Thermophile

grows at temperatures from TO to 55 ° C, the optimum temperature is 43 ° C.,

Relation to the acidity of the medium. It grows at pH 4.0-9.0, the optimum pH is 7.0.

Genopit. Poliauxotrof, does not contain plasmids, is resistant to kanamycin (10 μg / ml), is not pathogenic.

The product synthesized by the strain. Lactic acid.

Strain storage. The culture is stored in a lyophilized state or in glycerol at -20 ° C for 1 year.

An example of the use of a strain for the production of an enzyme / 3-galactosidase.

To obtain the starting seed, the culture of the strain Streptococcus thermophllus CM1351 was grown for 20 hours at 37 ° C on M21 medium containing the following components, g / l: peptone - 10, yeast extract - 5, lactose - 10.

Na2HPOo-8.5, KH2P04-2, MgS04-1.

The resulting cell suspension is used to inoculate M21 fermentation medium. The fermentation process is carried out in a Marublschi fermenter (1 liter volume),

M21 medium with 5% lactose at a constant pH of 8.0 and a temperature of 37 ° C. A constant pH value is maintained by automatic rubbing with alkali (NsOH). The fermentation process proceeds without artificial aeration with gentle stirring. After 12 hours, the resulting culture fluid was centrifuged to separate the cells. - / 2-galactosidase activity was determined by reaction with 0-nitrophenyl- / 3-galactopyranoside according to the methods of Pardi, Jacob and Mono. The unit of enzyme activity was taken as the amount of enzyme that hydrolyzes 1 μM of the substrate in 1 min at 37 ° C. Under these conditions, the activity of the enzyme in the culture fluid after 12 hours of fermentation was 34 units / ml (34,000 units / ml, if the amount of the enzyme that hydrolyses 1 M substrate for 1 min at 37 ° C is taken as the unit of activity). which corresponds to a specific activity of 33 u / mg protein.

Thus, the advantages of the proposed strain in comparison with the known strain are: short fermentation time, high enzyme activity and the ability to release D-galactosidase in the culture fluid. In addition, the proposed strain is easily integrated into the technological process of food production, as it is based on a strain already used in industry.

Formula a invented and

The bacterial strain Streptococcus thermophllus VKPM B-5392 is a producer of lectosidease.