RU1806193C - Strain of bacterium bacillus polymyxa - a producer of @@@-amylase - Google Patents

Abstract

Usage: biotechnology, the strain can be used for the production of enzyme preparations used for the production of maltoen products for medical purposes, baby and dietary nutrition, as well as for use in the food industry, in particular in the brewing, confectionery, and collapse industries. SUMMARY OF THE INVENTION: Bacillus polymyxa strain VKPM B-5690 is isolated from natural sources and is a producer with resistant morphological characters. On media with starch produces / -amylase at the level of 10-200 u / ml. The technological advantages of the strain are that, along with the synthesis of / -amylase, the strain is capable of synthesis on media with starch exopolysaccharide - polymyxan, which has a stabilizing effect against / 3-amylase. In the complex of concomitant enzymes, α-amylase and neutral protease are absent. which provides high quality maltose syrups. Preparations of β-amylase, obtained on the basis of the proposed producer, can be widely used in the hydrolysis of starch-containing raw materials in order to obtain maltose products, 1 ill., 2 tablets. ё

Description

The invention relates to biotechnology and provides a new strain of bacteria that can be used for the production of enzyme preparations used for the manufacture of maltose products for medical purposes, baby and diet food, as well as for use in the food industry, in particular in brewing, confectionery industry, starch industry.

The aim of the invention is a new bacterial strain that is capable of biosynthesis of / 3-amylase at the prototype level on media containing non-deficient and cheap components, the enzyme complex of which lacks a-amylase and protease, which improves the quality of maltose syrups.

This is achieved by the fact that a new strain of bacteria (stored in the All-Union Collection of Microorganisms - No. B-5690) of B. polymyxa F-12, isolated from natural sources (soil), is offered as a producer of / 3-amylase.

The advantage of the proposed strain B. polymyxa F-12 lies in the fact that the proposed producer, with high ability to biosynthesis of β-amylase (at the level of the prototype), on media containing

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non-deficient and cheap components, does not contain α-amylase and protease in the enzyme complex, which ensures high quality of maltose syrups obtained using the preparations / fr-amylase of the proposed producer. In addition, the producer synthesizes an ecopolysaccharide during cultivation, which provides a reduction in losses during the isolation of enzyme preparations.

The strain has the following characteristics.

Morphological characters: spore-bearing, mobile short thin straight or slightly curved rods, 0.6 ± 0.8 x 3-4 μm in size. Single or paired. The spores are oval, located terminally.

Cultural traits: grows on solid and liquid nutrient media, On synthetic media, growth is weak.

On potato agar, flat, creamy, mucous colonies. By 24 hours of growth - in the form of light plaque, by 48 hours - colonies are well developed, mucous.

On the braids (medium containing 2% soy flour, 5% starch, 2% agar), the colonies are flat, cream, mucous, and well developed.

On wort-agar- (8%) growth is difficult.

On a liquid medium - 2% soy flour, 5% starch, 0.3% CaNRSM grows in the entire volume of the medium (with stirring), sporulation begins from 40-48 hours.

Physiological signs: aerob, temperature limits of growth of 20-40 ° C, optimal growth temperature of 32-37 ° C; grows in the range of pH 5.5-9.0, the optimal pH is 6.0-7.5,

Starch dilutes, fiber does not decompose. Assimilates: glucose, maltose, raffinose, arabinose, xylose, mannitol, sucrose. Not assimilating: dulcite. Milk coagulates, slightly dilutes gelatin. It decomposes nitrates to nitrites, indole and HaS does not form. On media containing 3-10% starch, it is capable of biosynthesis of β-amylase at a level of 10-200 u / ml depending on the composition of the media and culturing conditions. On media with a high starch content (3-10%), it is capable of forming an exopolysaccharide (polimixan).

“-Amylase is absent, glucoamylaea is absent, neutral protease is absent, pollulanase is absent. The activity of hydrolytic enzymes was determined by the following methods:

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1. The activity of fi -amylase - according to Bernfeld (Methods In Enzymol ,, 1955, 1, No. 4, p. 149-150) under standard conditions: pH 7.0,, duration 20 min.

2. a-amylase activity - GOST 20264.4-74.

3. Proteolytic activity - GOST 20264.4-74.

4. Glucoamylase activity - GOST 20264.4-74.

5. Pullulanase activity - US Pat. England Mg 1466009, publ. 2.03.77 g.

To clarify the nature of the enzyme, a blue curve technique was used (Biotechnol. Lett., 1988, 10, No. 2.143-147). The blue curve of B. polymyxa F-12 is similar to the blue curve for B. polymyxa 3 and plant / -amylase (sweet potato from Sowa).

Example 1. The strain B. polymyxa F-12 was grown on medium composition,%: 5 - starch, 1.5 - soy flour, 0.3 - feHPO-i in a 10 l fermenter. The amount of seed 20 by volume. The cultivation was carried out at 37 ° C, the number of revolutions of the stirrer was 4000 rpm, air was supplied in a ratio of 151. The activity of / 3-amylase at 30 hours was 48 units / ml. The activity of α-amylase and neutral protease was absent. The yield of exopolysaccharide is 9 mg / ml. In the control (Bacillus subtilis 2011, grown under similar conditions on a similar medium), the activity of D-amylase is 50 units / ml, the activity of a-amylase is 0.2 units / ml, and the activity of the neutral protease is 0.2 units / ml.

PRI me R 2. The strain B. polymyxa F-12 was grown on rocking flasks with a volume of 750 ml with 50 ml of medium at a temperature of 37 ° C for 48 hours at 220 rpm The composition of the medium,%: 5 - corn starch, 2 - soy flour, 0.3 - KzNRSch initial pH of the medium is 7.0. A 48-hour culture of bacteria grown on potato agar served as seed. The amount of seed is 4% by volume. The activity of ft-amylase in the culture fluid is 200 u / ml, the activity of "-amylase and neutral protease is absent. The yield of exopolysaccharide is 17 mg / ml. In the control (B. subtHls 2011), grown on the same medium under similar conditions, 180 u / ml, o-amylase was 0.5 u / ml, and the neutral protease was 0.3 u / ml.

Example 3. The drug / -amylase B. polymyxa F-12 was obtained by precipitation with ethanol in a ratio of 1: 4 gtri pH 8.4, T ° 2-15 C. Controls were preparations B, subtilis 2011, obtained by precipitation in a ratio of 1: 4 . The results are presented in table. 1.

The preparations of β-amylase obtained from B. polymyxa F-12 culture fluid with a polysaccharide content of 5-17 mg / ml are white in color, easily dried, and have a loose, raw consistency. Preparations obtained from QL B.subtilis 2011 have a beige color, a smeared consistency, it is difficult to dry out, which leads to an increase in activity losses.

Optimum action of preparations of B. polymyxa F-12/3-amylase: pH 6.0-7.0; T ° opt 55-60 ° C. It works in the range of pH 4.5-9.0.

The optimum action of preparations of B. subtills 2011: pH 7.0-7.5; T ° opt 55-63 ° C. works in the range of pH 5.0-8.5.

Example 4. Alcohol deposition D-amylase preparations were used to hydrolyze native corn starch at a rate of 1 unit / g. The hydrolysis temperature of 50 ° C, pH 7.0; 35% solution of corn crash: mala was previously liquefied with a-amylase preparation (Amylosubtilin G-10X) at the rate of 1 unit / g. The liquefied substrate has the following characteristics: DE - 14.5%, maltotriose - 8.38%, maltose - 2.8%, glucose-0.12%.

The drawing shows the change in dextrose equivalent during the saccharification of corn starch with / -amylase preparations.

The qualitative and quantitative composition of maltose syrups evaluated by high performance liquid chromatography is presented in Table. 2.

The optimal duration of hydrolysis for B. polymyxa F-12 is 12-18 hours, for B. subtills 2011 60-72 hours, due to the high specificity of the enzyme ft. Amylase B. polymyxa F-12 for corn starch.

The absence of cz amylase in the complex of enzymes B, polymyxa 1-12, determines a low glucose content; the absence of a neutral protease provides a color index 5 times lower than when using B. subtllis 2011 preparations.

The proposed new strain of B. polymyxa F-12 compared with the prototype has the advantage that, along with the synthesis of / -amylase at the prototype level on starch media, the producer does not synthesize / -amylase and a neutral protease, which ensures high quality of obtained maltose products during the processing of native starch: low glucose content - traces against 2-8% when using the prototype, high maltose content - 36-50% and high dextrose equivalent 31-45%, against 18-35% maltose and 28-38% DE dl prototype and. The use of B. polymyxa 1-12 preparations reduces the hydrolysis time from 60-72 hours for the prototype to 12-18 hours. The ability of the proposed producer to synthesize exopolysaccharide on media with starch simultaneously with / -amylase, in contrast to the prototype, reduces the loss in activity during the isolation of drugs from 51% to 37-41%.

The new B. polymyxa 1-12 strain is a producer with persistent morphological traits. B. polymyxa F-12 preparations / -amylases obtained from a culture fluid with an activity of 10-200 u / ml may have an activity of 250-25000 u / g, depending on the purification method.

Claims (1)

SUMMARY OF THE INVENTION Bacillus polymyxa bacterial strain VKPM B-5690 - producer of β-amylase, .Table 1 table 2 Qualitative and quantitative composition of hydrolysates with duration hydrolysis 12 h