RU1799395C - Strain of bacterium acidocaldarius used for straw ensilage - Google Patents

Abstract

Usage: biotechnology and can be used in the production of bacterial biomass used as a starter culture for bioconversion (silage) of straw. SUMMARY OF THE INVENTION: Bacillus acidocaldarius bacterial strain obtained

Description

The invention relates to agriculture and can be used in fodder production for the microbial conversion of straw or other cellulose-containing substrates in order to obtain feed with increased eatability and nutritional value in comparison with the feedstock.

An object of the invention is to provide a new strain of cellulose-assimilating bacteria.

This is achieved by using Bacillus acidocaldarius 17, a strain that differs from the C.flavigena prototype strain by a higher population stability and processability, which helps to reduce costs and obtain stable results both in the production of starter culture and in the bioconversion of the substrate by the silage method. Advantage

The proposed strain before the previously known is the absence of polymorphic ™ and population resistance. The spore-forming ability of B. acidocaldarlus 17 SO 1, its sQ thermal stability and acid tolerance Јд} provides higher s survival under extreme conditions, in particular when dry preparations are prepared by spray or freeze drying method j, as well as in the process of silage maturation, when Јд} the temperature of the silt mass will increase to and above. This temperature, combined with the acidic pH of the medium, causes the death of the prototype strain and termination of the cellulose digestion process, while B. acidocaldarius 17 remains viable and active under these conditions. B. acididarius 17 differs from C. flavigena 22 in significantly higher

. indicators of growth rate, biomass accumulation activity and economic coefficient, which reduces the cost of producing bacterial starter cultures.

The high adaptability of the B. acidocaldarius strain, established in laboratory experiments, during the production of starter cultures at the pilot industrial installation of the Institute of Microbiology and Virology of the Academy of Sciences of the Republic of Kazakhstan, as well as in production tests, showed the promise of its widespread introduction in fodder production. This strain is recommended for preparing starter cultures under industrial production conditions. At the Stepnogorsk Scientific Experimental Industrial Base (SNOPB PO Progress), the experimental industrial regulations for producing its biomass have been worked out.

The proposed bacterial strain B.acidocaldarius 17 has the following morphological and physiological characteristics.

Gram-positive bacteria are both temperature- and acid-resistant. . These are sticks with rounded ends, size 0.8 x 3.5 microns; with age, they approach in shape to cocci. They are arranged singly, in pairs and in short chains. End spores. Movable, optional aerobic.

Enzymatic properties.

a) saccharolytic:

1. Break down glucose, arabinose, xylose, sucrose, maltose to acid. When carbohydrates are fermented, gases do not form,

2. Hydrolyze polysaccharides: starch, dextrin and cellulose.

3. From alcohols hydrolyze mannitol, no dulcite.

b) proteolytic:

1. On BCH, ammonia forms well, while indole and hydrogen sulfide are weak.

2. Gelatin is diluted. .

3. Cause alkalization and proteo. milk lysis.

1. Acetoin does not form.

2. Catalase form.,

3. Tyrosine is not cleaved.

4. Casein is not hydrolyzed.

5. Nitrates are reduced to nitrites.

6. There is no growth at 0.001% lysozyme,

7. On the BCH form click.

8. 1-10% NaCI growth is good.

9. On Saburo's environment, growth is good. Colo1 is not gray with a slight pink tint.

10. Growth on the BCH - the film is thin. The environment is transparent. The precipitate is medium, friable.

11. Growth on RPA - the surface of the brain is visible. Matova (the mealy). Beige gray. The edge of the colony is lobed. The structure is coarse-grained, the consistency is dense, it is easily removed from the agar by a film, and is not torn behind a loop (not a mucous membrane). The ability to emulsify is a suspension in the form of a piece of film.

12. Gelatin injection. Light beige surface growth. Lamination is layered, very slow.

The strain synthesizes a constitutive complex of cellulases characteristic of cellulolytic bacteria. At a concentration of 10 bacterial cells in 1 ml of medium, cellulolytic activity is 7–9 mg

Rv / ml. (on filter paper);

The culture is stored on mowed wort agar or 0.7% straw agar at 5 ° C. reseeding is required after two months. For the cultivation of the strain, a liquid medium of the following composition is used, g / l:

NaN03: 2.0 K2HP04 1.0 MgSO / j 0.3. NaCI 1.0 Yeast auto-release 1.0 Glucose or starch: South Cultivation 24 hours 30-35 ° C, pH 6-7. The strain is isolated from prelum straw by reseeding on a medium with straw agar. It is able to develop on straw under anaerobic conditions at silage pH values of 3.8-4.7.

at. .; ....:; - ;:.

. To enhance the bacterial conversion of cellulose, B. acidocaldarius 17 (CLB) is used in a mixed culture with Streptococcus lactis diastaticus (AMC).

which assimilates the metabolic products of cellulolytic bacteria, eliminates catabolite repression and stimulates its growth with some (unidentified) products of its metabolism, without exerting an antagonistic effect. The presence of prerequisites for symbiotic coexistence of these microorganisms allows their biomass to be used for preparation

starter cultures in one fermentation, which increases its economic efficiency.

PRI me R 1, Our proposed strain of cellulolytic bacteria of B. acidocaldarius 17 and the prototype strain

Cellutomonas flavigena 22 was grown in a laboratory fermenter with a working volume of 2 l on a modified Hutchinson medium (g / l): MaMOz 2.0; K2HPO / I 1.0; MgSO 0.3; NaCl 1.0; yeast eutolysate 1.0; GLUCOSE

10 g. The cultivation was carried out with stirring at 32 ° C, pH 6-7.

The specific growth rate of B. acidocaldarius was 0.42, and C. flavigena was 0.10. The stationary growth phase of the first strain is observed after 17 hours of cultivation, the maximum synthesis of cellulose complex enzymes after 24 hours. The amount of biomass was 4.1 g / l, the economic coefficient of processing the substrate was 0.51, and the productivity was 1.72 g / l per hour. Generation time 1 h 39 min.

The cultivation time of C. flavigena before the onset of the stationary phase is 6 hours, biomass accumulation 3.6 g / l, economic coefficient of substrate processing 0.40, productivity 0.36 g / l per hour, generation time 6 hours 56 minutes.

Using both strains of cellulolytic bacteria, solid-phase fermentation of wheat straw was carried out.

The straw is crushed to a size of 3-10 cm. A conventional feed silage technique is used. The straw is hydrated in layers with an aqueous solution to a moisture content of 65%. The solution is sprayed uniformly over the entire surface of the silage from the tank by means of a pump through a hose with a spray at the end. The working solution consists of the starter culture B and AMS (1: 1) at the rate of 3 g of dry preparation titer of 10 cells in ml per 1 ton of straw in 1.0% sodium chloride. After thoroughly tamping with a tractor, the trench is sealed with a film. ... Analysis of the obtained product was carried out after 30 days. The organoleptic characteristics of both silos are good. When using B. acidocaldarius 17, the silo has the smell of bread dough, while the silage of C. flavigena 22 fermented vegetables, pJ In either case, 4.3–4.4, the amount of organic acids 1.6 g / kg. Thus, we can conclude that the organoleptic characteristics that determine the eatability of silage when using various cellulolytic bacteria practically do not differ. Determination of the amount of hardly hydrolysable.x sex of isaccharides (cellulose) in the obtained silage samples showed that when using the proposed strain of .acidocaldarius 17 bacteria, their destruction process proceeds deeper. So, the cellulose content decreased by 10%, while in the case of C. flavigena by 8.5%.

Since the hydrolysis of difficultly hydrolyzable polysaccharides is carried out using the CBL, a way to improve

digestibility of feed, it is necessary to recognize the advantage of the claimed mmmm over the prototype strain.

Example 2. As our previous studies have shown, a favorable effect on the development of C. flavigena 22 is exerted by the lactic acid bacteria strain Lpentoaceticum B (b) -23. Growth B.acidocaldarius

17 is stimulated by another strain of lactic acid bacteria Str. lactis diastaticus. Solid phase fermentation of wheat straw was carried out using selected mixed cultures of cellulolytic and lactic acid bacteria.

. The cultures were grown under laboratory conditions (see Example 1) and mixed with cellulolytic bacteria before use. lactic acid. in the ratio 1: 1.

The results obtained showed that when mixed cultures of cellulolytic and lactic acid bacteria are used in both cases for siloing the straw, the resulting product is slightly

different from silage prepared with starter cultures from monocultures of cellulolytic bacteria. It determines only a little more lactic acid (0.1t 0.2%, depending on the type of experiment) and

slightly less bound butyric acid. Apparently, when using a monoculture of cellulolytic bacteria, organic acids are produced by representatives of spontaneous lactic acid flora abundantly found in benign straw. To confirm this assumption, in the next series of experiments, wheat straw was preliminarily subjected to alkaline pretreatment, which on the one hand promotes its delignification and, on the other hand, kills it. spontaneous microflora (Table 2). The data obtained showed that the use of

In this case, monoculture of cellulolytic bacteria leads to the production of negative organoleptic products. indicators (unpleasant smell and taste) that are not eaten

animals. In addition, under alkaline conditions, the development of putrefactive microflora is possible. It has been shown that in order to obtain a complete product from pre-treated alkali straw, the introduction of

in the composition of the ferment of lactic acid bacteria. As a result of their development, lactic and acetic acids accumulate, which reduce the pH of the medium and ensure the conservation of the product, as well as improve organoleptic characteristics.

The above results allow us to conclude that in order to ensure the desired process direction in the case of using non-standard raw materials for bioconversion, it is necessary to introduce into the composition of the starter culture 5 lactic acid bacteria in addition to cellulolytic.

The data below allow us to conclude that the mixed culture of CLB and AMS is advantageous. 10

Example 3. Biomass C. flavlgena (prototype), grown on a pilot plant of the Institute, its titer is 0.2 billion cells / g. Microscopy and sowing 15 cultures on nutrient media showed its high polymorphism. Cells are a mixture of cocci and Gram-positive and Gram-negative rods. The cellulolytic activity of the obtained biomass is difficult to control, because to manifest it in the medium, it is necessary to have an inductor (cellulose source), which causes a lower growth rate of the culture, but provides synthesis of 25 cellulase complexes. For this reason, C. flavigena biomass obtained on media containing digestible sugars as a carbon source often has very low cellulolytic activity or no activity at all.

When B. acidocaldarius is cultured on the medium described above, maximum cellulolytic activity is noted

after 24 hours of cultivation. at a temperature of 50 ° С, the activity of PS (CMCase) reaches 140 activity on a filter medium of 9 mg RV / ml, cellobiose - 3 m

Received in the industry. tera (SNOPB on Progress), starter culture (dry and pa B.acidocaldarius (Caldarin) and characteristic

The culture is homogeneous, numbered by presence, gram-positioned check, at the beginning of the stationary one a single

Cellulolytic active as in the presence of carbon sources in the medium; in monosugars. In B. acidocaldarjus, the prototype allows constituents to be active in any utilized substratum.

Thus, the offer of cellulolytic B. acidocaldarius 17 is characterized by a higher technological level. It allows the successful use of a stable production for printing high quality feed products.

Claims (1)

Claims The bacterial strain Bacillus VKPM B-5250 used no straw. Table 1 Comparative characteristics of the production-valuable properties of the proposed strain of bacteria and strain of the prototype after 24 hours of cultivation. At pM 6.0 and a temperature of 50 ° C, the activity of endoclucose (CMCase) reaches 140 mg of RV / ml. activity on filter paper (APB) - 9 mg RV / ml, cellobiesis - 3 mg glucose / ml. Obtained in industrial trusses. terach (SNOPB ON Progress) experimental batches, starter cultures (dry and pasty) B.acidocaldarius (Caldarin) has the following characteristic The culture is homogeneous, clean, represented by the presence of gram-positive rods, at the beginning of the stationary growth phase, the appearance of single spores is observed. Cellulolytic activity was found both in the presence of cellulose-containing carbon sources in the medium and in monosugars. The constitutive type of the enzyme in B. acidocaldarjus allows, in contrast to the prototype, to obtain active biomass on any utilized substrate. Thus, the proposed strain of cellulolytic bacteria B. acidocaldarius 17 differs from the known one by its higher manufacturability, which makes it possible to use it successfully for the stable production of starter cultures ensuring the high quality of the obtained feed product. . The claims The bacterial strain Bacillus acidocaldarius VKPM B-5250 used to bind straw. Na-carboxy methyl cellulose Ability to fail .-. peitoses (xylose and arabi nose) Cellulose complex enzyme activity: endoglphanase (CMCase) on filter paper celgoia The need for rst factors 0.32 Discard  mg Rv / ml - 13.1 e / cl 3 mg RV / ml - 0.83 units / mp 1 mg cont. / Ml-0.28 units / ml Not required Indicators of a biochemical analysis of silage from native wheat straw and from a pre-treated 0.5% silt solution Characterization of experimental batches of sourdough Table2 Table3