RU2555552C1 - STRAINS OF BACTERIA Bacillus subtilis - HIGHLY ACTIVE PRODUCER OF PECTOLYTIC ENZYMES MACERATING PLANT TISSUE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of bacterium Bacillus subtilis RNCIM B-11964 is proposed, which is a highly active producer of pectolytic enzymes macerating plant tissue.

EFFECT: strain exhibits high pectate-lyase and pectin-lyase activity with respect to pectins from different sources.

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Description

The invention is a new strain of Bacillus subtilis BN-135, relates to the microbiological industry and relates to a new strain producing pectolytic enzymes that macerate plant tissue, namely pectate lyase and pectin lyase, and can be used to obtain enzyme-containing drugs used for the treatment and prevention of diseases associated with disorders of the digestive process, as well as in the preparation of drugs used to increase yield and improve the quality of vegetable and fruit juices and drinks ie, a fruit wine clarification, essential and vegetable oils, tea during processing trudnousvoyaemyh vegetation and obtaining biological additives for agriculture.

The modern direction of dietetics is based on the use in the human diet of more than 2/3 of plant components (vegetables, fruits, cereals) to the total amount of food consumed. This leads to the creation of digestive drugs with the inclusion of enzymes that actively break down plant tissue, without affecting dietary fiber and without forming glucose.

From literary sources it is known that the macerating effect of plant tissue is maximally manifested when a complex of pectolytic enzymes, such as pectate lyase and pectin lyase, is combined, and a special role in the maceration of plant tissue is given to the enzyme pectate lyase, which cleaves pectin substances non-hydrolytically in including insoluble pectin - protopectin of plant tissue, which forms the basis of intercellular substances and "cementing" plant tissue, and the enzyme pectin lyase enhances matz riruyuschee action of pectate lyase (Dean, M. Cell wall degradation by a pectate trans-eliminase / M. Dean, R.K.S. Wood // Natura -. 1967. - №214 - P. 408-410).

In this case, the plant tissue breaks up into individual cells, forming a homogeneous easily digestible mass with preservation of dietary fiber. When protopectin is cleaved, the level of soluble pectin increases and oligogalacturonides are formed, which, like soluble pectin, are effective enterosorbents and remove heavy metal salts and toxins from the body. In addition, under the action of the enzyme pectate lyase, microcellulose fibers are preserved, which are extremely useful for normalizing intestinal motility and cleaning it of toxins.

The need for these enzymes necessitated the search for superproductive strains to obtain highly active macerating preparations that differ not only in high yield of the target product, but also in their ability to ferment on inexpensive and affordable raw materials.

To obtain pectolytic enzymes, bacterial and fungal producers are used in industry.

Pectolytic enzymes synthesized by microscopic fungi have an optimal zone of action in an acidic environment at pH 3.0-5.5 and a temperature of 28-35 ° C and are practically inactive in an alkaline environment.

The optimum action of digestive enzymes and the drug obtained on their basis should be active in a slightly alkaline pH zone, that is, at a pH characteristic of the small intestine, where the main processes of digestion of food nutrients occur. This group of enzymes includes pectate and pectin lyases synthesized by bacterial cultures and actively catalyzing the degradation of pectin at a pH of 6.5-10.0 and a temperature of 35-45 ° C.

In addition, alkaline pectolytic enzymes of macerating action of bacterial origin can be used in the treatment of cellulose and in the processing of bast fiber crops without violating the mechanical strength of the cellulose fiber due to the fact that the enzyme preparations based on them practically do not contain cellulolytic enzymes. Neutral and slightly alkaline pectolytic enzymes find their application in the processing of neutral vegetables and fruits in order to increase the yield and improve the quality of vegetable and fruit juices and purees, clarified fruit and berry wine, for use in the processing of oilseeds to increase the yield of essential and vegetable oils, during processing hardly digestible plant feed and obtaining biological additives for agriculture.

To obtain pectolytic enzymes, aerobic and facultative anaerobic bacterial producers are known.

Optional anaerobic producers are cultivated either under conditionally anaerobic conditions without mixing the nutrient medium, or under aerobic cultivation conditions when immersed during aeration of the nutrient medium. The aerobic process of cultivating producers is more technologically advanced.

A known strain of facultative anaerobic bacteria Bacillus macerans CMPM B-2692 is a producer of pectolytic enzymes (A.S. USSR No. 1162224. The strain Bacillus macerans CMPM B-2692 is a producer of pectolytic enzymes, 1984), which forms pectate under deep cultivation under aerobic conditions -transeliminase (according to the new classification of enzymes - pectate lyase) with an activity of 9000 units / ml, endopoligalacturonase with an activity of 6.7 units / ml, exo-polygalacturonase with an activity of 5.3 units / ml. Cultivation of the producer is carried out on a nutrient medium containing beet pulp as a carbon source, at a temperature of 32-35 ° C, pH 6.8-7.2 for 18-20 hours.

The disadvantages of this strain are the low activity of pectate transeliminase (pectate lyase) and the lack of synthesis of pectin lyase.

The bacterial strain Bacillus macerans UV-21 is known (RF Patent No. 2323974. The bacterial strain Bacillus macerans bkm b-2419d is a producer of pectatliase, pectin lyase and polygalacturonase and a complex of alkaline carbohydrases containing xylanase, beta-glucanase, galactomanazulose, arabine, 2006 ) - which under aerobic conditions when immersed at a temperature of 37-40 ° C, pH 7-8 for 24-48 hours on a nutrient medium containing one or more substrates - sources of carbon, nitrogen and other nutrients, which can be used nature insoluble substrates, for example, beet pulp, or partially soluble substrates — corn extract or starch hydrolyzate, synthesize pectate lyase with an activity of 120–210 units / ml and pectin lyase with an activity of 12–18 units after 36–48 hours of cultivation. / ml and alkaline carbohydrase complex - xylanase -10-18 units / ml, glucanase 12-15 units / ml, arabinase - 8-12 units / ml, xyloglucanase 6-10 units / ml and amylase 15-25 units ./ml.

The disadvantages of this strain include the low activity of macerating enzymes - pectate lyase and pectin lyase in the culture fluid and a long period of cultivation of the producer.

The closest in technical essence to the prototype analogue of the claimed strain is an aerobic strain of bacteria Bacillus subtilis CMPM B-2691 - producer of pectolytic enzymes (A. from the USSR No. 1160742. Strain Bacillus subtilis CMPM B-2691 - producer of pectolytic enzymes, 1985). Optimum cultivation conditions: pH 8.1–8.3; temperature 37-42 ° С. The producer synthesizes on a nutrient medium containing beet pulp, pectolytic enzymes: pectate-transeliminase (pectate-lyase) - 7500 units / ml, endo-polygalacturonase - 20 units / ml, exo-polygalacturonase - 2.0 units / ml per 18-20 hours of cultivation.

The disadvantages of the strain are the low activity of pectate transeliminase (pectate lyase) and the lack of synthesis of pectin lyase.

The aim of the invention is to obtain a bacterial strain Bacillus subtilis BN-135 - having a constitutive character of the synthesis of macerating enzymes and having an increased synthesis of pectate lyase and pectin lyase on media containing both pectin substances and starch-containing substrates exhibiting high pectate lyase and pectin-lyase activity in relation to pectins from various sources.

The technical result that can be obtained by carrying out the invention consists in obtaining a highly active enzyme preparation of macerating action containing highly active pectate lyase and pectin lyase for use in the medical industry and other industries.

To achieve the technical result, it was proposed to use the bacterial strain Bacillus subtilis BN-135, a highly active producer of pectolytic enzymes that macerate plant tissue, pectate lyase and pectin lyase, and use affordable and cheap raw materials for cultivating the producer.

The inventive strain of Bacillus subtilis BN-135 was isolated as a result of multiple screening on an agar medium during aerobic growth of the initial Bacillus subtilis strain CMMP B-2691 and isolation of monospore variants. The options selected on the plates were tested for the productivity of the synthesis of pectate lyase and pectin lyase when cultured in a liquid medium in flasks under conditions of active aeration.

The bacterial strain Bacillus subtilis BN-135 was deposited in the All-Russian Collection of Industrial Microorganisms FSUE GosNII Genetics under No. VKPMV-11964.

Storage conditions. The strain can be stored in a lyophilized state for several years and / or on jambs with potato agar at + 4 ° C with mandatory reseeding at least once for 3-6 months.

The bacterial strain Bacillus subtilis BN-135 has the following cultural, morphological and physiological characteristics.

Cultural and morphological characters.

Cells are straight, rod-shaped, 2.0-5.0 × 0.9-1.0 μm, motile, gram-positive, are found in long and short chains. Spores are ellipsoidal, without exosporium, 2.0-2.4 × 1.0-1.2 μm, located centrally or subterminally.

On potato agar: good growth, the colonies are light beige, round, shiny, the edges are even, the size of the colonies is from 4 to 11 mm in diameter. The pigment does not diffuse on Wednesday.

On wort agar: the colonies are light beige, round, shiny, with a lighter and denser, slightly convex middle, the edges are even, the size of the colonies is from 2 to 6 mm.

On Czapek’s environment with glucose: the colonies are homogeneous, convex milky white, shiny, transparent, the edges are even, the growth is plentiful, the size of the colonies is from 2 to 5 mm.

On meat and peptone agar: weak growth, dotted, beige colonies.

Physiological and biochemical characteristics of the strain

Attitude to carbohydrates: assimilates glucose, sucrose, xylose, arabinose, maltose and mannitol with the formation of acid and gas, hydrolyzes starch.

Forms catalase, acetone, crystalline dextrins. Nitrates are reduced to nitrites. Phenylalanine does not deamine; tyrosine does not cleave. Ammonia and hydrogen sulfide does not form. Hippurates do not hydrolyze. Gelatin dilutes 1 cm at the top. Milk peptones with the formation of a clot and gas. It grows on a medium with 0.001% lysozyme, does not grow on a medium with 5% sodium chloride.

It grows well on slices of carrots, potatoes, turnips, white cabbage, zucchini, pumpkin, apple (non-acidic varieties), pears with complete maceration of the substrate; on hydrolyzed starch, various types of grain flour.

Attitude to oxygen - aerob. The minimum growth temperature is + 10 ° C, the maximum + 45 ° C, the optimal 37-42 ° C, pH 6.8-7.6.

This type of bacteria is not listed as pathogenic in the sanitary-epidemiological rules of SP 1.3.2322-08 / SP 1.3.2518-09 “Safety of work with microorganisms of the III-IV pathogenicity (danger) groups and pathogens of parasitic diseases”.

The resulting strain of Bacillus subtilis BN-135 in its morphological characteristics during growth on potato agar differs from the original strain of Bacillus subtilis CMMP B-2691 in the large size of the colonies and the later entry into the stage of spore formation.

The obtained strain, by its biochemical properties, unlike the initial strain, has the constitutive character of the synthesis of pectate lyase and pectin lyase, differs from the initial one in increased oxygen demand and increased ability to enzyme biosynthesis during deep cultivation under aerobic conditions on liquid nutrient media containing a carbon nutrition source, both pectin substances and starch-containing substrates.

The cultivation of the strain Bacillus subtilis BN-135 is carried out under aerobic conditions in a submerged state at a temperature of 37-42 ° C, pH 6.8-7.6 for 18-24 hours.

The activity of pectate lyase was determined by the ability to cleave pectins of a low degree of esterification: beetroot - with an esterification degree of 35-37% and citrus - with an esterification degree of 20-34%, pectin lyase - by the ability to cleave pectins of a high degree of esterification: apple - with a degree of esterification 70% and citrus - with an degree of esterification of 66%. The unit of activity is such an amount of enzyme that for 1 hour at 40 ° C and a pH of 8.4 causes the formation of unsaturated degradation products of pectin, increasing the optical density by 0.1. The method is based on direct spectrophotometric determination of unsaturated decomposition products of pectin having a maximum optical density in the spectrum at a wavelength of 235 nm, in quartz cuvettes with an absorbing layer thickness of 10 mm.

The determination of macerating ability was carried out according to a test based on determining the change in the mass of a plant substrate as a result of the action of an enzyme preparation obtained from the culture fluid of the claimed producer in the amount of 450 pectate lyase units per 1 g of substrate for 4 hours. As a plant substrate, crushed carrots were used in Tris-hydrochloric acid buffer with a pH of 8.4. The degree of maceration of carrots was determined by the decrease in the mass of the substrate treated with the enzyme in comparison with the control variants and was expressed as a percentage.

The possibility of using the invention is illustrated by examples, which do not limit the scope and essence of the claims associated with it.

Example 1

The culture of Bacillus subtilis BN-135 was grown in flasks on a liquid nutrient medium of the following composition, g / l:

Ground beet pulp - 40.0 Disubstituted Ammonium Phosphate - 75.0 Potassium phosphate monosubstituted - 10.0 Corn extract - 5.0 a piece of chalk - 5.0 Tap water - the rest

The nutrient medium was poured into 50 ml into rocking flasks with a capacity of 750 ml and sterilized at a pressure of 0.1 MPa for 45 minutes. An aqueous suspension of spores of the Bacillus subtilis BN-135 strain, which are formed on the surface of mowed potato agar after inoculation and growth for 48 h at 40 ° C, in an amount of 5% by volume of the nutrient medium, was used as a seed. The pH of the medium before cultivation was set at 7.0 using solutions of hydrochloric acid or potassium hydroxide. Cultivation was carried out under aerobic conditions on a rocking chair with a rotation frequency of 200-220 rpm, a temperature of 40 ° C and for 18 hours.

Example 2

The culture of Bacillus subtilis CMMP B-2691 (prototype) was grown on a nutrient medium and under the conditions corresponding to example 1.

Example 3

The culture of Bacillus subtilis BN-135 was grown for 20 hours under conditions corresponding to the conditions of example 1. The composition of the nutrient medium differed from the composition of example 1 by the content of rye flour in an amount of 25 g / l instead of beet pulp.

Example 4

The culture of Bacillus subtilis CMPM B-2691 (prototype) was grown for 24 hours on a nutrient medium and under the conditions corresponding to example 3.

Example 5

The culture of Bacillus subtilis BN-135 was grown for 22 hours under conditions corresponding to the conditions of example 1. The composition of the nutrient medium was different from the composition of example 1 with barley flour in an amount of 25 g / l instead of beet pulp.

Example 6

The culture of Bacillus subtilis CMPM B-2691 (prototype) was grown for 24 hours on a nutrient medium and under conditions corresponding to the conditions of example 5.

At the end of producer culture, enzyme activity was determined in a centrifugate obtained by centrifuging the culture fluid at 10,000 rpm for 10 minutes.

These results of experiments on the cultivation of the claimed strain and prototype are presented in table 1.

To determine the macerating ability at the end of the fermentation process, dry enzyme preparations were obtained from the culture fluids of the claimed strain and prototype obtained on a nutrient medium using beet pulp (example 1, 2), and preparations of the claimed strain obtained using rye flour (example 3) and barley flour (example 5). For this, the culture fluid was separated from the biomass by centrifugation, and the centrifugate was concentrated by ultrafiltration on a UPM-50 membrane with a pore size of 50,000 Da and dried on a freeze dryer. The resulting preparations were used for maceration of carrots.

The enzymatic activity of the preparations obtained and the macerating ability of carrots are presented in table 2.

The degree of maceration of carrots with the preparations of the claimed strain was close and amounted to: using beet pulp - 38.2%, using rye flour - 38.4%, using barley flour -37.9%. The degree of maceration of carrots with the drug obtained from the culture fluid of the prototype using beet pulp was lower and amounted to 30.5%.

Thus, the use of the inventive strain of Bacillus subtilis BN-135 compared with the prototype has the following advantages:

1. Increased synthesis of pectate lyase - up to 18600 units / ml (in the prototype up to 8560 units / ml) on a nutrient medium containing beet pulp, and up to 26700 units / ml on a nutrient medium containing starch-containing substrate (in the prototype up to 1400 units / ml).

2. High synthesis of pectin lyase - up to 5200 units / ml (in the prototype up to 260 units / ml) on a nutrient medium containing beet pulp, and up to 6900 units / ml, on a nutrient medium containing starch-containing substrate (in prototype - missing).

3. Higher macerating activity of plant materials (carrots).

The inventive strain of Bacillus subtilis BN-135, in contrast to the prototype, has a constitutive character of synthesis of enzymes macerating plant tissue — pectate lyase and pectin lyase, has the ability to synthesize pectate lyase and pectin lyase on nutrient media containing pectin as a carbon source substances and starch-containing substrates, has a high activity against pectins from various sources, and preparations obtained using the inventive strain have a higher macerating ability.

To achieve high activity of the strain, the use of complex and expensive nutrient media is not required, which will make it possible to obtain highly active macerating preparations containing pectate lyases and pectin lyases for its industrial use for use in the medical industry, as well as in the food industry and agriculture.

Claims (1)

The bacterial strain Bacillus subtilis VKPM B-11964 is a highly active producer of pectolytic enzymes that macerate plant tissue.