RU2571945C1 - Nutrient medium to cultivate bacteria bacillus subtilis bn-135- producent of pectate lyase - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: nutrient medium for depth cultivation of bacteria Bacillus subtilis BN-135 contains hydrolyzed rye flour or hydrolyzed barley flour, corn extract, di-basic ammonium phosphate, potassium dihydrogen phosphate, calcium chloride, sodium carbonate and tap water at the specified ratio of components.

EFFECT: invention makes it possible to increase activity of pectate-lyase.

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Description

The invention relates to the microbiological industry and relates to a nutrient medium for the cultivation of the bacterium Bacillus subtilis BN-135, a producer of pectate lyase.

A preparation based on pectate lyase can be used in the preparation of enzyme-containing drugs used to treat and prevent diseases associated with digestive disorders, as well as in the preparation of drugs used to increase the yield and improve the quality of vegetable and fruit juices and mashed potatoes, fruit - clarified berry wine, essential and vegetable oils, tea, in the processing of difficult to assimilate vegetable feed and obtaining biological additives for agriculture.

The enzyme pectate lyase belongs to enzymes of macerating action and carries out non-hydrolytic cleavage by trans-eliminative means of pectin substances, including insoluble pectin - protopectin of plant tissue, which forms the basis of intercellular substances and "cementes" plant tissue. In this case, the plant tissue breaks up into individual cells, forming a homogeneous 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.

The composition of the culture media for the cultivation of pectate lyase producers has been well studied at the laboratory and semi-industrial levels. In their composition, sources of pectin are most often used as a carbon source, in particular beet pulp or its hydrolyzate, and sometimes starch-containing raw materials.

Known nutrient medium for deep cultivation of the producer of macerating enzymes Bacillus circulans 31 (AS USSR 1026405. Nutrient medium for deep cultivation of the macerating enzymes Bacillus circulans 31 - producer of macerating enzymes, 1981), containing, g / l: alkaline hydrolyzed beet pulp - 26-80 , corn extract 4-20, urea - 3-10, calcium carbonate - 0.5-5.0, potassium sulfate - 0.5-3.0. The producer is cultivated under aerobic conditions for 18 hours in a slightly alkaline zone with an initial pH of the nutrient medium of 8.2-8.5. The proposed nutrient medium allows to obtain the activity of pectate-trans-eliminase (according to the new classification of enzymes - pectate-lyase) up to 60 units / ml for 18 hours of growth.

A significant drawback of this nutrient medium is the low activity of pectate-trans-eliminase (pectate-lyase) in the culture fluid.

A known nutrient medium for the cultivation of the facultative anaerobic bacterium Bacillus polymyxa in a method for producing an enzyme that destroys plant material is pectate trans eliminase (pectate lyase) (German Patent No. 137585. An enzyme that destroys plant material, the process for its preparation and use in compositions for digestion, 1974), which provides for the cultivation of the strain under aerobic conditions using a nutrient medium containing starch, sugarcane, molasses, glucose or a mixture of these components as a carbon source nents, and as a source of nitrogen - soybean flour, corn extract, yeast extract, peptone, nitrates, ammonium salts, or a mixture of two or more of these components together with organic and / or inorganic salts, especially calcium carbonate. It is proposed to carry out cultivation of the producer in a batch manner with constant stirring and aeration and maintaining the pH at 5.2-7.8 for 2 to 4 days. When the enzyme was isolated from the culture fluid, a yield of pectate-trans-eliminase (pectate-lyase) was obtained up to 30,000 units / ml.

The disadvantages of this nutrient medium can be attributed to a fairly high activity of pectate-trans-eliminase (pectate-lyase) for a long period of cultivation of the producer.

The closest analogue of the prototype to the claimed nutrient medium is a nutrient medium for cultivating the aerobic bacterium Bacillus subtilis CMPM B-2691 - producer of pectolytic enzymes (A.C. USSR No. 1160742. Strain Bacillus subtilis CMPM B-2691 - producer of pectolytic enzymes, 1985). The culture medium contains, g / l: ground beet pulp - 40.0, disubstituted ammonium phosphate - 75.0, monosubstituted potassium phosphate - 10.0, corn extract - 5.0, chalk - 5.0, tap water - rest. Optimum cultivation conditions: pH 8.1–8.3; temperature 37-42 ° C. The maximum content of pectate-trans-eliminase (pectate-lyase) in this nutrient medium is reached by 18-20 hours of cultivation and is 7500 units / ml. Additionally, the strain synthesizes endopoligalacturonase with an activity of 20 units / ml and exopoligalacturonase with an activity of 2.0 units / ml.

The disadvantage of this nutrient medium is the low activity of pectate transeliminase (pectate lyase) in the culture fluid.

For large-scale production of enzymes, in addition to the presence of superproductive producer strains, the composition of the nutrient medium, which primarily affects the yield of the target product and, accordingly, the profitability of the whole process, is decisive.

Nutrient media based on beet pulp are fairly depleted in carbohydrates. The bulk of the pulp are pectin substances, the content of which can reach up to 40%, and fiber (up to 22%). Beet pulp as a waste product from the production of sucrose from sugar beets does not have a standard composition, therefore, each batch of pulp must be characterized by chemical composition and checked in the laboratory during experimental cultivation. Beet pulp requires preliminary grinding and strict sterilization modes, which increases energy consumption in the production of the enzyme. The culture fluid obtained using beet pulp is poorly filtered. In addition, the current volumes of beet pulp cannot fully satisfy the needs of agriculture for feeding agricultural animals and the microbiological industry to obtain enzyme preparations (Gracheva, I.M., Krivova A.Yu. Technology of enzyme preparations / 3 -th ed., revised and enlarged. - M.: “Elevar”, 2000. - 512 p.).

The technical problem to which the present invention is directed is to develop a new, more productive and technological composition of the nutrient medium containing affordable and cheap raw materials and providing high synthesis of pectate lyase.

The technical result that can be obtained by carrying out the invention is to increase the activity of pectate lyase in the culture fluid to values not lower than 40,000 units / ml in 18-22 hours of cultivation.

To achieve the technical result for the deep cultivation of Bacillus subtilis BN-135, a producer of pectate lyase, it was proposed to use a nutrient medium consisting of a carbon source, corn extract, ammonium phosphate disubstituted, potassium phosphate monosubstituted, nutrient salts and tap water. In order to increase the activity of pectate lyase in the culture fluid, it was proposed to use a starch-containing substrate, rye or barley flour, previously hydrolyzed with mushroom cellulase, instead of beet pulp, as calcium chloride and sodium carbonate in the following ratio of components, weight. %:

hydrolyzed rye flour or hydrolyzed barley flour 2.5-4.0 corn extract 0.4-0.6 disubstituted ammonium phosphate 0.6-1.0 monosubstituted potassium phosphate 0.3-0.5 calcium chloride 0.05-0.15 sodium carbonate 0.15-0.2 tap water rest

Rye and barley flour used in the nutrient medium, in addition to starch, also contains fiber, hemicellulose and pentosans. The quantitative content of carbohydrates in this type of starch-containing raw materials is presented in table 1.

It is proposed to use cellulolytic enzyme preparations of fungal origin, for example, Celloviridin or CellloLux-F, which contain, in addition to cellulase, glucanase and xylanase enzymes as an enzyme preparation. The enzymatic composition of these preparations is presented in table 2.

The hydrolysis of starch-containing substrate is proposed to carry out fungal cellulase at the rate of 5 units. cellulase per 1 g of raw material for 1.0-2.0 hours at a temperature of 50 ° C.

The use of rye or barley flour, previously hydrolyzed with a mushroom cellulolytic enzyme preparation, allows one to obtain a nutrient medium containing reducing sugars in an amount that does not cause repression of pectate lyase synthesis. The presence in the environment of easily metabolizable sugars in optimal concentrations in the form of flour carbohydrate degradation products helps to reduce the initial unproductive cultivation stage (lag phase), more rapid growth of the producer and the accumulation of an increased amount of biomass, which leads to a significant increase in the level of enzyme biosynthesis in the culture fluid.

The claimed nutrient medium has a pH close to neutral (about 7.0), which is optimal for the biosynthesis of pectate lyase by the bacterium Bacillus subtilis BN-135. Producer cultivation in this medium is carried out at a natural pH of the nutrient medium, which excludes additional operations to adjust the pH of the medium before inoculation and during cultivation of the producer.

In the present invention, pectate lyase activity was determined by the ability to cleave beet pectin with a low degree of esterification (degree of esterification 35-37%). The unit of activity is such an amount of an 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 at a wavelength of 235 nm, in quartz cuvettes with an absorbing layer thickness of 10 mm.

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 rocking flasks with a capacity of 750 ml, containing 50 ml of culture medium of the following composition, weight. %:

hydrolyzed rye flour 2,5 corn extract 0.6 disubstituted ammonium phosphate 0.6 monosubstituted potassium phosphate 0.3 calcium chloride 0.1 sodium carbonate 0.15 tap water rest

Rye flour was previously hydrolyzed with the enzyme preparation Celloviridin at the rate of 5 units. cellulase per 1 g of raw material for 1.0 h at a temperature of 50 ° C. The culture medium was sterilized at a pressure of 0.1 MPa for 45 minutes.

An aqueous suspension of producer culture spores, which form on the surface of mowed potato agar after inoculation and growth for 48 h at 40 ° C, was used as a seed. To obtain inoculum, 20 ml of sterile tap water was added to the test tube with slanted agar and rinsed from the jamb using a microbiological loop under aseptic conditions. Spore seed was made in an amount of 5% by volume of the fermentation medium. Cultivation was carried out under aerobic conditions on a rocking chair with a rotation frequency of 200-220 rpm at a natural pH of the nutrient medium and a temperature of 40 ° C. The activity of pectate lyase in the culture fluid after 20 hours of cultivation of the producer was 41,600 units / ml.

Example 2

The cultivation of the bacteria Bacillus subtilis BN-135 was carried out for 22 hours under conditions corresponding to the conditions of example 1, on a nutrient medium of the following composition, weight. %:

hydrolyzed barley flour 3.0 corn extract 0.4 disubstituted ammonium phosphate 1,0 monosubstituted potassium phosphate 0.5 calcium chloride 0.05 sodium carbonate 0.15 tap water rest

Hydrolysis of barley flour was carried out with the enzyme preparation Celloviridin at the rate of 5 units. cellulase per 1 g of raw material for 2.0 hours at a temperature of 50 ° C. The activity of pectate lyase in the culture fluid after 22 hours of cultivation of the producer was 42300 u / ml.

Example 3

The cultivation of Bacillus subtilis BN-135 was carried out in a 10 liter BIOSTAT fermenter (fill factor 0.5) on a nutrient medium of the following composition, weight. %:

hydrolyzed rye flour 4.0 corn extract 0.5 disubstituted ammonium phosphate 0.8 monosubstituted potassium phosphate 0.4 calcium chloride 0.15 sodium carbonate 0.2 tap water rest.

Hydrolysis of rye flour was carried out with the enzyme preparation CelloLux-F at the rate of 5 units. cellulase per 1 g of raw material for 1.5 hours at a temperature of 50 ° C. The culture medium was sterilized at a pressure of 0.1 MPa for 45 minutes.

For inoculation of the nutrient medium, an aqueous suspension of spore inoculum of the Bacillus subtilis BN-135 culture was used in an amount of 5% by volume of the nutrient medium.

Cultivation of the culture in the fermenter was carried out under the following conditions: temperature 40 ° C, continuous aeration (air supply - 1 vol. Per 1 vol. Medium per min) and stirring with a stirrer speed of 200 rpm at a natural pH value.

The duration of the fermentation is 18 hours.

The activity of pectate lyase in the culture fluid after 18 hours of culture of the producer was 48,400 units / ml.

Thus, the proposed nutrient medium for the cultivation of the bacterium Bacillus subtilis BN-135 - producer of pectate lyase contains affordable and cheap raw materials, and its use allows to increase the activity of pectate lyase in the culture fluid in comparison with the prototype 5.5-6.5 times (from 7500 units / ml to 41600-48400 units / ml) for 18-22 hours of cultivation.

Claims (1)

The nutrient medium for the deep cultivation of bacteria Bacillus subtilis BN-135 is a pectate lyase producer, consisting of a carbon source, corn extract, ammonium phosphate disubstituted, potassium phosphate monosubstituted, nutrient salts and tap water, characterized in that the nutrient medium as a carbon source contains starch-containing substrate - rye or barley flour, previously hydrolyzed with fungal cellulase, calcium chloride and sodium carbonate as trace nutrients the ratio of components, wt.%:
hydrolyzed rye flour or hydrolyzed barley flour 2.5-4.0 corn extract 0.4-0.6 disubstituted ammonium phosphate 0.6-1.0 monosubstituted potassium phosphate 0.3-0.5 calcium chloride 0.05-0.15 sodium carbonate 0.15-0.2 tap water rest