RU2107694C1 - Method for production of exopolysaccharides - Google Patents

Abstract

FIELD: biotechnology, namely, production of capsule polymers of carbohydrate nature. SUBSTANCE: natural association of bacteria which colonise on surface of glass plates being poured into sea water is used as raw materials. Raw materials are treated with active chlorine (its concentration being 0.5-1.5 mg/l) within 4 ± 0.2 h. EFFECT: improved efficiency of the method. 2 cl, 4 dwg

Description

 The invention relates to biotechnology and can be used to obtain capsular polymers of carbohydrate nature, widely used in medicine, food, cosmetic, textile industry, metallurgy, and also when drilling wells.

 The search for strains producing these compounds, as well as methods and means that intensify their synthesis, is of undoubted interest to the national economy.

 It is known to obtain polysaccharides from bacterial strains (Ed. St. USSR N 1366528, class C 12 P 19/04, 1988). A disadvantage of the known methods is, firstly, the lack of a means of intensifying the process of formation of polysaccharides; secondly, technological complexity is the need for special microbiological equipment.

 It is known (Sentsova O.Yu., Maksimov VN The effect of heavy metals on microorganisms. // Advances in microbiology. 1985. Issue 20. S. 227-251.), Which in response to adverse effects, in particular on the presence of ions heavy metals, bacteria activate the synthesis of exopolysaccharides that bind metals to form low-toxic complexes (prototype).

 The disadvantage of this method is the limited use of the obtained polysaccharides due to the presence of heavy metals in the form of organometallic complexes.

 The aim of the invention is to optimize and increase the efficiency of the process of producing exopolysaccharides.

 This goal is achieved by the fact that in a method that includes pretreatment of biological raw materials with ethanol and water, followed by extraction with 2% sulfuric acid and 2% alkali in combination with heat treatment and isolation of the target product, natural associations of bacteria are used as the feedstock, settling on the surface of glass plates immersed in the marine environment, and before pre-treatment the raw materials are exposed to "active chlorine" (AHL) and in concentrations of 0.5-1.5 mg / l for 4 ± 0.2 hours

 In the invention, as a negative agent initiating the synthesis of protective fractions of bacterial polysaccharides, a complex of "active chlorine" compounds formed during the electrolysis chlorination of sea water is proposed. This concept includes a group of compounds, in particular dissolved molecular chlorine, chlorine dioxide, chloramines, etc. At pH values of 8.0 ± 0.5 typical of sea water, the main components of “active chlorine” are hypochlorite ion, hypochlorous acid. Electrolysis products quickly decompose and turn into components usually found in seawater. It was experimentally established that the intensity of the synthesis of exopolysaccharides depends on the concentration of the reagent, the duration of exposure and the nature of the starting biological material.

 In FIG. 1 shows the layout of biological material; in FIG. 2 - movements of chlorinated water in experimental containers; in FIG. 3 is a diagram of an experimental setup for treating biological material with “active chlorine”; in FIG. 4 - data on changes in the content of exopolysaccharides that perform a protective function (a - acid-soluble fraction; b - alkali-soluble fraction,% of control).

 The method is implemented as follows.

Glass plates are fixed in metal frames painted with a neutral composition and immersed in sea water to a depth of 1-1.5 m. In the summer months (May-August) at a water temperature of 20 ± 3 ^o C for 10 days in natural conditions, bacteria form on their surface attributed to the genera Pseudomonas, Vibrio, Bacterium, Micrococcus (Brief determinant of Bergi bacteria. M: Mir, 1980. 495 p.). At the same time, protozoa that feed on bacteria, in small amounts, diatoms, and macrophyte seedlings were noted on the plates.

 Upon completion of the formation of the biological system, the plates are delivered to the aquarium and immersed in experimental vessels with a capacity of 5-10 l, into which chlorinated sea water is supplied (see Fig. 1).

 In FIG. 1 marked: 1 - capacity; 2 - plates with natural associations of bacteria; 3 - chlorinated water supply pipe.

 Plates placed in vessels with running seawater that did not contain Achl were used as control plates.

 "Active chlorine" is obtained by the method of non-diaphragm electrolysis of sea water (see Fig. 2).

 In FIG. 2 are indicated: 4 - a pipe supplying sea water, 5 - electrolyzers, 6 - a device for measuring and adjusting the flow velocity, 7 - containers for placing experimental material, 8 - a cuvette table.

,
где
C - концентрация активного хлора, мг/л;
K_э - электрохимический эквивалент хлора, K_э!=1,32 г/А•ч;
η - выход по току активного хлора, η = 0,65;
I - ток, А;
W - расход воды, м³/ч.Sea water continuously enters the electrolysis cell 5, where Achl is formed in the process of electrolysis, and flows into the experimental vessel 7, where the biological material is placed. The specified concentration of active chlorine is supported by the current load on the cell 5 and is calculated by the formula

,
Where
C is the concentration of active chlorine, mg / l;
K _e - the electrochemical equivalent of chlorine, K _e ! = 1.32 g / A • h;
η — current output of active chlorine, η = 0.65;
I - current, A;
W - water consumption, m ³ / h

 The concentration of "active chlorine" was determined by standard methods (Unified methods of water analysis. - M .: Chemistry, 1973). The determination was carried out immediately after sampling.

 The constant concentration of "active chlorine" in the experiment is ensured by stable values of the current load and flow rate of sea water. Indications are monitored by ammeter and rotameter, respectively.

 After a 4-hour exposure of the plates in the biocide solution, the biomass is scraped from them, which is steamed sequentially with 80% ethanol and distilled water (1: 100 ratio of biomass and extractant) to remove carbohydrates included in the energy processes (monosaccharides, spare polysaccharides of the type glycogen and starch). The remainder of the sample is treated sequentially with 2% sulfuric acid and 2% sodium hydroxide, in acid and alkaline hydrolysates, the volumes of which are fixed, exopolysaccharides are contained, their quantities are determined with an anthrone reagent (Trevelayn WE Marrison jz Fractionation and microdetermination of cell carbohydrates - j Biochem, 1952, v. 50, p. 3-7).

 Example 1. The effectiveness of various concentrations of active chlorine on the intensity of the synthesis of exopolysaccharides performing protective functions by natural associations of attached bacteria is studied. Already at a concentration of "active chlorine" of 0.25 mg / l, the content of acid polysaccharides exceeds the control level of 60% (Fig. 3, a). The maximum (approximately three-fold) stimulation of the synthesis of this fraction of carbohydrates is observed at an AHl concentration of 1 mg / L. A further increase in the intensity of the negative effect on bacteria leads to a decrease in the effect of stimulating the synthesis of exopolysaccharides (Fig. 3, a) to 140% of the control. In the presence of 0.25 mg / l of "active chlorine" compounds, the amount of carbohydrates contained in the alkaline hydrolyzate of the sample exceeds the control level by 40%. A twofold increase in the concentration of the agent (0.5 mg / L) is accompanied by an intensification of the synthesis of this carbohydrate fraction by about 2.5 times in comparison with the control. In the presence of 2 mg / l of “active chlorine”, their synthesis is inhibited (the amount of alkali-soluble carbon decreases to the control level, Fig. 3, b).

 Thus, under the influence of “active chlorine” compounds at a concentration of 0.25–1.5 mg / L and a duration of exposure of 4 hours, changes occur in the metabolic processes of fixed natural bacterial associations: the synthesis of polysaccharides fulfilling a protective function of national economic importance is stimulated .

 Example 2. The effect of the duration of continuous exposure to AHl at a concentration of 1 mg / L (see table) on the synthesis of protective polysaccharides by organisms of the studied biological system is tested. When exposed for 2 hours, the amount of carbohydrates of each fraction (acidic and alkaline) decreases very slightly. With an increase in the contact time with the biocide by a factor of 2, a 15–20% increase in their content is noted. Further lengthening of the exposure (8, 16, 24 hours) is accompanied by the same focus of the processes: the amount of acid polysaccharides increases, reaching a maximum (170% of the control level) at 24 hours of exposure (see table).

 The highest content of alkali-soluble carbohydrates is observed with an exposure duration of 8 hours. Elongation of exposure to 16 and 24 hours causes a slight decrease in their amount in the sample (see table).

 It should be noted that the intensification of the synthesis of protective polysaccharides largely depends on their level in the control. If it is high enough, which can be largely determined by the outbreak of macrophyte seedlings on the fouling glasses, and the protective mechanisms are already on, then an increase in their content in response to the action of Achl will be slightly less pronounced.

 A comparative analysis of the obtained data indicates that a more distinct stimulation of the synthesis of bacterial polysaccharides is achieved by varying the concentrations of the biocide. With an increase in the duration of exposure, the same orientation of the processes persists, although its quantitative estimates are lower.

 The proposed method has the following advantages compared with the known.

 1. The method involves controlling the synthesis of exopolysaccharides, significantly increasing the efficiency of the process.

 2. The availability of raw materials and the relative simplicity of its growth.

 3. The compounds that make up the "active chlorine" are formed from the components of sea water and, due to their instability, decompose with the formation of starting products, which allows the reuse of water.

 4. When returning wastewater to a reservoir, the used concentrations of unstable compounds that make up “active chlorine” will not harm the environment.

Claims (1)

 A method of producing exopolysaccharides, including pretreatment of biological raw materials with 80% ethanol and distilled water, followed by extraction with 2% sulfuric acid and 2% alkali in combination with heat treatment and the isolation of the target product, characterized in that they are used as biological raw materials natural associations of bacteria predominantly of the genera Pseudomonas, Vibrio, Bacterium, Micrococcus, settling on the surface of glass plates exposed to the marine environment in the summer, and before pretreatment at biological raw materials are exposed to “active chlorine” in a concentration of 0.5-1.5 mg / l for 4 ± 0.2 g.

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