RU2310685C1 - Bacterium serratia marcescens strain producing lipilytic enzymes for preparation for waste water from fats - Google Patents

Abstract

FIELD: biotechnology, food processing industry.

SUBSTANCE: invention relates to bacterium Serratia marcescens strain which is lipase producer.

EFFECT: increased lipase yield.

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Description

The invention relates to microbiology and biotechnology and relates to a strain of bacteria - producer of the lipase enzyme.

Lipases catalyze the hydrolysis of triacylglycerides to form fatty acids and glycerol. Food enterprises pollute the environment with effluents containing high concentrations of vegetable and animal fats. The search for new strains of microorganisms - lipase producers that are active in the destruction of fats of plant and animal origin, is an urgent task.

The main producers of lipases are strains of the fungi Rhizopus [1, 2], Fusarium [3], some yeast (Jarrowia) [4]. Known, for example, a strain of fungi Fusarium oxysporum - lipase producer [3]. This strain is grown at a temperature of 30 ° C for 150-200 hours on a nutrient medium containing, g / l:

MgSO ₄ · 7H ₂ O 0.5 KN ₂ PO ₄ 1,0 NaNO ₃ 3.0 Peptone 30,0 Yeast extract 1,0 Olive oil 10.0

After 150 hours of cultivation, the maximum accumulation of specific lipase activity in the culture fluid is achieved - 150 units / mg of biomass (by dry weight). A disadvantage of the known strain is the duration of cultivation (6-7 days), the complexity of the nutrient medium and the complexity of the procedure for obtaining culture fluid (release from fungal mycelium).

The use of bacteria producing lipases has an advantage over fungi in accelerating the cultivation process. This may make the lipase manufacturing process more advantageous. Known, for example, a strain of Serratia marcescens 345 [5], which is grown at a temperature of 30 ° C in a nutrient medium (pH 7.0), containing, g / l:

Soya flour decoction 50,0 Urea 0.1 MgSO ₄ · 7H ₂ O 0.1 KH ₂ PO ₄ 0.2

After 24 hours, the maximum amount of lipase with a specific activity of 95 units / mg of biomass (by dry weight) accumulates in the culture fluid. The disadvantage of this strain is the low specific activity of the produced enzyme.

Closest to the claimed strain, the prototype is a strain of Serratia marcescens B-10 L-1 [6], which is grown at a temperature of 28-30 ° C in a nutrient medium (pH 7.0), containing, g / l:

MgSO ₄ · 7H ₂ O 0.25 KN ₂ PO ₄  3.0 Na ₂ HPO ₄ · 2H ₂ O 7.0 NH ₄ Cl 1,0 CaCl ₂ · 2H ₂ O 0.02 NaCl 0.5 Soya flour decoction 50,0

The maximum accumulation of lipase is characterized by a value of 800 units.act./mg biomass (dry weight).

The disadvantage of this strain is the relatively low specific activity of the produced enzyme.

An object of the invention is to obtain a new bacterial strain that provides a higher yield of lipase, which is active in the destruction of solid fats of animal and vegetable origin.

The task is achieved by the proposed strain of Serratia marcescens IB 2-2, isolated from a wastewater sample taken in the aeration tank of the biological treatment facilities of the Perm meat processing plant. The strain is maintained in the collection of microorganisms of the Institute of Biology of the Ufa Scientific Center of the Russian Academy of Sciences. The strain of Serratia marcescens IB 2-2 is characterized by the following features.

Morphological signs. The cells are rod-shaped, straight, 0.5-0.8 × 1.0-3.0 μm in size, motile, single, paired or in chains, do not form spores. Gram-negative.

Colony morphology was determined after 4-5 days of growth at 28 ° C. On meat-peptone agar (MPA) - round with smooth edges, smooth, slightly convex, opaque, shiny, cream, soft colony consistency, diameter 3-5 mm.

Physiological and biochemical characteristics. The strain is a facultative anaerobic, has both respiratory and fermentative types of metabolism, is not picky about growth factors. The optimum growth temperature is 28-30 ° С; they grow at 5 ° С and 40 ° С. It dilutes gelatin, peptones and coagulates milk, hydrolyzes lecithin. Able to grow on Simmons medium with citrate, the Voges-Proskauer reaction is positive. The test with methyl red is positive. It gives negative oxidase, positive catalase tests. Ferments with the formation of acid glucose, xylose, arabinose, sucrose, mannose, galactose, fructose, lactose, maltose, starch, sorbitol, acetate. It grows on MPA with 8% NaCl.

The strain is stored on the jambs of the MPA in the refrigerator with reseeding on fresh jambs after 2-4 months, as well as in the freeze-dried state.

The strain is identified by the identifier Bergey′s Manual of Determinative Bacteriology. The analysis of the strain by sequencing two fragments of the 16S RNA gene and the subsequent comparison of these fragments with all known sequences deposited in the World Genes Database (GenBank) confirmed the validity of the identification.

For the cultivation of the strain Serratia marcescens IB 2-2 in order to obtain lipase, a nutrient medium of the following composition is used, g / l: soy flour - 5; yeast extract - 60; K ₂ NRA ₄ - 5.0; (NH ₄ ) ₂ SO ₄ - 1.0.

The amount of accumulated biomass of strain IB 2-2 is determined by counting the number of viable cells in the culture fluid. For this, the bacterial titer in 1 ml of culture fluid is determined by the inoculation method and converted to mg of biomass (by dry weight) based on the fact that 10 ⁹ bacterial cells weigh 0.28 mg [6].

Lipase activity is determined as follows. 0.5 ml of culture fluid supernatant is added to a reaction mixture containing 4.5 ml of 0.05 M phosphate buffer (pH 8.0) and 5 ml of a 40% emulsion of olive oil in 2% polyvinyl alcohol. The test sample is incubated for 30 min at a temperature of 37 ° C, after which 10 ml of a mixture of ethanol: acetone (1: 1) are added. The degree of cleavage of olive oil is determined by titration of the hydrolysis products with a solution of sodium hydroxide (0.05 M NaOH), in the presence of 1% thymolphthalein, until a blue color occurs. The control sample is treated similarly to the test sample, but the lipase is applied immediately before titration. The specific activity of lipase is expressed in micromoles of oleic acid released in 1 hour by hydrolysis of the substrate with 1 ml of culture fluid, calculated on the amount of biomass contained in 1 ml of culture fluid. Calculation of specific lipase activity (LA), in units act. / mg of biomass (dry weight), produced by the formula:

LA = (VV _k ) · K / t · V _E · C,

where V is the amount of 0.05 M NaOH spent on titration of the test sample, ml;

V _k - the amount of 0.05 M NaOH consumed in the titration of a control sample, ml;

K is the amount of oleic acid titrated with 1 ml of 0.05 M NaOH (K = 50), mcmol;

t is the reaction time, hour;

V _E is the volume of the culture fluid added to the reaction mixture, ml;

C is the concentration of biomass (by dry weight), mg / ml.

The results of determining the lipolytic activity of the culture fluid obtained by culturing a strain of Serratia marcescens IB 2-2 are shown in the table. These data indicate a high activity of lipase produced by the proposed strain of microorganisms. So, the maximum specific activity of the enzyme strain Serratia marcescens IB 2-2 is 1018 μm oleic acid / mg biomass, which is 1.27 times higher than the maximum specific activity (800 units act./ mg biomass) of the strain according to the prototype.

The ability of lipolytic enzymes produced by the strain of Serratia marcescens IB 2-2 to the destruction of solid fats of plant and animal origin was determined as follows.

Example 1. Utilization of cooking oil is carried out by periodically growing a strain of Serratia marcescens IB 2-2 in flasks on a shaker (200 rpm), the volume of the medium is 100 ml. The composition of the nutrient medium: soy flour - 5; yeast extract - 60; K ₂ HPO ₄ - 5.0; (NH ₄ ) ₂ SO ₄ - 1.0; the pH of the medium is 7.0, the temperature of cultivation 28 ° C. Growing time 40 hours. The initial content of cooking oil is 1 wt.%. Bacterial growth was evaluated by microscopy on a solid (agarized) growth medium. The degree of destruction of solid fats was determined by the gravimetric method using the extraction process. At the end of the fermentation process of the Serratia marcescens strain IB 2-2, the bacterium titer in the culture fluid was 3.0 · 10 ⁹ CFU / ml, and the degradation of cooking oil was 100%.

Example 2. Analogously to example 1, the utilization of pork fat is carried out by periodically growing the strain Serratia marcescens IB 2-2 in flasks on a rocking chair with the process parameters and the composition of the nutrient medium shown in example 1. Growth time 40 hours. The initial content of pork fat is 1 wt.%. At the end of the fermentation process of the Serratia marcescens strain IB 2-2, the bacterium titer in the culture fluid was 4.0 · 10 ⁹ CFU / ml, and the destruction of pork fat was 100%.

Example 3. Analogously to example 1, the utilization of beef fat is carried out by periodically growing the strain Serratia marcescens IB 2-2 in flasks on a rocking chair with process parameters and the composition of the nutrient medium shown in example 1. Growing time 51 hours. The initial content of beef fat is 1 wt.%. At the end of the fermentation process of the Serratia marcescens strain IB 2-2, the bacterium titer in the culture fluid was 3.0 · 10 ⁹ CFU / ml, and the destruction of beef fat was 100%.

The experimental results shown in examples 1-3 indicate the high efficiency of the biodegradation process of solid fats of animal and vegetable origin under the action of lipolytic enzymes produced by the Serratia marcescens strain IB 2-2.

The experimental results shown in examples 1-3 indicate the high efficiency of the biodegradation process of solid fats of animal and vegetable origin under the action of lipolytic enzymes produced by the Serratia marcescens strain IB 2-2.

Thus, a strain of Serratia marcescens IB 2-2 was obtained, which has high lipolytic activity, which can be used as the basis of a biological product for biological treatment of wastewater or sewage systems from fats.

Bibliography

1. A.S. USSR 1581742 A1, publ. 07/30/90.

2. A.S. USSR 1726513 A1, publ. 04/15/92.

3. P. Rapp. Production, regulation and some properties of lipase activity from Fusarium oxysporum f. sp. vasinfectum // Enzyme and Microbial Technology. - 1995. - V.17. - P.832-838.

4. A.S. USSR 1454852 A1, publ. 01/30/89.

5. Bashkatova N.A., Severina L.O. The influence of cultivation conditions on lipase biosynthesis in Serratia marcescens // Microbiology. - 1979.- T.68, issue 5. - S.826.

6. Patent RU 2148645 C1, publ. 05/10/2000.

Lipolytic activity of the strain Serratia marcescens IB 2-2 The cultivation time, h Lipolytic activity, μM oleic acid / mg biomass (dry weight) Strain Serratia marcescens IB 2-2 Strain Serratia marcescens B-10 L-1 (prototype) 5 0 91 10 53 800 fifteen 188 300 twenty 393 160 25 446 42 thirty 1018 31 35 439 thirty 40 188 thirty fifty thirty thirty

Claims (1)

The bacterial strain Serratia marcescens KM IB UC RAS RAS IB 2-2 is a lipase producer.