RU2760288C1 - Methylococcus capsulatus mc19 strain - producer of protein mass - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, microbiological industry and relates to a methanotrophic strain of Methylococcus capsulatus. The Methylococcus capsulatus MC19 strain, which has the ability to produce a protein mass, has been deposited at the Federal Publicly Funded Institution of Science G.K. Scriabin of the Russian Academy of Sciences (FPFI RAS) under the registration number VKM B-3493D.

EFFECT: strain Methylococcus capsulatus VKM B-3493D can be used in animal husbandry as a feed additive, aquaculture, in the food industry as a food additive, in the microbiological industry as a component of nutrient media, as well as for use in sports and dietary nutrition.

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Description

Technology area

The invention relates to biotechnology, microbiological industry and concerns a methanotrophic strain of Methylococcus capsulatus , which allows to obtain biomass, which, in addition to protein, contains antioxidants and B vitamins, which increase the biological value of protein, which can be used in animal husbandry as a feed additive, aquaculture, in the food industry in as a food additive, in the microbiological industry as a component of nutrient media, as well as for use in sports and dietary nutrition.

State of the art

Protein is a beneficial source of minerals, vitamins, fats and carbohydrates. As a source of protein, proteins of animal origin (fish meal, bone meal) are often used. Microbial protein can be an alternative to animal protein. For many important indicators, the biomass of microorganisms has a very high nutritional value. Large-scale cultivation of industrial microorganisms and the use of their biomass is one of the main sources of protein for humans and animals. The raw material base of microorganisms is practically inexhaustible, the growth of biomass is intense, the protein composition of unicellular organisms is very constant, and it, as a rule, is balanced in amino acid composition.

The use of bacteria as a producer of fodder protein is more effective, for example, prokaryotic organisms form at least 70% of protein by weight, compared to yeast, which form no more than 60% of protein from biomass. Various types of microorganisms are producers of complete protein and vitamins, including methanotrophic bacteria - microorganisms growing on natural or associated gas, such as Methanomonas carbonatophila, Methanomonas methanooxidans, Methylomonas methanica, Methylocystis parvus, Methylococcus capsulatus. The quality of the protein obtained using methanotrophic bacteria is equivalent to proteins of animal and vegetable (based on soybeans) origin. This allows it to be widely used as a substitute for both animal and vegetable flour.

The most active natural gas-fired protein biomass producer is the species Methylococcus capsulatus . The disadvantages of this type of methanotroph include the instability of its growth on natural gas during long-term cultivation under flow-through conditions, which reduces the productivity of the process and the yield of the finished product. This problem is solved by strains Methylococcus capsulatus GBS-15, Methylococcus capsulatus CONCEPT-8, Methylococcus capsulatus LBTI 028 which have a number of properties that provide more stable growth during cultivation (RF patent No. 2613365, RF patent No. 2706074, RF patent No. 2728345).

It is known that bacteria, yeast and yeast-like microorganisms can be producers of antioxidant pigments. Natural antioxidants (inhibitors of oxidation processes) include ubiquinones (UC) (coenzyme Q) (Mosichev M.S., Matveev D.A., Belov A.P. // Applied Biochemistry and Microbiology 1994 - vol. 3. - issue .3 - p. 430-435., Landi L., Cabrini L., Sechi AM et al. // Biochem. J. 1984 - V.222 - No. 2 -P.463-466., Landi L., Carbini L., Tadolini B. et al // Ital J. Biochem 1985 - V.34 - V.5 -P.356-363.

Ubiquinone is a substance necessary to maintain biological functions as a component of the electronic transport system, and is also known to be involved in the activation of ATP production, antioxidant activity in the body and stabilization of membranes, and can be a tool for enhancing energy production in mitochondria, increasing the antioxidant function of the body. and maintaining homeostasis. The compounds stabilize membranes and proteins, protect cells from UV radiation, absorb heavy metals, and are provitamins, immunostimulants and adaptogens for humans and animals. The production and use of feeds containing ubiquinones, which have a therapeutic and prophylactic effect, makes it possible to increase the level of the body's adaptive defense to the effects of unfavorable environmental factors.

Known methods for producing ubiquinones Q ₇ , Q ₈ , Q ₁₀ using purple phototrophic microorganisms (Inventor's Certificate USSR 1398398) and Q ₉ (Inventor's Certificate USSR 1353808), followed by purification at concentrations of 2.2. - 4.3 mg / g dry weight. Despite the fact that mineral media of simple composition are used for the cultivation of phototrophic microorganisms, the production of protein on an industrial scale is associated with significant energy consumption, which affects the cost of the resulting biomass.

An additional advantage of the protein of unicellular organisms in comparison with proteins of animal origin is the presence of B vitamins (this is especially true for vitamin B12) in quantities that meet the needs of animals. In particular, the use of lactic acid, propionic acid bacteria and bifidobacteria is known as a protein feed of microbial origin containing vitamins of group B (for example, RF patent No. 2244000, RF patent No. 2270248, RF patent No. 2390554, RF patent No. 2250258, RF patent No. 2392312) ... However, for the production of biomass from these bacteria, it is necessary to use expensive substrates, while the accumulated biomass is characterized by an insufficiently high concentration, and, therefore, an insufficient amount of protein (no more than 50%) that could be formed by the culture.

Thus, of the microorganisms listed above, for the industrial production of biomass, it is most preferable to use methanotrophic bacteria, which, under suitable conditions, actively process natural gas, multiply rapidly and build up their biomass, rich in valuable protein, vitamins and other biologically active substances. The biomass of Methylococcus capsulatus is characterized by a high level of crude protein (at least 70%), as well as a ratio of amino acids similar to fishmeal, which allows it to be used as a source of protein mass. Tests of the protein premix with the inclusion of biomass of methane-oxidizing bacteria, carried out on farm animals, birds and fish, have confirmed the high biological value of this biomass as a source of protein , vitamins and other nutrients. The harmlessness of using biomass of methane-oxidizing bacteria has long been proven. Veterinary and sanitary-hygienic studies have established the good quality and harmlessness of livestock, poultry and fish farming products obtained using feed with biomass of methane-oxidizing bacteria.

Closest to the claimed invention is the strain of Methylococcus capsulatus GBS-15, deposit number VKPM B-12549 (RF Patent No. 2613365). This strain has a number of properties that provide a more stable growth during cultivation, its phage resistance was shown in water from various external sources and when grown in an atmosphere of natural gas of different composition (with a methane content from 85% vol. To 99.9% vol.) ). Strain Methylococcus capsulatus GBS-15 grows on mineral media, while a biomass concentration of 32 g / l and a flow rate of 0.25 h ^{-1 are} achieved. The possibility of the synthesis of ubiquinone (mainly Q7 and Q8) is mentioned.

However, the qualitative and quantitative composition of the coenzymes synthesized by the strain has not been experimentally confirmed. In addition, there is no data on the presence and quantitative content of B vitamins in the resulting biomass, which increase the nutritional value of the protein produced by microorganisms.

The technical problem solved by the claimed invention is to obtain a strain containing antioxidants (ubiquinones / ols) in concentrations comparable to natural producers of yeast cultures and phototrophic microorganisms, taking into account the peculiarities of cultivation and protein content in the latter; as well as B vitamins, namely B2, B3 and B12, not lower than that of natural strains of lactic acid, propionic and bifidobacteria, capable of synthesizing vitamins, but inferior in growth rate and cultivation conditions. This is achieved by culturing Methylococcus capsulatus MC19 on media of different compositions.

Disclosure of invention

The technical result of the claimed invention is to obtain a strain with increased biological activity, which is achieved:

- high content of crude protein;

- the ability for intracellular biosynthesis of vitamins B2, B3, B12;

- antioxidant activity due to the synthesis of coenzymes Q8, Q9.

The inventive strain allows you to obtain biomass, which, in addition to protein, contains antioxidants and B vitamins, which increase the biological value of the protein mass. StrainMethylococcus capsulatus MC19 converts monocarbon compounds and contains at least 80% crude protein in biomass, at least antioxidants: ubiquinones / tins Q8 / Q8-2H - 9.0 / 8.0 and Q9 / Q9-2H - 0.6 / 0.2 μg / g dw, B vitamins in biomass not less than: B2 –3 mg / g dw, B3 –15 mg / g dw, B12 - 1.0 mg / g d.v. The strain stably produces B vitamins and coenzymes on media of various compositions. Moreover, the strainMethylococcus capsulatus MC 19 maintains a high growth rate - not less than 0.25 hours^-1, the ability to accumulate protein in concentrations of at least 80%.

All of the above indicates that the use of methanotrophs based on the Methylococcus capsulatus MC19 strain is promising. This allows you to create a full-fledged source of microbial protein mass with increased biological value for the national economy.

The technical result is achieved by a strain obtained by selection from a culture sample of methane-oxidizing microorganisms based on the Methylococcus capsulatus strain, under conditions of continuous cultivation in a bioreactor in a methane-air atmosphere with a constant flow of a nutrient medium deposited at the V.I. G.K. Skryabin of the Russian Academy of Sciences (IBFM RAS) with the assigned identifier VKM B-3493D (VKM B-3493D).

Implementation of the invention

The Methylococcus capsulatus strain MC19 was obtained as a result of selection for the ability to biosynthesize in significant amounts of antioxidants and B vitamins during continuous cultivation from the Methylococcus capsulatus strain VSB-874 and samples of associations of methane-oxidizing microorganisms, which were preserved and maintained in scientific laboratory collections 770 (Patent No. 200) ... The Methylococcus capsulatus MC19 strain within the microbial community has been continuously cultivated for 6 years. The process of continuous cultivation took place in laboratory fermenters, with a nutrient medium flow of 0.2-0.3 h ^-1 , in a methane-air atmosphere (methane: air ratio 25-30: 75-70), on a mineral medium with a phosphorus source in the form orthophosphoric acid, with a nitrogen source in the form of an aqueous solution of ammonia, used as a titrant. The nutrient medium of the following composition (g / l): MgSO ₄ * 7H ₂ O 0.12, KCl 0.07, K ₂ SO ₄ 0.03; trace elements, mg / l: CuSO ₄ * 5H ₂ O 2.375, FeSO ₄ * 7H ₂ O 10, MnSO ₄ * 5H ₂ O 5, H ₃ BO ₃ 2.5, ZnSO ₄ * 7H ₂ O 1, NiSO ₄ * 7H ₂ O 0.25, CoSO ₄ * 7H ₂ O 0.18, Na ₂ MoO ₄ * 2H ₂ O 0.18, CaCl2 - 0.083 g / l; H ₃ PO ₄ 85% - 0.2 ml / l. Dissolved oxygen was maintained in the range of 0.3–1.0 mg / l; dissolved carbon dioxide was at the level of 48 - 60 mg / l. The productivity reached 4.5 g of absolutely dry weight / l * h. The cultivation took place in bioreactors of 25 l and 12 l volumes with an ideal stirrer and in a bioreactor with a volume of 20 l under high-performance conditions.

The strain was identified to species by 16S RNA analysis.

The strain is characterized by the following features:

Cultural and morphological characteristics. Methylococcus capsulatus MC19 gram-negative immobile cocci, 0.8x1.0 microns in size, usually occurring in pairs. Capable of forming a capsule. They have a resting stage in the form of cysts. Culture on agar medium forms small colonies with a diameter of 1.0-1.5 mm, transparent, convex with a smooth edge. The color of the colonies ranges from cream to light orange. The color in a liquid medium is creamy pink.

Physiological and biochemical features. The strain belongs to thermotolerant microorganisms with a temperature optimum of 42 ° C, although growth can occur at temperatures from 37 to 52 ° C. Aerobe. It has a strictly respiratory type of metabolism using oxygen as the final electron acceptor. It uses methane or methanol as the only source of carbon and energy. Ammonium salt is preferred as nitrogen source, although it can grow on nitrates. Possesses nitrogenase activity. Capable of autotrophic fixation of CO _2. Metal ions of copper, cobalt, nickel, iron, and to a lesser extent zinc at a concentration of 10 μM have an inhibitory effect on NADH oxidoreductase, which can serve as a mechanism of toxic action in relation to bacteria (Saratovskikh et al., 2005, 2007). Intracellular membranes are arranged in the form of stacks of vesicular discs characteristic of type I methanotrophs (according to the Whittenbury. R classification).

The strain is non-pathogenic.

Methylococcus capsulatus MC19 is a genetically stable organism with no known plasmids. Genetic manipulations with the strain were not carried out.

The area of industrial application of the strain is a feed product for animal husbandry, poultry farming and aquaculture, food additive, microbiological industry.

The strain can be recommended for the development of effective methods for enriching feed with vitamins, antioxidants, protein and other biologically active products of microbial origin.

The invention is confirmed by the following examples.

Example 1. For the cultivation of Methylococcus capsulatus MC19 VKM B-3493D, nutrient media of the following composition are used, g / l:

AMS medium: MgSO ₄ 1.0, Na ₂ HPO ₄ 0.717, KH ₂ PO ₄ 0.272, NH ₄ Cl 0.5, CaCl ₂ 0.02;

NMS medium: MgSO ₄ 1.0, Na ₂ HPO ₄ 0.717, KH ₂ PO ₄ 0.272, KNO ₃ 1.0, CaCl ₂ 0.02.

A 0.1% solution of trace elements is added to the media, containing (g / l): EDTA 5.0; FeSO ₄ ⋅ 7H ₂ O 2.0; ZnSO ₄ ⋅ 7H ₂ O 0.1; MnCl ₂ ⋅ 4H ₂ O 0.03; CoCl ₂ ⋅ 6H ₂ O 0.2; CuCl ₂ ⋅ 5H ₂ O 0.1; NiCl ₂ ⋅ 6H ₂ O 0.02; Na ₂ MoO ₄ 0.03. pH up to 5.6-6.0.

The cultivation is carried out as follows. In a flask with a volume of 500 ml, add 50 ml of medium and inoculate with 5%. Cultivation conditions: 41.5-42 ° C, methane-air 1: 1, stirring speed 160 rpm.

After 10 days of cultivation, the biomass contains crude protein on NMS media 82% and AMS 79%.

Example 2. Strain Methylococcus capsulatus MC19 VKM B-3493D was evaluated for the content of ubiquinones (CoQ8-10) / ubiquinols (CoQ8-9H ₂ ) by culturing it on liquid nutrient media.

The cultivation conditions and the nutrient media used are as in example 1. The cultivation time is 3 days.

Identification of ubiquinones / olv is carried out by HPLC-MS / MS on a C18 column, isocratic elution with 100% 5 mM ammonium formate in methanol with 0.1% formic acid. Detection is carried out by ion transitions (MRM), precursor ions are adducts of ubiquinones (tins) with NH⁴⁺; the ionization conditions are optimized according to the Q10 standard. Qualitative identification Q8.9 carried out according to the mass of ionic fragments, which are the same for homologues, and differ in the mass of the precursor ion. Also, the qualitative identification of substances without standards is carried out by the retention time on the column. Sample preparation for the determination of ubiquinones (tins) is carried out as follows. Different volumes of culture liquids, depending on the concentration of the culture, are centrifuged, the sediment is weighed, 5 parts of acetone are added, and disintegrated in an ultrasonic bath for 15 minutes. Then mix on a multishaker for 15 minutes. The homogenesis is cooled at -20 ° C for 20 minutes and precipitated by centrifugation at + 4 ° C at 3000 rpm for 3 minutes, taken into a plate for analysis.

Content of ubiquinones (CoQ8-10) / ubiquinols (CoQ8-9H2) in biomass μg / g dw:

Ubiquinones / Ubiquinols Wednesday AMS NMS environment Q8 9.287 2.495 Q8-2H 8,043 5.676 Q9 0.614 - Q9-2H 0.249 - Q10 - -

Example 3. Strain Methylococcus capsulatus MC19 VKM B-3493D was evaluated for its ability to synthesize B vitamins by cultivating it on liquid mineral media under the conditions indicated in example 1.

The quantitative determination of vitamins B1, B2, B3 nicotinamide, B3 nicotinic acid, B5, B6, B7, B12 in the culture liquid (supernatant and biomass) is carried out by HPLC-MS / MS method.

Chromatographic separation was performed on a YMC-Triart C18 column, 50x2.0 mm, 3 μm (YMC, Japan) with a built-in SecurityGuard ULTRA C18 2.1mm guard column (Phenomenex, USA) at 40 ° C. Mobile phase A was a 10 mM ammonium formate solution with the addition of 0.1% formic acid; B — acetonitrile with the addition of 0.1% formic acid. Gradient elution is used. The mobile phase speed and the stab volume are 350 μL / min and 1 μL, respectively. The total analysis time is 6 minutes. The work uses electrospray ionization (ESI) in a positive mode. Sample preparation for the determination of vitamins B1, B2, B3 NAC, B3 NAM, B6, B7, B12 is carried out as follows. 4 - 30 ml of culture fluid, depending on the concentration of the culture in the sample, are transferred into 15/50 ml vials, centrifuged for 15 minutes, 3900 rpm. 1 ml of the supernatant is taken into an Eppendorf volume of 1.5 ml, sent for repeated centrifugation for 15 minutes, 14680 rpm. The resulting solution was poured into a 96 well plate for HPLC-MS / MS analysis to determine the concentration of vitamins in the culture supernatant. To determine the concentration of vitamins in the biomass, the culture settled after the primary centrifugation is decanted, weighed, resuspended in 5 parts of deionized water. The resulting suspension is poured into a 2 ml polypropylene tube with a tightly screwed screw cap with a rubber gasket, centrifuged for 15 minutes, 14680 rpm, the supernatant is taken for HPLC-MS / MS analysis to determine the concentration of residual vitamins in the intercellular space. The twisted sample is re-resuspended and sent to a thermo-shaker for 60 minutes, 800 rpm, 100 ° C to extract vitamins from bacteria. After the sample is sent to centrifugation for 15 minutes, 14680 rpm and HPLC-MS / MS analysis.

The content of B vitamins during cultivation of the strain on NMS medium:

B vitamins Concentration of vitamins in the supernatant, ng / ml Concentration of vitamins in biomass, μg / g d.w. B1 <10 <10 B2 <50 2 842.50 B3 NAC (nicotinic acid) <50 <50 B3 NAM (nicotinamide) <20 15,050.00 B6 <5 <5 B7 <200 <200 B12 <20 1,097.50

The content of B vitamins during cultivation of the strain on the AMS medium:

B vitamins Concentration of vitamins in the supernatant, ng / ml Concentration of vitamins in biomass, μg / g d.w. B1 <10 <10 B2 <50 <50 B3 NAC (nicotinic acid) <50 <50 B3 NAM (nicotinamide) <20 2 610.00 B6 <5 <5 B7 <200 <200 B12 <20 98.30

Cultivation of a culture of Methylococcus capsulatus MC19 VKM B-3493D in an ejection-type fermenter with a volume of 40 liters (working volume - 25 liters) with a continuous supply of liquid mineral media under the conditions specified in example 1 at an increased pressure of 6 atm. made it possible to obtain a comparable content of protein, vitamins B2, B3, B12 and ubiquinones Q8, Q9 in biomass, as well as when grown at atmospheric pressure.

The presented examples characterize the strain Methylococcus capsulatus MC19 VKM B-3493D, as a producer with a high protein content, vitamins B2, B3, B12 and ubiquinones Q8, Q9 in biomass on any kind of media.

Claims (1)

Strain Methylococcus capsulatus MC19 VKM B-3493D - producer of protein biomass.