Method for preparing exopolysaccharides

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

 

The object of the present invention is a method of producing exopolysaccharides by fermentation using microorganisms. In particular, the invention relates to a method for production of exopolysaccharides by fermentation of microorganisms in a nutrient medium containing at least one source of carbon assimilated by microorganisms, and at least one organic nitrogen source, derived from plants of the legume family and has a high protein content.

In the framework of the present invention, the term "exopolysaccharides" refers to polysaccharides produced by microorganisms.

The exopolysaccharides with high molecular weight are increasingly used in various industries due to their thickening, viscosity, emulsifying, stabilizing properties, in particular, in aqueous media. So, xanthan gum is used due to its exceptional rheological properties in such diverse areas as construction, production of paints, paper, textile, cosmetic, food industry, agriculture, water management, drilling, oil and other

These exopolysaccharides have a high molecular weight, typically above 1· 106g/mol (measured by gel-filtration) and consist of chains of glucose, mannose, galactose, is amnesy, glucuronic acid mannurone acid, guluronic acid, possibly with acetate and pyruvate derivatives, their special structure and properties are described, for example, in:industrial Gums - Whistler - 2nd edition - Chapters XXI-(XIII(1973).

The exopolysaccharides preferably are produced by aerobic culture of the microorganism in an aqueous nutrient medium.

Xanthan gum produced by bacteria of the genus Xanthomonas. The exopolysaccharides of the same type can be produced by a variety of microorganisms, among the most famous of which are the microorganisms of the genera Agrobacterium, Arthrobacter, Alcaligenes (actinopyga), Pseudomonas (Levan), Rhizobium, Sclerotium (scleroglucan).

The aqueous nutrient medium usually contains, in addition to various elements of growth, the carbon source and the nitrogen source. In industrial fermentation choice source of carbon and/or nitrogen source simultaneously based on its availability, cost and ability to provide high productivity.

So, in SU 1838417 A3 describes a method that includes receiving exopolysaccharides xanthan type the cultivation of the microorganism-producer of polysaccharides on a nutrient medium containing sources of carbon, organic nitrogen and growth factors in aerobic conditions. In the particular case as a microorganism-producing bacteria are used anthomonas campestris.

In some areas about what islenet, for example in the food or cosmetic industry, there are additional conditions. In these areas the sources of carbon and nitrogen should be selected, in addition, therefore, to get the exopolysaccharides satisfy the necessary sensory, sensory and visual requirements.

Among the commonly used sources of carbon and nitrogen is not easy to find those that would satisfy all these requirements.

For example, when the microorganism is not able to absorb all of the nitrogen source at the completion of fermentation remain insoluble residues, which, on the one hand, the environment can develop third-party strains, can ruin the wort for the Department of exopolysaccharides, and, on the other hand, which can lead to staining of exopolysaccharides when possible thermal sterilizing and bleaching treatment. In some ways fermentation to eliminate this drawback using enzymes. Other methods include the stages of filtration and/or centrifugation. What methods did not remove insoluble residual postfermentation products, this leads to the appreciation of the whole process.

Some sources of carbon and/or nitrogen have the disadvantage that significantly lengthen the cycle of fermentation, which results in the company and, to infection and, consequently, to the damage of the wort before the separation of exopolysaccharide and decreased productivity.

The nature of the source of nitrogen is especially important in case we need to get the exopolysaccharide with good organoleptic, sensory and visual properties. It is also responsible for the high output of exopolysaccharides.

It was found that some sources, obtained from the seeds of some legumes, such as caruba represent a valuable source of organic nitrogen for the fermentation of microorganisms. These fractions are in fact satisfy all the above requirements.

Among the fractions obtained from the seeds of karubi, the best results, particularly in regard to productivity, give those that have high protein content. On the so-called standard environments, such as those described, for example, in Industrial Gums - Whistler - 2nd edition - Chapters XXI-XXIII (1973) for the control of fermentation productivity is of the order of 0.3-0.4 g/(kg· h); for fractions, obtained from the seeds of karubi, this productivity is more than 0.4 g/(kg· h).

The present invention aims to provide a method of producing exopolysaccharides by fermentation of microorganisms, which would be simple and economical.

Another object of the invention is to offer with the persons receiving the exopolysaccharides by fermentation of microorganisms, which would have eliminated the above problems of environmental pollution.

Thus, the object of the invention is a method of producing exopolysaccharides by fermentation of microorganisms, characterized in that the fermentation is carried out in a nutrient medium containing at least one source of carbon assimilated by microorganisms, and at least one organic nitrogen source, and the specified source comes from the fraction of seeds karubi.

Other advantages associated, in particular, with the choice of nitrogen source are reduced fermentation time, the absence of insoluble residual postfermentation products and improved performance.

In addition, this method allows to obtain the exopolysaccharide with good organoleptic, sensory and visual properties.

In addition, the rheological properties obtained by this method of exopolysaccharide stored and even in some cases be improved.

The method according to the invention can be applied to achieve any of exopolysaccharide by fermentation using microorganisms. Many microorganisms, such as bacteria, yeast, fungi, algae, and are able to produce exopolysaccharides. Among others are:

bacteria belonging to the genus Xanthomonas, and, in particular, VI is s, described in Bergey''s Manual of Determinative Bacteriology (8th edition - 1974 - Williams N. Wilkins Co. Baltimore), such as Xanthomonas begoniae, Xanthomonas campestris, Xanthomonas carotae, Xanthomonas hederae, Xanthomonas incanae, Xanthomonas malvacearum, Xanthomonas papavericola, Xanthomonas phaseoli, Xanthomonas pisi, Xanthomonas vasculorum, Xanthomonas vesicatoria, Xanthomonas vitians, Xanthomonas pelargonii;

bacteria belonging to the genus Arthrobacter, and in particular to the species Arthrobacter stabilis, Arthrobacter viscosus;

bacteria belonging to the genus Erwinina;

bacteria belonging to the genus Azotobacter, and in particular, to mean Azotobacter indicus;

bacteria belonging to the genus Agrobacterium, and in particular to the species Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium tumefaciens;

bacteria belonging to the genus Alcaligenes, in particular, Alcaligenes faecalis;

bacteria belonging to the genus Pseudomonas, in particular Pseudomonas methanica;

bacteria belonging to the genus Corynebacterium;

bacteria belonging to the genus Bacillus, in particular Bacillus polymyxa;

fungi belonging to the genus Sclerotium, and in particular to the species Sclerotium glucanicum, Sclerotium rolfsii or Plectania occidentalis;

fungi belonging to the genus Aspergillus, in particular Aspergillus species itaconicus, Aspergillus terreus;

yeast belonging to the genus Hansenula, such as species Hansenula casulata.

Preferably, the used microorganism is a bacterium of the genus Xanthomonas and, in particular, of the species Xanthomonas campestris.

The main object of the invention is a method of producing exopolysaccharides by fermentation of microorganisms, characterized in that farm, the rotation is carried out in a nutrient medium, containing at least one source of carbon assimilated by microorganisms, and at least one organic nitrogen source, and the specified source comes from the fraction of seeds karubi.

The fruit produced by the trees karubi, consists of two parts: peel and seed. Seed karubi and, in particular, the endosperm of the seed, is already widely used under the name "resin karubi". With endosperm touches germ by-product produced in large quantities when selecting resin karubi.

It turned out that those fractions of seed karubi, which have high protein content, are best suited for implementing the method according to the present invention.

Thus, the fraction of seeds karubi mainly contains at least 45%, preferably at least 50%, and more preferably at least 60% of the protein relative to the dry weight of dry matter.

The protein content is determined on the basis of measurements of nitrogen released during combustion at 950° C in an atmosphere of oxygen under the conduction current of helium. As a measuring apparatus using LECO FP 428.

These proteins consist of both essential and nonessential amino acids.

A special variant of the method according to the invention consists in the use of fractions of seeds to ruby, in which proteins are characterized by, preferably, a high content of arginine, glutamine and/or glutamic acid and lysine.

In this particular embodiment, the content of arginine is preferably from 9 to 20% and preferably from 12 to 14%, weight/weight, relative to the total number of amino acids.

The content of glutamine and/or glutamic acid is mainly from 18 to 30%, preferably from 22 to 27%, weight/weight, relative to the total number of amino acids.

Lysine is preferably from 18 to 30%, preferably from 12 to 14%, weight/weight, relative to the total number of amino acids.

The content of amino acids determine the usual well-known specialist of ways.

In addition to the proteins used fractions may also contain lipids. The exopolysaccharides obtained by fermentation of microorganisms in a nutrient medium containing at least one organic nitrogen source derived from the fraction of seeds karubi containing lipids are especially superior organoleptic, visual and sensory properties. These lipids also prevent foaming in the phases of preculture.

Preferably the lipid content of these fractions is at least 4%, more preferably at least 5% and even more preferably from 7 to 15% is the compared to the weight of dry matter.

Lipids are specified in relation to the total fat content. It is determined by extraction with hexane in a Soxhiet extractor. The sequence of operations is as follows:

- in cartridge extractor weigh approximately 10 g of milled germ material karubi, i.e. E grams, and the cartridge clog hydrophilic cotton swab;

in pre-tared (P0 g) the cylinder capacity of 250 ml injected with 150 ml of hexane;

- carry out the extraction in a period of about 6 hours;

the solvent is evaporated using rotary evaporators and complete drying of sludge in a drying Cabinet at 105° C for 1 hour;

after cooling in a desiccator, weigh the container containing the precipitate, i.e. P1 grams.

The fat content and, therefore, lipids, determined by the following formula:

Fat content (%)=100× (P1-P0)/E.

Of the characteristic components included in the composition of these lipids, it is possible, in particular, to call palmitic, stearic, oleic and linoleic acid.

In addition to protein and lipid fractions of the seed karubi can also contain carbohydrates.

According to a special variant of the invention, this fraction represents the embryo of the seed karubi.

In this embodiment, a specified fraction of pre-purified from fractions of endosperm through the Yu conventional known methods.

Used fraction of seeds karubi can be preferably presented in the form of flour. This flour receive conventional methods of grinding such as grinding in the mills of the following types:

- roller mill for flour with an average grading of the mesh type 100, i.e. flour, containing not more than 1 wt.% particles larger than 80 mesh and not more than 10 wt.% particles smaller than 200 mesh;

mills with spit (pin mills) for flour with finer granularity:

- type mesh 200, i.e. flour, does not contain particles larger than 80 mesh and containing not more than 60 wt.% particles smaller than 200 mesh, and

- type mesh 175, i.e. flour, containing not more than 1 wt.% particles larger than 80 mesh and not more than 75 wt.% particles smaller than 200 mesh.

Flour can be used in its original form or after processing by suitable enzymes, such as alkaline, acidic and/or neutral proteases; lipases; pitsani; alkaline, acidic and/or neutral phosphotase; amylases. Processing enzymes is carried out using conventional known methods.

The grading specified flour can range from 10 to 150 microns. In the case of processed flour the grain size distribution is, in particular, from 20 to 60 microns, preferably from 30 to 50 microns.

Measurement of particle size can be made by laser granulometry using granulometry MALERN, supplied by the company Malvern Instruments S.A.

You can also use the fraction of seeds karubi unprocessed form, i.e. after separation of the endosperm in the form of plates or in the form of a primary aqueous dispersion or suspension.

Although the invention is described for seed karubi, it can also be applied to other legumes, for example, guar, Cassia, containers. These pulses are given as examples and do not limit the invention.

In another particular embodiment of the invention the fermentation is carried out with a mixture of organic and mineral sources of nitrogen.

In this case, mineral nitrogen source may be selected from the nitrates of ammonium or of sodium, phosphates or silicates, ammonium, magnesium sulfate, potassium sulfate or sodium, individually or in a mixture.

The concentration of organic and, if necessary, mineral nitrogen sources in the fermentation medium is from 1 to 80 g/l, preferably from 3 to 50 g/l and even more preferably from 5 to 30 g/L.

The fermentation medium contains a source of carbon assimilated by microorganisms.

As a constitutive source of carbon in the fermentation medium include glucose, sucrose, fructose, galactose, trehalose, mannose, melibiose, raffinose, maltotriose, maltose, lactose, lactulose, methyl-β galactopyranoside, methyl-α -galacto ransid, cellobiose, gentiobiose, methyl-β -D-glucopyranoside, methyl-α -D-glucopyranosid, esculin, ribose, arabinose, xylose, palatinose, rhamnose, fucose, melezitose, D(+)Arabic, L(-)Arabic, xylitol, dulcet, tagatose, glycerol, myo-inosit, mannitol, ▫ maltitol, turanose, sorbitol, adonit, lyxose, aritra, starch, preferably hydrolyzed, hydrolysates of starch, mixtures of these sugars and mixtures containing at least one of these sugars. Preferred sugars are glucose and sucrose.

The concentration of assimilated carbon source is from 1 to 100 g/l, preferably from 15 to 80 g/L.

The fermentation medium may additionally contain trace elements, such as trace amounts of inorganic salts, such as sulphates, chlorides of iron, calcium, manganese, magnesium, sodium, potassium, Nickel, cobalt, copper, zinc or mixtures thereof, as well as vitamins, nucleotides and/or other conventional additives, such as agents, regulation of pH and protivovspenivayushchie agents.

The method of producing exopolysaccharides according to the invention by fermentation of microorganisms can be carried out in the presence of enzyme (enzymes, such as alkaline, acidic and/or neutral protease; polishers; amidase; peptidases; amyloglucosidase; phosphatase; phytase.

However, one of the main advantages of the method according to the invention the making is in fact, it is possible to carry out the fermentation of the microorganisms in the absence of enzymes. Unexpectedly, it was found that in the absence of enzymes do not change neither the duration nor the productivity of the fermentation. Moreover, the rejection of the enzyme leads to the accumulation of insoluble and undissolved residual postfermentation products, which could lead to the development environment third-party strains that can cause damage to the wort before the separation of the exopolysaccharide.

The very cultivation of microorganisms can be carried out in the usual way. Specialist, depending on the microorganism will be able to make a choice conditions, in particular temperature and time of incubation, as well as the nature of the environment for the specified microorganism.

For conservation of the microorganism, it is preferable to carry out the stage of preculture. Under precultural understand phase, consisting in the development and multiplication of bacterial strain without the production of exopolysaccharide.

The microorganism is introduced into the fermentation medium in a known manner by means of inoculum or intermediate crops.

Fermentation can take place under pressure from 0 bar to 4 bar. The fermentation can be carried out at a temperature of from 15 to 100° C, preferably from 25 to 80° S, and even more preferably from 25 to 35° C.

The pH value of the fermentation medium can costal shall be from 5 to 9, preferably from 6 to 8. The pH value can be set at a desired level, depending on the case, using a base such as sodium hydroxide, potassium hydroxide or ammonium hydroxide, or with acid, such as sulfuric acid, phosphoric acid, hydrochloric acid or nitric acid.

The fermentation medium, placed in a fermentation tank or container may be subjected to agitation and aeration. This mixing can be carried out, for example, by using reciprocal vzbaltyvaya, rotating vzbaltyvaya, one or more vzbaltyvaya blocks or bubble columns. The duration of fermentation is usually more than 30 hours, typically from 40 to 100 hours.

Performance is measured based on the number of received exopolysaccharide, in grams, in relation to kg wort for one hour fermentation. Thanks to the method according to the invention see better performance by 3-15%, preferably 5-10%.

Upon completion of the fermentation the exopolysaccharide can be separated from the wort and purified according to known methods, such as filtration, concentration, crystallization or solvent extraction.

The invention also extends to the exopolysaccharides obtained or that may be received by the specified method. In particular, it is the soap which includes xanthan gum, obtained by the method according to the invention.

Xanthan gum obtained by the method according to the invention, in aqueous solution at 1% in distilled water, has a high transparency, i.e. transparency about 70-95%, or about 80-95%. The coefficient of transparency of the aqueous solution is measured by spectrophotometry at 600 nm.

The following examples serve to illustrate the present invention without limiting its scope.

Examples

Example 1

This example describes a phase preculture 1 and 2 for Xanthomonas campestris.

All these components are dissolved in 1 liter of drinking water, homogenized using a magnetic stirrer and pour into the vessels of Erlenmeyer 500 ml fractions 112 ml.

Drugs are placed in the autoclave for 30 minutes at 120° C.

Strain initially preserved by freezing in the tube at -196° method of freezing vapor of liquid nitrogen.

For freezing with liquid nitrogen prepare preculture in a special medium of the following composition:

To obtain environment all these ingredients dispersed in spring water. Set pH 6.5 using H2SO4(10%). The medium is sterilized in an autoclave at 120° C for 20 minutes.

After 24 hours incubation at 28° With the rotating vesbaltawati at 220 rpm and an amplitude of 50 mm in Kul is ur add 10% vol. clean and sterile glycerol. After this culture spread on the cryovials with the capacity from 1 ml to 10 ml, preferably from 2 ml to 4 ml

These tubes can in the vapor of liquid nitrogen. Preculture 1 sown by cryoprobes, pre-thawed in air at room temperature. All or 50% of the content of cryoprobes sterile injected into the vessel of Erlenmeyer capacity of 500 ml, the medium was sterilized by autoclaving as described above.

Sowed thus the medium is incubated for 24 hours at 28° With the rotating vesbaltawati at 220 rpm and an amplitude of 50 mm

After 24 hours incubation get preculture with a pH of 7 to 7.5, a viscosity of 50 to 500 MPa· and containing a population of bacteria Xanthomonas campestris above 1010/ml.

Preculture 2:

Preculture 1 is used for vysielanie in preculture 2. The composition of the environment preculture 2:

- Sucrose 10 g/l Eurosucre

- Flour from the germ karubi 4 g/l Meyhall AG

- Na2HPO43 g/l Europhos

Potable or softened water qsp 1 litre

- the pH adjusted to 10%sulphuric acid to a value of 6.5 before sterilization.

All these components are suspended in 1 liter of drinking water and adjusted pH to 6.5. Complete medium was dispensed into vessels of Erlenmeyer 500 ml fractions with a volume of 112 ml and autoclave at 120° C for 30 minutes.

The contents of the vessels of Arlena the EPA sow with 0.1-0.2 ml of preculture 1. Incubated vessels of Erlenmeyer from 24 to 30 hours at 28° With the rotating vesbaltawati at 220 rpm with an amplitude of 50 mm

After 24-30 hours of incubation get preculture with a pH from 5.8 to 7.1, a viscosity of 100 to 1000 MPa· and containing a population of bacteria Xanthomonas campestris more than 109/ml.

Example 2

This example describes the preparation and issuance of the exopolysaccharide two ways fermentation: organic source of nitrogen and a mixed source of organic nitrogen and mineral nitrogen.

In this example, resort to two stages of preculture. This stage is carried out in vessels of Erlenmeyer capacity of 500 ml, which corresponds to 100 ml of medium (see examples 1 and 2).

Stage production, which corresponds to the stage at which bacterial strain produces a polysaccharide that occurs in the fermenter with a capacity of 20 litres, including 15 liters useful volume.

Stage preculture 1 and 2:

Stage preculture 1 and 2 operate in the same manner as in example 1.

Stage production:

Wednesday 1:

The last stage is the stage of production of the exopolysaccharide.

The environment of the fermenter 1 has the following composition:

- Sucrose 42 g Eurosucre

- Flour from the germ karubi 6 g Meyhall Hell

- gSO4·7H2O 0.25 g Bittersalz

- Na2HPO42 g Prolabo

- Organic protivovospalitel 0.5 ml

- Drinking or smahc the fair water qsp 1 l

Obtaining sources of nitrogen and carbohydrates carried out separately.

Sucrose ⇒ Qsp grams of glucose dissolved in qsp 3 l of demineralized water or drinking water in a vessel Mariota. Lower the pH to a value of 5.2 with N2SO4(10%). The solution is sterilized in a vessel Mariota for 45 minutes at 120° in the autoclave. Flour from the germ karubi + salt ⇒ Qsp grams of flour from the germ karubi, 30 g of Na2HPO4, 3.75 g MgSO4·7H2O and 7.5 ml of anti-foaming agent is dissolved in qsp 7 l of demineralized water. Bring the pH to a value of 6 using H2SO4(10%). The mixture is sterilized in situ for 45 minutes at 120° C.

1N sodium hydroxide ⇒ 40 g of solid NaOH is dissolved in qsp 1 l of distilled water. The solution is sterilized in a vessel Mariota for 30 minutes at 120° in the autoclave.

On reaching all the ingredients of a temperature of 28° they are mixed in the fermenter. Then in the fermenter sown qsp of preculture 2.

The conditions of fermentation in the fermenter are as follows:

Mixing ⇒ 200 rpm from 0 to 20 hours, then 400 rpm to complete the fermentation.

Aeration ⇒ 400 l/h from 0 to 18 hours, then 825 l/h with 24 hours to complete the fermentation.

The temperature is 28° C.

pH is adjusted to a value of 6.8 using 1N NaOH.

The pressure is atmospheric.

Wednesday 2:

Wednesday 2, which is set alternative environment 1, has the following composition:

- Sucrose 42 g/l (Eurosucre)

- NH4NO31,15 g/l (Atochem)

- MgSO4·7H2O 0.25 g/l (Bitter salz)

- (NH4)2HPO40,217 g/l (Europhos)

- Soluble drug 36 g/l (Meyhall AG)

flour from the germ karubi

- Organic protivovospalitel 0.2 ml

- Softened water qsp 1 l

Sucrose ⇒ Qsp grams of glucose dissolved in qsp 3 l of demineralized water. Set pH to 5 using H2SO4(10%). The solution is sterilized in containers Mariota for 30 minutes at 120° in the autoclave.

Nitrogen + salt⇒ 17,25 g NH4NO3, 3.75 g MgSO4·7H2O, 3,22 g (NH4)2HPO4, 525 g of soluble drug flour from the germ karubi and 3 ml protivovspenivayushchie agent dissolved in qsp 7 l of demineralized water. Set the pH of the solution at level 6 with N2SO4(10%). The mixture is sterilized in situ for 45 minutes at 120° C.

1N sodium hydroxide ⇒ 40 g of solid NaOH is dissolved in qsp 1 l of distilled water. The solution is sterilized in containers Mariota for 30 minutes at 120° in the autoclave.

Soluble preparation of flour from the germ karubi prepared by dilution of flour softened water to a concentration of 6-15%. This solution may, if desired, be processed by the enzymes of the type alkaline, acidic and/or neutral proteases, lipases; Fitz; Melo the data acidic and/or neutral phosphatase, amylase before optional stage decanting in a horizontal rotary decanter, which is carried out to remove impurities that can degrade the quality of the final product.

For achieving all these ingredients temperature 28° With them stirred fermenter (Wednesday 1 or 2). In the fermenter then sown qsp of preculture 2.

The conditions of fermentation in the fermenter 2 are as follows:

Mixing ⇒ 200 rpm from 0 to 20 hours, then 400 rpm to complete the fermentation.

Aeration ⇒ 400 l/h from 0 to 24 hours, then 825 l/h with 24 hours to complete the fermentation.

The temperature is 28° C.

pH is adjusted to a value of 6.8 using 1N NaOH.

The pressure is atmospheric or is from 0.5 to 4 bar.

The results of fermentation.

According to the study of culture medium duration of the fermentation is from 45 to 65 hours of dry substance, which may be precipitated by isopropanol, is from 20 to 30 g/kg, and the yield of a product by weight, relative to the used carbon source is from 50 to 70%. The obtained fermentation wort has a lightness and Shine exceeding ever received with other sources of nitrogen.

Example 3

This example describes a phase preculture 1 and 2 for bacteria of the genus Agrobacterium tumefaciens.

All the components are mixed in 1 liter of drinking water, the resulting solution was homogenized by stirring on a magnetic stirrer and placed in an Erlenmeyer flask 500 ml portions at 112 ml. Withstand autoclave for 15 to 30 minutes at a temperature of 120° C.

Strain initially remain in test tubes, frozen at minus 196° by way of freezing in liquid nitrogen.

For freezing in liquid nitrogen receive preculture on the environment RYS-10, having the following composition:

To prepare the environment all the ingredients displaytouch in spring water. The medium is sterilized for 15 to 30 minutes at a temperature of 120° in an autoclave at natural pH level.

After incubation duration from 24 to 30 hours at a temperature in the range of from 28 to 32° under stirring at vibromassage with a speed of 220 rpm and an amplitude of 50 mm, in culture, add 10%. clean and sterile glycerol. Then the culture is placed in cryoprobes capacity from 1 to 10 ml, preferably from 2 to 4 ml of the Above test tubes stored in liquid nitrogen.

Preculture 1 seeded from previously thawed on the air cryoprobes. The entire volume at 50% of the content of cryoprobes placed in sterile conditions in Erlenmeyer flask with a capacity of 500 ml, the contents of which withstand crashes-auto St is t in an autoclave and sterilized as stated above.

Planted so the environment is kept in the incubator for 24 to 30 hours at a temperature of from 28 to 32° under stirring at vibromassage with a speed of 220 rpm and an amplitude equal to 50 nm. After 24 hours incubation get preculture, the pH of which varies from 7 to 7.5, a viscosity which ranges from 50 to 500 MPa· and the density of the bacteria Agrobacterium tumefaciens above 1010colony forming units (CFU) per ml

Preculture 2:

Preculture 1 required for seeding preculture 2.

The composition of the medium in g/l

sucrose 10

flour from the seed embryo of karubi 1,875

ammonium nitrate 1,0

magnesium sulfate 7H2About 0,25

the disodium salt of phosphoric acid 3,0

rapeseed oil 2 ml/l

softened spring water in the required amount of dooshima 1 l

All components are suspended in 1 liter of soft spring water, and the pH was adjusted to 6.8±0,1. The final environment is placed in the Erlenmeyer flask 500 ml portions at 112 ml and incubated in an autoclave for 15 to 30 minutes at a temperature of 120° C.

Then in these flasks Erlenmeyer sewout from 0.1 to 0.2 ml of preculture 1. The flask was placed in an incubator for a period of from 24 to 30 hours at a temperature of from 28 to 32° under stirring at vibromassage with a speed of 220 rpm and an amplitude of 50 mm After incubation for 24 to 30 hours are preculture, the pH of which is Oh varies from 6.8 to 7.5, the viscosity is from 100 to 1000 MPa· s, and the density of the bacteria Agrobacterium tumefaciens is more than 109CFU/ml

Example 4

This example describes the preparation and obtaining of exopolysaccharides by fermentation in three ways:

in Erlenmeyer flasks 500 ml

- in laboratory fermenters with a capacity of 20 liters

- in the pilot fermenters with a capacity of 1000 liters

Example 4-1

Stage preculture 1 and 2:

This Example describes two stage “preculture”. These stages are carried out in Erlenmeyer flasks 500 ml, and use 100 ml of medium (see Example 1).

Stages of preculture 1 and 2 are also carried out as in Examples 1 and 2.

The last stage is the stage of receipt of exopolysaccharides.

The acquisition phase:

Wednesday 1: Erlenmeyer flask 500 ml (100 useful ml)

The medium has the following composition (brought to volume 1 l):

sucrose 25,00 g Eurosucre

flour from the seed embryo of karubi 1,82 MeyhallAG

ammonium nitrate 0,69 g Prolabo 21280293

To2NRA44,00 Prolabo 26927292

lactic acid 2,20 Prolabo

MgS4that· 7H2About 0.25 to Bittersalz

organic antifoam 0,50

softened water in the required amount to 1 liter

The pH of the final medium was adjusted to 6.9. Sterilized Erlenmeyer flask 500 ml, containing 100 ml of medium in the autoclave for 15 to 30 minutes when the temperature 120° C.

Spend incubation at a temperature of from 28 to 32° on vibroshaker with an amplitude of 50 mm and a speed of 220 rpm

The end of the incubation determined by the analysis on sucrose content, which must be less than or equal to 1.5 g/l).

Example 4-2

Stage preculture 1 and 2:

This Example describes two stage “preculture”. These stages are carried out in Erlenmeyer flasks 500 ml, containing 100 ml of medium (see Example 1).

Stages of preculture 1 and 2 operate in the same way as in Examples 1 and 2.

The last stage is the stage of receipt of exopolysaccharides.

The acquisition phase:

Wednesday 2: laboratory fermenters with a capacity of 20 liters (15 useful liters)

The last stage is the stage of obtaining ekzopolisakharidov.

Wednesday 2, which can be an alternative environment 1, has the following composition:

sucrose 32.00 g/l Eurosucre

soluble fraction

flour from embryo

seed karubi 2.30 g/l Meyhall AG

- NH4NO30.86 g/l Prolabo 21280

- K2NRA40.20 g/l Prolabo 26927

- MgS4, 7H2About 0.25 g/l Prolabo 25165

- lactic acid 2.00 g/l Prolabo 20536

organic antifoam 0.2 ml/l

softened water in the required amount to 1 liter

preparation of sources of nitrogen and carbohydrates carried out separately.

Sucrose ⇒ Required number of grams of sucrose are dissolved in the flask MA is iota in softened water in a quantity required to obtain 3 l of a solution. The pH is reduced to 5.2 10%solution of sulfuric acid. The solution is sterilized in a flask of MARRIOTT from 30 to 45 minutes at a temperature of 120° in the autoclave.

Flour germ seed karubi + salt ⇒ Required number of grams of soluble fractions of the flour from the seed embryo of karubi, 12,9 g NH4NO3, 3.75 g MgS4, 7H2O and 3 ml of antifoam dissolved in soft water amount necessary for the formation of 7 l of a solution. The pH was adjusted to 6.5 10%solution of sulfuric acid. This mixture is sterilized, without selecting components, from 30 to 45 minutes at a temperature of 120° C.

The soluble fraction of the flour from the seed embryo of karubi prepared by dissolving the flour into the softened water to obtain a solution with a concentration of from 6 to 10%. In this solution possible before conducting decanting on the horizontal rotary decanter to eliminate impurities that can degrade the quality of the final product, if necessary add enzymes type alkaline, acidic and/or neutral proteases; lipase; phytase; alkaline, acidic and/or neutral phosphatase; amylase; organic acid.

Odnokorennye solution of sodium hydroxide ⇒ 40 g of pellets of NaOH dissolved in distilled water required to form 1 liter of solution. the shown solution is sterilized in a flask of MARRIOTT 30 minutes at a temperature of 120° With the autoclave.

When all the ingredients reach a temperature of from 28 to 32° With, they are mixed in the fermenter. Then in the fermenter put the required number of preculture 2.

The conditions of fermentation in the fermenter following:

- Mixing ⇒ 200 rpm for from 0 to 20 hours, then 400 rpm before the fermentation

- Aeration ⇒ 400 l/h from 0 to 18 hours, then 825 l/h for 24 hours to complete the fermentation

- The temperature of the support in the range of from 28 to 32°

- PH regulate odnomomentnym solution of sodium hydroxide to a value of 6.7±0,2.

Example 4-3

Stage preculture 1 and 2:

This Example describes two stage “preculture”. Preculture 1 prepared in Erlenmeyer flasks 500 ml, containing 100 ml of medium, and Preculture 2 is prepared in the fermenter with a capacity of 20 liters, containing 15 liters of medium (see Examples 1 and 2).

Stages of preculture 1 and 2 are also carried out as in Examples 1 and 2.

The last stage is the stage of receipt of exopolysaccharides.

The acquisition phase:

Wednesday 3: experimental fermenters with a capacity of 1000 liters (800 useful liters).

Wednesday 3, which may be an alternative environments 1 and 2, has the following composition:

sucrose 32.00 g/l (Eurosucre)

NH4NO30,94 (Hydro)

MgS4·7H2About 0.12 g/l (Bitter salz)

K2NRA40.10 g/l (Rhodia)

Rast is ARIMA fraction of the flour

from the germ seed karubi 26,60 g/l (Meyhall AG)

Lactic acid 2.50 g/l Purac

organic antifoam 0,20 ml

softened water in the required amount to 1 liter

Sucrose ⇒ Required number of grams of sucrose are dissolved in softened water in an amount necessary to obtain 320 l of solution. The pH was adjusted to 5.00 10%solution of sulfuric acid. The solution is sterilized in a sterilizer with a capacity of 600 l from 30 to 45 minutes at a temperature of 120° C.

Nitrogen + salt ⇒ 750 g NH4NO3, 96 g MgS4, 7H2O, 80 g K2HPO421.3 kg soluble fractions of the flour from the seed embryo of kaburi and 150 ml of antifoam dissolved in softened water in an amount necessary to obtain 230 l of solution. The pH level of the specified solution was adjusted to 6.00 with concentrated sulfuric acid. This mixture is sterilized, without selecting components, within 30 to 45 minutes at a temperature of 120° C.

The soluble fraction of the flour from the seed embryo of karubi prepared by dissolving the flour into the softened water to obtain a solution with a concentration of from 6 to 10%. In this solution possible before conducting decanting on the horizontal rotary decanter to eliminate impurities that can degrade the quality of the final product, if necessary add enzymes type alkaline, acidic and/linetraining proteases; lipase; phytase; alkaline, acidic and/or neutral phosphatase; amylase.

When all the ingredients reach a temperature of from 28 to 32° With, they are mixed in the fermenter. Then in the fermenter put the required number of preculture 2.

The conditions of fermentation in the fermenter 3 the following:

- Mixing ⇒ 110 rpm from 0 to 20 hours, then 200 rpm before the fermentation

- Aeration ⇒ 25 Nm3/h from 0 to 24 hours, then 45 Nm3/h for 24 hours to complete the fermentation

- The temperature of the support in the range of from 28 to 32°

The pH regulating solution of 300 g/l NaOH to 6.7±0.2

- The pressure is atmospheric or higher and may be in the range from 0.5 to 4 bar.

The results of fermentation:

In accordance with the test environment culture duration of fermentation may vary from 55 to 100 hours, the solids that precipitated with isopropanol, vary from 15 to 25 g/kg, and the weight of the output with respect to the carbon source ranging from 50 to 70%. The resulting wort fermentation has a lightness, Shine, never observed no other source of nitrogen.

1. The method of producing exopolysaccharides by cultivation of microorganisms in a nutrient medium containing at least one source of carbon assimilated by microorganisms, and, IU the greater extent, one organic nitrogen source, characterized in that the nitrogen source is from the faction seed karubi.

2. The method according to claim 1, characterized in that the protein content in fractions of seed karubi is at least 45%, preferably at least 50%, even more preferably at least 60% relative to the weight of dry matter.

3. The method according to claim 2, characterized in that the proteins preferably have a high content of arginine, glutamine and/or glutamic acid and lysine.

4. The method according to one of claims 1 to 3, characterized in that the lipids in the fraction of seeds karubi is at least 4%, preferably at least 5%, more preferably 7-15% relative to the weight of dry matter.

5. The method according to one of claims 1 to 4, characterized in that the specified fraction is the embryo of the seed karubi.

6. The method according to one of claims 1 to 5, characterized in that the fraction of seeds karubi is in the form of flour.

7. The method according to claim 6, characterized in that the grain size distribution of flour 10-150 microns.

8. The method according to one of claims 1 to 7, characterized in that the fermentation is carried out in a nutrient medium, additionally containing at least one mineral source of nitrogen.

9. The method according to claim 8, characterized in that the mineral nitrogen source is selected from ammonium nitrate or sodium phosphate or self the tov ammonium individually or in a mixture.

10. The method according to one of claims 1 to 9, characterized in that the concentration of organic and, if necessary, mineral nitrogen source in the fermentation medium is 1-80 g/l, preferably 3-50 g/l, even more preferably 5-30 g/l

11. The method according to one of claims 1 to 10, characterized in that the assimilated carbon source selected from glucose and sucrose.

12. The method according to one of claims 1 to 11, characterized in that the concentration of assimilated carbon source is 1-100 g/l, preferably 15-80 g/l

13. The method according to one of claims 1 to 12, characterized in that the fermentation is carried out at a temperature of 15-100°C., preferably 25-80°S, even more preferably 25-35°C.

14. The method according to one of claims 1 to 13, characterized in that the microorganism is chosen from the group of bacteria of the genus Xanthomonas, the genus Alcaligenes, the genus Agrobacterium, the genus Arthrobacter, the genus Azotobacter, Pseudomonas, the genus Corynebacterium, fungi of the genus Sclerotium, the genus Aspergillus and yeasts of the genus Hansenula.

15. The exopolysaccharide obtained by the method according to one of claims 1 to 14, with 1%of the first aqueous solution in distilled water transparency about 70-95%.



 

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