RU1438235C - Method of growing microorganisms for producing l-lysine - Google Patents

Abstract

The invention relates to the microbiological industry and relates to a method for cultivating microorganisms using emulsifiers of antifoam agents, including polypropylene glycol ether, stabilizer and water, and can be used in lysine biosynthesis. The aim of the invention is to reduce the cost of the process. The method consists in using saponified animals and microbial fats in the amount of 0.2-1% by weight of the emulsion as stabilizer. The resulting stable emulsion of polypropylene glycol ether, for example, B-400 propinol, is introduced into the nutrient medium in an amount containing 0.015-0.15% antifoam from the volume of the medium, and then, during periods of increased foam formation, sterile the emulsion is served in portions - / L mi containing 0.0002-0.02% of the foam-Y system from the volume of the medium. 3 tab. VMM

Description

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Joint venture

HloOpflTCIHK RTNPSIGSG to GLP, pO () IP log ches to o G; kag jggspspyyub cult pyrope and microorganism of the low-sea of the city of ig.use of g, intnchny1khkh dehumidifiers and can be used with 6HocnFfTP4e L-lneiiia.

The aim of the invention is to reduce the cost of the process.

The way the goth was used is as follows.

Microorganisms eliminate gamma aeration on a nutrient medium into which a sterile emulsion of polypropylene glycol ester, for example, propcnol B-AOO, is added, stabilized with omshchen zkir in an amount of 0.2-1.0 wt%.

Saponification of animal or microbial hspre is carried out with a 40% aqueous solution of potassium or sodium hydroxide for 1-1.5 hours, depending on the type of fatty raw materials while stirring the reaction mixture. .

The sterile emulsion is added in such an amount that the content of polypropylene glycol ether in the nutrient medium is 0.015-0.15%. During cultivation, the sterile nucc emulsion is added during periods of intensification of foaming in portions containing 0.0002- 0.02% polypropylene glycol ether from the volume of the medium.

Example 1. Lysine biosynthesis was carried out using a microorganism culture Brevibacterium sp.22L in a 100 m fermenter. The growth of the microorganism is carried out in 50 m of a sterile gestational medium containing 10 tons (20% by volume) of sugar beet molasses; 1 m (2%) of corn extract; 5.3 m (10.6%) of BBKj fermentalizate 0.9 t (1.8%) of ammonium chloride 55 kg (0.11%) of disubstituted ammonium phosphate and 30 kg (0.015% of propanol B-400 to the volume of medium ) A 25% emulsion of propinol B-400 stabilized by saponified fish oil in a co-nitrate of 0.5 wt.%.

The emulsion is prepared as follows.

3 tons of water and 20 kg of saponified fish ira are loaded into a stirred reactor. After complete dissolution of the stabilizer, 1 ton of polypropylene glycol propylene ether is added to the reactor, the mixture is stirred and passed through a dispersing device. 4020 kg of a 25% emulsion of foams and gels are obtained, which is 0.5 m; and:,%, nMiri.iiRMHOfo fish. The resistance of the emulsion to cop. Lg of spensia is determined by means of iitrifu1 and zop - nor its function in the rifuge at 000 rpm for 15 min. Veg1 1 the cause of stability in npoiieHTax p is calculated using the formula

 V.

. 100%,

where V is the volume of polypropylene glycol ether in the emulsion, Y is the volume of the precipitated phase,

cm

For this emulsion, X 99%. After steaming by steam for 2 hours, the emulsion is used to add to the fermenter. The stability of the emulsion after sterilization X 95%.

Saponification of fish oil is carried out as follows.

A fish oil with a sludge number of 178.2 mg KOH / G in an amount of 25 L is loaded into a reactor equipped with a low-speed stirrer and a heating jacket, and 5.1 l saponified with a saponification number of 5.1 L of a 40% aqueous solution of sodium hydroxide are added. Alkaline hydrolysis is carried out at 80-85 C for 1.5 hours with periodic stirring. The process is stopped when the reaction mass reaches pH 8.

A 5-m producer 24-hour culture grown on culture medium with 4% molasses, 7% corn extract, 1% ammonium chloride was used as seed.

Fermentation is carried out under ayeptic conditions at 30-32 s, continuous azration and stirring with compressed air in an amount of 1 m / min per 1 m of culture liquid, at a pH of between 7.3-7.5; pressure 1-h in the apparatus 0.2-0.3 ati. Foam control is carried out by adding defoamers in 0.4 L portions (0.0002% of propinol B-400 from the volume of medium) to the fermenter from the flasks of the sterile emulsion during periods of sharp increase in foaming. The biosynthesis process is carried out for 59 hours. 50 m of culture fluid is obtained with a lysine content of 32.5 g / l. Biomass yield 36 g / l.

The total consumption of stabilized emulsion propipol B-400 pl operation

gost; sewed 298 l, which in terms of ml, the propypol B-400 is Ik, kg.

EXAMPLE 2 Lysine biosynthesis is carried out using a microorganism culture Micrococcns glutamicuin fr 95 D with a fermenter with a capacity. 100 conditions and x, similar to those described in example 1.

Before sterilization, 300 kg (0.15% propinol B-400 of the volume of medium) of a 25% propinol B-400 emulsion prepared as follows are introduced into the growth medium.

3 tons of water and 40 kg of saponified microbial fat are loaded into a stirred reactor. After the stabilizer is completely dissolved, 1 ton of propinol is added to the reactor, the mixture is stirred and passed through a dispersant. Get 4040 kg of a 25% emulsion of propinol B-400, stabilized with 1 wt.% Saponified microbial fat. The stability of this emulsion is, After steam sterilization, as in example 1, the stability of the emulsion is equal and it is used to add to the fermenter and introduce into the nutrient medium,

Saponification of microbial fat is carried out as follows.

Microbial fat with a saponification number of 179.2 kg KOH / G in an amount of 20 L is loaded into a reactor equipped with a silent mixer and a heating jacket and 4.5 l of a 40% aqueous solution of potassium hydroxide calculated from the saponification number are added. Alkaline hydrolysis is carried out at 80-85 ° C for 1.5 hours with periodic stirring. The process is stopped when the reaction mass reaches pH B,

Fermentation is carried out as indicated in

Example 1, I regulate foaming by adding a sterile emulsion of propinol B-400 stabilized with saponified microbial fat to the fermenter via pipelines containing 0.02% polypropylene glycol ether based on the volume of the medium. The duration of the fermentation is 64 hours. 49 m of culture fluid is obtained with a lys content of 34.2 g / l. The biomass yield of 40 g / l. The total consumption of stabilized emulsion of propylene B-400 on. the operation was 250 liters, which is calculated on

 ,

SOBsUGG Ave. - lOl.) (.-ОГТ; Ч Ч | (-; 62,. -j Ki-;

Fl rimer, C. Ocyiuec ri j; 4; f Ppo-gnte lipinp with tymoshchirt ku. microsip 1P a11ppm lU twibncLefi iini p. E-53I r fermenter with; mkost.ch1 100 m m for 72 h, both opistmo and primro 1. Into the nutrient medium before stsrilpz 1I injected 100 l (0.08% propcnol (- from the volume of medium) 40% - Sing out the propylol B-400 propylol, staschlizirochlmnon with mild fish oil in the amount of 1 wt.%. Less than (fish oil. And emulsion attenuation gtrovodpt according to example 1. Stability of the original emulsifier, after sterilization emulsion stability.

The mode of foaming is carried out by feeding this sterile emulsion into portions of the fermenter in portions containing 0.008% propinol from the medium. 52 m of culture liquid with a lysine content of 35.4 g / l is obtained. Biomass yield 36 g / l. The total consumption of stabilized emulsion for the operation was 166 l, which, in terms of B-400 propinol, was 70.5 kg,

PRI me R 4, Biosynthesis was carried out using a culture of the microorganism Brevibacterium sp. E-531 in a fermenter with a capacity of 100 m for 69 hours, as described in example 1. 300 kg (0.15% of propinol B-400 from the volume of medium) of 25% propinol B-400 emulsion are fed into the nutrient medium cooked as follows. .

3 tons of water and 10 kg of C-20 sorbitol are loaded into a reactor equipped with a stirrer and the system is stirred until the stabilizer is completely dissolved. In another, equipped with a stirrer and a heating jacket, 1 ton of B-400 propipol is loaded. and 10 tons of sorbitan C, which is a solid. After heating and stirring until sorbitan C is completely dissolved, the pinol B-400 is transferred to the first reactor, the mixture is mixed and passed through a dispersant. Get 4020 kg of a 25% emulsion of propinol B-400, stabilized with 0.5 wt.X mixture of sorbitan C-20 / sorbitan C. The stability of the emulsions, after sterilization,

The mode of foaming is carried out by feeding this sterile emulsion into the fermenter in portions, co5i .i lH.DS

 C1, P, l. 1Г1к) J-j- iOO from 2 put m d (15lp; im) and F3

m.p.ml g, 1) glm. Tln.iiy tnKvi S6 m kulg-tu-cp pipeline sterile emulsion

rlpioio zhnl.k () sti with a lysi-peo-extinguisher. At the end of the fermeptan U, 5 g / l. The yield of GP omassa is 38 g / l.

The total sum of the pulsation of the pulsation H; I of the opera-pulp-fluid and the biopsy yield is 3 (0 l, which is in terms of mass, as well as the flow rate of the desiccant on

those on propnnol sost.o. 85kg defoaming. Cultivation data Semi-aromatic results are given in sij-no producers of lysine using a table of contents, 1.10 km of emulsion according to Table 2 are given in

As can be seen from the table, when using the floor table 3. . yawning as a stabilizer

emulsions polypropylene of glycolic ether. The analysis of Tables 2 and 3 shows ipa, for example, propinol B-AOO, the immense advantage of saponified veins of IHbtx lipoproteins, there is an increase of 15 rubles compared to soap as a stabilization compared to -zator emulsions of polypropylene glycol gzmulsin with the same con-zfirs. Using stabilizer saponified fats reduces cooking time. Examples 5-10. The emulsin is carried out, the emulsin is obtained. GP synthesis of lysine using a culture 20 with a smaller droplet size than in the case of the host microorganism Brevibacterium sp., And the next or other culture in the enzyme and with a larger number of them on a nutrient medium, as described by centering. This leads to age- example 1. As an anti-foaming agent, it has an anti-foaming efficiency and, as l use 25% propulsion emulsions, to reduce the consumption of foam-alcohol B-400 or polypropylene glycol-sieve. The latter, as can be seen from this ether of the alcohols of the С-р-С fraction, co, table 3, favorably affects the average mol. m. 1000, stabilized-growth culture and lysine biosynthesis. They are mixed with saponified fish or microbial, in O1 saponified fats it has mesiron and, for comparison, it has 30 combination of properties of components, which allows to obtain stable emulsion. The emulsion is prepared as follows. . Loaded into a stirred reactor; 3t

Claims (1)

; water and 20 kg of emulsion stabilizer of the Formula of the invention I and fix the time of its complete dissolution - 35  ; Reni. After complete dissolution, the method of culturing the microorganizer in the reactor is added 1 tnism - producers of L-lysine, pre-. antifoam, mixed with antifoam, and a pre-absorbing feeder, is passed through a dispersing oral medium and emulsifiers of antifoam from the food. After stirring, 40 polypropylene glycol ether is taken from the emulsion sample to be determined as a medium-diluent, adding it to the droplet feeding diameter, and then, using a good medium, inoculation and subsequent dispersion, the fermentation process is selected from control samples for analysis of variance .leam of the level of foaming and fractional The stability of the emulsion before and after -45 filing of the emulsion defoamer, about sterilization is determined, as in the example and with the fact that, with Re 1., in order to reduce the cost of the process, as the results of determining the parameters of the stabilizer, animals use emulsions with various stabilizers or microbial veins, which are preliminary (the content of antifoam in the emulsion is 50 saponified at 80 ... 85 s for 25 vol.%) are given in Table 2. 60 ... 90 min before setting Fermentation is carried out in aseptic pH 8, while the emulsion is prepared from Kifx conditions at 20-32 ° С, continuous calculation of content in it aeration and mixing with compressed air 0.2 ... 1 wt.% and in nutrient in quantity (1 m / min per 1 m 55QP AV is introduced at the rate of 0.015 cultural fluidity, at a pH of 0.15 wt.% polypropylene glycol within the range of 7.3–7, 5, and .p.involutions in the appa-ether of the volume of the medium, and a fractional dose of 0.2-0.3 ati. Pension-J; a4y is regulated at the rate of 0.0002 imaging entities, as in pr-02 wt.%. tj in yes go vD 1G | f Ch and oh oh g 04 oh oh 1L cm about " about 00 th  with 1L m ™ P "L with g4 4 VO v about s .-Si m m cx "oh and cm M g f 4J OO rt yu de p 1-1 s 5) . m about. PQ L Brevibacterium I 38235 12 table 3