CN113046398A - Fermentation method for stably and efficiently producing L-isoleucine and fermentation stabilizer - Google Patents

Fermentation method for stably and efficiently producing L-isoleucine and fermentation stabilizer Download PDF

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CN113046398A
CN113046398A CN202110539756.3A CN202110539756A CN113046398A CN 113046398 A CN113046398 A CN 113046398A CN 202110539756 A CN202110539756 A CN 202110539756A CN 113046398 A CN113046398 A CN 113046398A
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fermentation
isoleucine
stabilizer
stably
efficiently producing
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高鹏
吴涛
常利斌
龚华
李岩
赵津津
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TONGLIAO MEIHUA BIOTECHNOLOGY CO Ltd
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TONGLIAO MEIHUA BIOTECHNOLOGY CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound

Abstract

The invention discloses a fermentation method and a fermentation stabilizer for stably and efficiently producing L-isoleucine, wherein 0.8-1.2mL of fermentation stabilizer is supplemented to 1L of fermentation liquor once every 1h during 22-42h of fermentation, and each liter of fermentation stabilizer contains 2-4g of cobalamin, 1-3g of pyridoxine, 1-3g of choline chloride and 2-4g of folic acid, so that key nutrients which are actually lacked and are important to the anabolism of L-isoleucine are compensated after the fluctuation of a nitrogen source, the yield is increased, the aim of stable production is fulfilled, and the stability of an acid production level is increased; the acid production by horizontal fermentation of a 50L tank can stably reach more than 48.0 g/L. The method is simple and easy to implement, has low investment but remarkable effect, does not need to add any additional equipment or manpower input, and is suitable for large-scale industrial production.

Description

Fermentation method for stably and efficiently producing L-isoleucine and fermentation stabilizer
The technical field is as follows:
the invention relates to the technical field of L-isoleucine fermentation, in particular to a fermentation method for stably and efficiently producing L-isoleucine and a fermentation stabilizer.
Background art:
l-isoleucine, also known as L-isoleucine, belongs to Branched Chain Amino Acids (BCAA), has various physiological functions, is a raw material for synthesizing human hormones and enzymes, has the effects of promoting protein production and inhibiting protein decomposition, and has wide application and commercial value in the food and medicine industries.
At present, a plurality of enterprises in China have produced L-isoleucine by fermentation methods, and laboratories in various colleges and universities are also developing the research subject of L-isoleucine fermentation processes. In the existing L-isoleucine fermentation process, corn steep liquor, hydrolysate or bean cake powder and the like are often used as main nitrogen sources. 10g/L corn steep liquor in the fermentation medium as disclosed in patent CN 201910980511; the patent CN201910134832 discloses that the fermentation medium contains soybean meal hydrolysate. The nitrogen source has a wide application in the field of fermentation because of its rich nutrition, relatively low cost and readily available raw materials, but it has a disadvantage of poor quality stability. Taking corn steep liquor as an example, the corn steep liquor is a concentrated solution of a soaking solution obtained by soaking corn with sulfurous acid, contains abundant amino acids, reducing sugar, organic acid, phosphorus, trace elements and growth factors, and is influenced by differences in production places, seasons, soaking processes and the like of raw materials, and the nutritional composition of the corn steep liquor has large fluctuation, so that the acid production level is large fluctuation. Often, at this moment, technical staff adopts the mode of increasing main nitrogen source quantity, or adding other more stable nitrogen sources to improve, and the result has increased the auxiliary material cost on the one hand, and on the other hand probably because the increase of feed liquid nutrient substance concentration, the thallus growth produces adverse effect in the early stage of fermentation, even brings the difficulty for later stage extraction.
If substances which are really important to the anabolism of L-isoleucine in nitrogen sources such as corn steep liquor, hydrolysate or bean cake powder can be determined, the substances can be supplemented in a targeted manner, and the effect of achieving twice the result with half the effort can be achieved, but related research and reports are not available at present.
The invention content is as follows:
in order to solve the above technical problems, the first object of the present invention is to provide a fermentation method for producing L-isoleucine stably and efficiently with low investment, which is simple and easy to implement.
The second object of the present invention is to provide a fermentation stabilizer for stable and efficient production of L-isoleucine.
The first purpose of the invention is implemented by the following technical scheme: a fermentation method for stably and efficiently producing L-isoleucine is characterized in that when the yield is reduced due to the quality fluctuation of a main nitrogen source, 0.8-1.2mL of fermentation stabilizer is supplemented to 1L of fermentation liquor once every 1 hour during fermentation for 22-42 hours, and each liter of fermentation stabilizer contains 2-4g of cobalamin, 1-3g of pyridoxine, 1-3g of choline chloride, 2-4g of folic acid and the balance of deionized water.
Furthermore, the main nitrogen source is any one or the combination of more than one of corn steep liquor, corn steep liquor hydrolysate and bean cake powder.
The second purpose of the invention is implemented by the following technical scheme: a fermentation stabilizer for stably and efficiently producing L-isoleucine comprises, per liter, 2-4g of cobalamin, 1-3g of pyridoxine, 1-3g of choline chloride, 2-4g of folic acid, and the balance of deionized water.
The invention mainly aims at the fermentation production of the L-isoleucine by taking corn steep liquor, hydrolysate or bean cake powder and the like as main nitrogen sources, when the yield is reduced due to the quality fluctuation of the main nitrogen sources, by supplementing proper amount of nutrient substances which are contained in the main nitrogen sources and are important relative to the anabolism of the L-isoleucine, including cobalamin, pyridoxine, choline chloride and folic acid, the adverse effect of the quality fluctuation of the main nitrogen sources is resisted, the actually lacking key nutrients which are important relative to the anabolism of the L-isoleucine behind the quality fluctuation of the nitrogen sources are compensated, the proportion among the cobalamin, the pyridoxine, the choline chloride and the folic acid is reasonably and optimally designed, the yield is improved, and the purpose of stable production is achieved.
The invention has the advantages that:
(1) according to the method, 0.8-1.2mL of fermentation stabilizer is supplemented into 1L of fermentation liquor at intervals of 1 hour during fermentation for 22-42 hours, and each liter of fermentation stabilizer contains 2-4g of cobalamin, 1-3g of pyridoxine, 1-3g of choline chloride and 2-4g of folic acid, so that key nutrients which are actually lacked and are important to the anabolism of L-isoleucine are compensated behind the quality fluctuation of a nitrogen source, the yield is improved, the aim of stable production is fulfilled, and the stability of the acid production level is improved; the acid production by horizontal fermentation of a 50L tank can stably reach more than 48.0 g/L.
(2) The method is simple and easy to implement, has low investment but remarkable effect, does not need to add any additional equipment or manpower input, and is suitable for large-scale industrial production.
The specific implementation mode is as follows:
the present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. Appropriate modifications to the process parameters, which may be apparent to those skilled in the art in view of the disclosure herein, are also considered to be within the scope of this patent.
Comparative experimental example 1:
the cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The culture medium used by the seeds comprises: 30g/L of cane sugar, 60g/L of corn steep liquor, 1.0g/L of magnesium sulfate, 1.5g/L of dipotassium hydrogen phosphate, 0.2mL/L of defoaming agent, 1.5mg/L of biotin, 12.5 mg/L of VBs, and the balance of water. The culture medium used for fermentation comprises the following components: 50g/L glucose, 40g/L corn steep liquor, 15g/L ammonium sulfate, 1.5g/L dipotassium hydrogen phosphate, 1.0g/L magnesium sulfate, 0.2mL/L defoaming agent, 2.0g/L valine, 1.0g/L methionine, 0.1mg/L biotin, 10.2mg/L VBE and the balance of water. The fermentation conditions were: the pressure in the tank is 0.06-0.08Mpa, the ventilation quantity is 10-20L/min, the stirring is carried out at 600rpm, the temperature is 30-33 ℃, the pH value is 6.8-7.2, and the dissolved oxygen is 30-50%.
The comparative experiment example was repeated 3 times, each time using a different batch of corn steep liquor, the fermentation was completed, and the isoleucine content in the fermentation broth was determined by HPLC, with the acid production level shown in table 1 below.
Comparative experiment example 2: and (3) during fermentation for 22-42h, supplementing 1.0mL of fermentation stabilizer into 1L of fermentation liquor at intervals of 1h, wherein the fermentation stabilizer contains 3g/L of cobalamin, 2g/L of pyridoxine, and the balance of deionized water.
The cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
The comparative experiment example was repeated 3 times, each time using a different batch of corn steep liquor, and after fermentation, the content of L-isoleucine in the fermentation broth was determined by HPLC, and the acid production level was shown in table 1 below.
Comparative experiment example 3: fermenting for 22-42h, and supplementing 1.0mL of fermentation stabilizer once every 1h to 1L of fermentation liquor, wherein the fermentation stabilizer contains 2g/L of choline chloride, 3g/L of folic acid and the balance of deionized water.
The cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
The comparative experiment example was repeated 3 times, each time using a different batch of corn steep liquor, and after fermentation, the content of L-isoleucine in the fermentation broth was determined by HPLC, and the acid production level was shown in table 1 below.
Comparative experiment example 4: fermenting for 22-42h, supplementing 1.0mL of fermentation stabilizer to 1L of fermentation liquor at intervals of 1h, wherein the fermentation stabilizer contains 2g/L of pyridoxine, 2g/L of choline chloride, 3g/L of folic acid, and the balance of deionized water.
The cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
The comparative experiment example was repeated 3 times, each time using a different batch of corn steep liquor, and after fermentation, the content of L-isoleucine in the fermentation broth was determined by HPLC, and the acid production level was shown in table 1 below.
Comparative experiment example 5: and (3) once supplementing 1.0mL of fermentation stabilizer into 1L of fermentation liquor every 1h during fermentation for 22-42h, wherein the fermentation stabilizer contains 3g/L of cobalamin, 2g/L of pyridoxine, 2g/L of choline chloride and the balance of deionized water.
The cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
The comparative experiment example was repeated 3 times, each time using a different batch of corn steep liquor, and after fermentation, the content of L-isoleucine in the fermentation broth was determined by HPLC, and the acid production level was shown in table 1 below.
Comparative experiment example 6: fermenting for 22-42h, supplementing 1.0mL of fermentation stabilizer to 1L of fermentation liquor at intervals of 1h, wherein the fermentation stabilizer contains 5g/L of cobalamin, 0.5g/L of pyridoxine, 2g/L of choline chloride, 3g/L of folic acid and the balance of deionized water.
The cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
The comparative experiment example was repeated 3 times, each time using a different batch of corn steep liquor, and after fermentation, the content of L-isoleucine in the fermentation broth was determined by HPLC, and the acid production level was shown in table 1 below.
Comparative experiment example 7: and (3) once supplementing 1.0mL of fermentation stabilizer into 1L of fermentation liquor every 1h during fermentation for 22-42h, wherein the fermentation stabilizer contains 3g/L of cobalamin, 4.5g/L of pyridoxine, 4.5g/L of choline chloride, 3g/L of folic acid and the balance of deionized water.
The cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
The comparative experiment example was repeated 3 times, each time using a different batch of corn steep liquor, and after fermentation, the content of L-isoleucine in the fermentation broth was determined by HPLC, and the acid production level was shown in table 1 below.
Example 1: a fermentation method for stably and efficiently producing L-isoleucine comprises the steps of fermenting for 22-42h, supplementing 0.8mL of fermentation stabilizer to 1L of fermentation liquor at intervals of 1h, wherein the fermentation stabilizer contains 2g/L of cobalamin, 1g/L of pyridoxine, 1g/L of choline chloride, 2g/L of folic acid and the balance of deionized water;
the cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
This example was repeated 3 times, using different batches of corn steep liquor each time, the fermentation was completed, and the content of L-isoleucine in the fermentation broth was measured by HPLC, and the acid production level is shown in table 1 below.
Example 2: a fermentation method for stably and efficiently producing L-isoleucine comprises the steps of fermenting for 22-42h, supplementing 0.8mL of fermentation stabilizer to 1L of fermentation liquor at intervals of 1h, wherein the fermentation stabilizer contains 3g/L of cobalamin, 2g/L of pyridoxine, 2g/L of choline chloride, 3g/L of folic acid and the balance of deionized water;
the cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
This example was repeated 3 times, using different batches of corn steep liquor each time, the fermentation was completed, and the content of L-isoleucine in the fermentation broth was measured by HPLC, and the acid production level is shown in table 1 below.
Example 3: a fermentation method for stably and efficiently producing L-isoleucine comprises the steps of fermenting for 22-42h, supplementing 1.0mL of fermentation stabilizer to 1L of fermentation liquor at intervals of 1h, wherein the fermentation stabilizer contains 3g/L of cobalamin, 2g/L of pyridoxine, 2g/L of choline chloride, 3g/L of folic acid and the balance of deionized water;
the cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
This example was repeated 3 times, using different batches of corn steep liquor each time, the fermentation was completed, and the content of L-isoleucine in the fermentation broth was measured by HPLC, and the acid production level is shown in table 1 below.
Example 4: a fermentation method for stably and efficiently producing L-isoleucine comprises the steps of fermenting for 22-42h, supplementing 1.2mL of fermentation stabilizer to 1L of fermentation liquor at intervals of 1h, wherein the fermentation stabilizer contains 4g/L of cobalamin, 3g/L of pyridoxine, 3g/L of choline chloride, 4g/L of folic acid and the balance of deionized water;
the cultured Corynebacterium glutamicum seed liquid is inoculated into a 50L fermentation tank according to the inoculum concentration of 10 percent, and the initial volume is 18L. The seed, the composition of the medium used for fermentation and the fermentation culture conditions were the same as those in comparative experiment example 1.
This example was repeated 3 times, using different batches of corn steep liquor each time, the fermentation was completed, and the content of L-isoleucine in the fermentation broth was measured by HPLC, and the acid production level is shown in table 1 below.
TABLE 1 comparative index of acid production for Experimental examples 1-7 versus examples 1-4
Figure BDA0003071208480000081
As can be seen from Table 1, 0.8-1.2mL of fermentation stabilizer is supplemented into 1L of fermentation liquor at intervals of 1 hour during 22-42 hours of fermentation, each liter of the fermentation stabilizer contains 2-4g of cobalamin, 1-3g of pyridoxine, 1-3g of choline chloride and 2-4g of folic acid, targeted nutrition supplementation is realized, the acid production difference between different corn steep liquor batches in the same process is obviously shortened, the acid production level is improved, the fermentation cost is reduced, the 50L tank horizontal fermentation acid production is stabilized to be more than 48.0g/L, 1.0mL of fermentation stabilizer is supplemented into 1L of fermentation liquor at intervals of 1 hour during 22-42 hours of fermentation, 3g/L of cobalamin, 2g/L of pyridoxine, 2g/L of choline chloride and 3g/L of folic acid are contained in the fermentation stabilizer, the effect is most remarkable. Meanwhile, it can be seen from table 1 that the purpose of improving yield and stabilizing production can be achieved only when the components and the proportion in the fermentation stabilizer are reasonably designed.
Interpretation of terms:
1. the fermentation process comprises the following steps: the method is a whole process for accumulating a target product by utilizing microbial strains, and comprises the steps of culture medium formula and configuration, strain rejuvenation, expanding culture, control of the culture process, control of key parameters such as temperature, pH, dissolved oxygen and the like, nutrition supplement, control of culture ending and the like.
2. The existing fermentation process comprises the following steps: refers to publicly reported or widely known fermentation processes.
HPLC determination: the high performance liquid chromatography detection method is a chromatographic process using liquid under high pressure as a mobile phase. So-called column chromatography, thin layer chromatography or paper chromatography are classical liquid chromatography. The stationary phase used is an adsorbent (silica gel, alumina, etc.) of greater than 100 um. The fixed phase used by the traditional liquid chromatography has large granularity and slow mass transfer diffusion, so the column efficiency is low, the separation capability is poor, and only simple mixture separation can be carried out. The stationary phase used by the high performance liquid chromatography detection method has small granularity (5-10 um), fast mass transfer and high column efficiency.
4. The invention adopts an Agilent Technologies 1200 to detect the content of L-isoleucine accumulated in fermentation liquor, and the specific method is as follows:
4.1 column: ZORBAX Eclipse-AAA column (3.5 μm, 4.6X 75 mm).
4.2 mobile phase A: 6.24g NaH was weighed2PO4·2H2O, transferred into a 1000mL glass beaker. 1000mL of ultrapure water was added and stirred until all crystals were completely dissolved. The pH of the solution was adjusted to 7.80 with NaOH.
4.3 mobile phase B: acetonitrile methanol water 45:45:10 (V/V).
4.4 flow rate: 2 ml/min.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (3)

1. A fermentation method for stably and efficiently producing L-isoleucine is characterized by comprising the following steps: when the yield is reduced due to the quality fluctuation of a main nitrogen source, 0.8-1.2mL of fermentation stabilizer is supplemented to 1L of fermentation liquor once every 1h during fermentation for 22-42h, wherein each liter of the fermentation stabilizer contains 2-4g of cobalamin, 1-3g of pyridoxine, 1-3g of choline chloride, 2-4g of folic acid and the balance of deionized water.
2. The fermentation method for stably and efficiently producing L-isoleucine according to claim 1, wherein: the main nitrogen source is any one or the combination of more than one of corn steep liquor, corn steep liquor hydrolysate and bean cake powder.
3. A fermentation stabilizer for stably and efficiently producing L-isoleucine is characterized in that each liter of the fermentation stabilizer contains 2-4g of cobalamin, 1-3g of pyridoxine, 1-3g of choline chloride, 2-4g of folic acid and the balance of deionized water.
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