阅读说明：本技术 一种采用复合菌株发酵制备i+g的方法 (Method for preparing I + G by fermenting compound strains ) 是由 孔凡明 吴涛 高鹏 常利斌 龚华 李岩 赵津津 于 2021-10-21 设计创作，主要内容包括：本发明公开了一种采用复合菌株发酵制备I+G的方法,利用了解淀粉芽孢杆菌菌株B.amyloliquefaciens G10和B.amyloliquefaciens 8311,以及大肠杆菌工程菌株AP-03,步骤包括S1.斜面培养；S2.一级种子培养；S3.二级种子培养；S4.复合培养；S5.大肠杆菌培养；S6.转化。本发明通过采用两株解淀粉芽孢杆菌菌株复合发酵同时生产肌苷和鸟苷,进一步通过大肠杆菌酶转化生成IMP和GMP,且通过控制合适的接种比、温度阶段控制策略,将发酵液中IMP和GMP比例控制在合适的水平,简化了常规I+G生产工艺和工序投入,省时省力,极大降低生产过程的设备投入和生产成本,经济效益显著。(The invention discloses a method for preparing I + G by adopting composite strain fermentation, which utilizes Bacillus amyloliquefaciens strains B.amyloliquefaciens G10 and B.amyloliquefaciens8311 and an escherichia coli engineering strain AP-03 and comprises the steps of S1. slant culture; s2, primary seed culture; s3, secondary seed culture; s4, performing composite culture; s5, culturing escherichia coli; and S6, converting. According to the invention, two bacillus amyloliquefaciens strains are adopted for composite fermentation to simultaneously produce inosine and guanosine, IMP and GMP are further generated through enzyme conversion of escherichia coli, and the proportion of IMP and GMP in the fermentation liquor is controlled at a proper level by controlling a proper inoculation ratio and a temperature stage control strategy, so that the conventional I + G production process and process investment are simplified, time and labor are saved, the equipment investment and production cost in the production process are greatly reduced, and the economic benefit is remarkable.)

1. A method for preparing I + G by adopting composite strain fermentation is characterized in that two strains of inosine and guanosine producing bacillus amyloliquefaciens and an engineering strain of escherichia coli for inosine/guanosine phosphorylation are subjected to the following treatment, wherein:

the processing steps are as follows: s1, slant culture; s2, primary seed culture; s3, secondary seed culture; s4, performing composite culture; s5, culturing escherichia coli; s6, converting; wherein:

s1, slant culture: respectively inoculating two bacillus amyloliquefaciens strains and an escherichia coli strain to a slant culture medium, wherein the slant culture medium comprises the following components: 2-5g/L of yeast extract, 5-10g/L of peptone, 5-10g/L of sodium chloride, 15-20g/L of agar powder and 7.0-7.2 of pH; culturing in an incubator at 35-37 deg.C for 24-30 h;

s2, primary seed culture: scraping a loopful of bacteria from two oblique culture media of the bacillus amyloliquefaciens which are subjected to oblique culture in the S1. and respectively inoculating the loopful of bacteria into two shake flasks, wherein the liquid culture media in the shake flasks comprise: 3-10g/L of tryptone, 2-5g/L of yeast extract, 2-10g/L of sodium chloride and 7.0-7.2 of pH; the culture conditions are 100-300rpm, 35-37 deg.C, and OD₆₀₀When the ratio is 10-15;

s3, secondary seed culture: s2, respectively inoculating two first-stage seed solutions of the bacillus amyloliquefaciens obtained in the first-stage seed culture into two second-stage seed tanks according to the inoculation amount of 5-10 vol% for culture, wherein the culture medium used by the second-stage seeds comprises the following components: glucose 20-100g/L, yeast extract2-10g/L of powder, 3-10g/L of corn steep liquor, 1-5g/L of peptone, 2-5g/L of sodium glutamate, 1.0-2.5g/L of magnesium sulfate, 0.5-2.0g/L of urea, 0.2-1.0g/L of dipotassium hydrogen phosphate and 0.2-0.5mL/L of defoaming agent; the secondary seeding tank culture conditions are as follows: the pressure in the tank is 0.05-0.1Mpa, the ventilation rate is 10-15L/min, the culture temperature is 35-37 ℃, the rotation speed is 200-₆₀₀When the ratio is 20-40;

s4, composite culture: s3, inoculating two secondary seed liquids of the bacillus amyloliquefaciens obtained in the secondary seed culture into the same fermentation tank according to the inoculation proportion of 5-15 vol% for composite culture, wherein the composite fermentation culture medium comprises the following components: 30-100g/L of glucose, 5-30g/L of corn steep liquor, 2-15g/L of yeast extract powder, 2-10g/L of corn steep liquor hydrolysate, 3-15g/L of ammonium sulfate, 2-10g/L of sodium glutamate, 1.0-4.0g/L of magnesium sulfate, 1.0-4.0g/L of dipotassium phosphate, 0.5-2g/L of potassium dihydrogen phosphate, 0.5-2g/L of calcium chloride, 0.02-0.1g/L, VB 10.01.01-0.05 g/L of biotin, 0.001-0.005g/L of manganese sulfate, 0.001-0.005g/L of ferrous sulfate and 0.2-0.5mL/L of defoaming agent; controlling fermentation conditions: pH6.6-7.2, temperature 30-37 deg.C, tank pressure 0.05-0.1MPa, ventilation 10-30L/min, stirring at 200rpm, circularly adjusting tank pressure, ventilation and stirring in sequence to maintain dissolved oxygen at 30-40%, controlling the concentration of base sugar at 10-15g/L by adding sugar, and culturing for 60-80h to obtain fermentation liquid containing inosine and guanosine;

s5, culturing escherichia coli: s1, scraping a ring from a slant culture medium of the Escherichia coli strain subjected to slant culture, and inoculating the ring into a shake flask, wherein a liquid culture medium in the shake flask comprises: 2-10g/L of tryptone, 1-5g/L of yeast extract, 2-10g/L of sodium chloride and 7.0-7.2 of pH; the culture conditions are that the rotating speed is 100-300rpm and the temperature is 32-35 ℃; when OD is reached₆₀₀When the culture medium is 10-15, inoculating the shake flask into a fermentation tank for culture, wherein the culture medium comprises the following components: 20-100g/L of glucose, 2-10g/L of yeast extract powder, 5-30g/L of corn steep liquor, 1-5g/L of ammonium sulfate, 1.0-4g/L of dipotassium phosphate, 0.1-0.6g/L of magnesium sulfate, 0.02-0.1g/L, VB 10.01, 10.01-0.05 g/L of biotin and 0.2-0.5mL/L of defoaming agent; controlling fermentation conditions: pH6.6-7.2, temperature 28-35 deg.C, tank pressure 0.05-0.1MPa, ventilation 10-20L/min, stirring at 200rpm, and circulating and adjusting tank pressure, ventilation and stirring sequentially to maintain dissolved oxygen at 40-50%; when OD is reached₆₀₀When equal to 20-30Expression of acid phosphorylase by Induction with IPTG, OD₆₀₀Finishing the fermentation at 80-120 to obtain an escherichia coli fermentation liquid;

s6, transformation: s4, inoculating fermentation liquor containing inosine and guanosine obtained in the composite culture into a fermentation tank, and adjusting the pH value to 3.5-5.5; then adding polyphosphate in a fermentation tank according to 1-3 times of the total weight of inosine and guanosine, finally inoculating the fermentation tank with S5. escherichia coli fermentation liquor according to the inoculation proportion of 10-20 vol%, and converting at 28-37 ℃ for 10-15h to obtain a conversion solution containing I + G.

2. The method for preparing I + G by fermentation using a complex strain according to claim 1, wherein in the S6. transformation, the pH of the fermentation broth containing inosine and guanosine is adjusted using hydrochloric acid or sulfuric acid.

3. The method for preparing I + G through fermentation by using a compound strain as claimed in claim 1, wherein in the S6. conversion, the polyphosphate is any one or a combination of sodium pyrophosphate, sodium tripolyphosphate and sodium hexametaphosphate.

The technical field is as follows:

the invention relates to a preparation method of I + G, in particular to a method for preparing I + G by fermenting a composite strain.

Background art:

the flavour development nucleotide disodium is a nucleotide food freshener, is prepared by mixing inosine disodium and guanylate disodium according to a certain proportion, and can be directly added into food to play a role in enhancing freshness. Through experiments, the disodium flavour development nucleotide is found to show a synergistic effect in the using process, namely, when the two flavour enhancers are used in a mixed way, the umami degree of the disodium flavour development nucleotide is far greater than the sum of the umami tastes generated when the two flavour enhancers are used separately. In the current society, flavor-developing nucleotide becomes one of essential seasonings in daily life. The types of delicious seasonings prepared by using the flavor-developing nucleotide are increasing, such as monosodium glutamate, chicken essence, chicken powder, specially fresh monosodium glutamate and the like, and the food seasonings play an important role in food processing and manufacturing. In practical application, the two substances are usually in the form of sodium salts and are mixed in a specific ratio to form the flavour nucleotide disodium (I + G).

The prior production methods of the flavor-developing nucleotide comprise an enzymolysis method and a fermentation method, wherein the enzymolysis method comprises an enzymolysis RNA method and a thallus autolysis method, and the fermentation method comprises a direct fermentation method and a fermentation-transformation method. The method for producing the flavor-developing nucleotide by the RNA enzymolysis method and the thallus autolysis method has the advantages of simple process, rich raw material sources, low cost, higher enzyme reaction yield and capability of producing the nucleotide to obtain a mixture of four kinds of nucleotides at one time. However, in the post-extraction process, the difficulty in separating and purifying to obtain four high-purity products is high, so that the production period is long, the extraction process is complicated, and the product purity is not high. The direct fermentation method for producing nucleotide not only needs to consider the relief of the feedback inhibition in the nucleotide metabolic process, but also needs to consider how to change the permeability of cells to inosinic acid, and has the problems of long production period, difficult yield improvement and the like. Since nucleotidase for degrading nucleotide into nucleoside generally exists in thalli, and nucleoside can directly pass through cell membrane, and basically does not cause feedback inhibition to purine synthesis process. Inosine or guanosine is produced by a fermentation method and becomes an important source for producing flavor-developing nucleotide through subsequent phosphorylation.

At present, most manufacturers adopt a chemical phosphorylation method for inosine and guanosine to produce flavor development nucleotide, namely inosine and guanosine are firstly converted into IMP/GMP by the chemical phosphorylation method, and the generated IMP/GMP is further purified and mixed according to a certain proportion to prepare I + G finished products. The method has the advantages of strong specificity, few byproducts and high phosphorylation yield, but the cost of raw materials used in the process is high, the raw materials are harmful to human bodies and the environment, and a large amount of energy and labor cost are also input in the processes of fermentation, extraction, purification and the like.

In recent years, with the popularization of environmental protection concepts and the continuous attention of people to green development, the enzyme conversion method for producing nucleotides is increasingly concerned by technologists, and the enzyme used for producing nucleotides by nucleoside phosphorylation is mainly acid phosphatase. At present, more researches on the production of flavor-developing nucleotide by an enzyme conversion method are carried out, and the researches mainly focus on the construction of enzyme-producing engineering strains, the optimization of an enzyme conversion process and the like. However, more processes are still focused on the research of single flavor-developing nucleotide, and after obtaining a single IMP/GMP finished product, I + G is compounded in proportion, and the process has partial process superposition, so that the production cost and equipment investment still have a larger optimization space. Under the background of advocating energy conservation and emission reduction green development, the production level is improved by improving the conventional I + G production process, the production process is further shortened, the limitation on energy requirements is reduced, and the method has very important significance on the promotion of the international competitiveness of I + G products.

The invention content is as follows:

the invention aims to provide a method for preparing I + G by adopting composite strain fermentation, which simultaneously produces inosine and guanosine by adopting two bacillus amyloliquefaciens strains through composite fermentation, further generates IMP and GMP through enzyme conversion of escherichia coli, and controls the proportion of the IMP and the GMP in fermentation liquor to be at a proper level by controlling a proper inoculation ratio and a temperature stage control strategy so as to simplify the conventional I + G production process and process investment.

The invention is implemented by the following technical scheme: a method for preparing I + G by adopting composite strain fermentation comprises the following steps of carrying out treatment on two strains of bacillus amyloliquefaciens for producing inosine and guanosine and an engineering strain of escherichia coli for inosine/guanosine phosphorylation, wherein the treatment comprises the following steps:

the strain information is as follows: two strains of bacillus amyloliquefaciens for producing inosine and guanosine are respectively numbered as B.amyloliquefaciens G10 and B.amyloliquefaciens8311, and one engineering strain of escherichia coli for inosine/guanosine phosphorylation is numbered as AP-03, and the strains are presented to a corridor plum blossom biotechnology development limited company, wherein the corridor plum blossom biotechnology development limited company and an applicant are both subsidiary companies belonging to a plum blossom biotechnology group, and the corridor plum blossom biotechnology development limited company is mainly responsible for research and development of strains.

The processing steps are as follows: s1, slant culture; s2, primary seed culture; s3, secondary seed culture; s4, performing composite culture; s5, culturing escherichia coli; s6, converting; wherein:

s1, slant culture: respectively inoculating two bacillus amyloliquefaciens strains and an escherichia coli strain to a slant culture medium, wherein the slant culture medium comprises the following components: 2-5g/L of yeast extract, 5-10g/L of peptone, 5-10g/L of sodium chloride, 15-20g/L of agar powder and 7.0-7.2 of pH; culturing in an incubator at 35-37 deg.C for 24-30 h; the step is used for purifying and activating the microbial strains, and ensures that the strains can recover and recover the activity from the preservation state as soon as possible.

S2, primary seed culture: scraping a loopful of bacteria from two slant culture media which are subjected to slant culture in S1, respectively inoculating the loopful of bacteria into two shake flasks, wherein the shake flasks are filled with 30-100ml of liquid culture medium, and the liquid culture medium comprises the following components: 3-10g/L of tryptone, 2-5g/L of yeast extract, 2-10g/L of sodium chloride and 7.0-7.2 of pH; the culture conditions are 100-300rpm, 35-37 deg.C, and OD₆₀₀When the ratio is 10-15; the step is a small amount of amplification culture process of strains.

S3, secondary seed culture: s2, the primary seed liquid of the two strains of the bacillus amyloliquefaciens obtained in the primary seed culture is inoculated in an amount of 5-10 vol% (namely the volume of the seeds)Accounting for the percentage of the total fermentation volume) are respectively inoculated into two secondary seed tanks for culture, and the culture medium used by the secondary seeds comprises the following components: 20-100g/L of glucose, 2-10g/L of yeast extract powder, 3-10g/L of corn steep liquor, 1-5g/L of peptone, 2-5g/L of sodium glutamate, 1.0-2.5g/L of magnesium sulfate, 0.5-2.0g/L of urea, 0.2-1.0g/L of dipotassium hydrogen phosphate and 0.2-0.5mL/L of defoaming agent; the secondary seeding tank culture conditions are as follows: the pressure in the tank is 0.05-0.1Mpa, the ventilation rate is 10-15L/min, the culture temperature is 35-37 ℃, the rotation speed is 200-₆₀₀When the ratio is 20-40; the step is a process of amplifying and culturing strains in a large scale.

S4, composite culture: s3, inoculating two strains of bacillus amyloliquefaciens secondary seed liquid obtained in the secondary seed culture into a fermentation tank according to an inoculation proportion of 5-15 vol% for composite culture, wherein a composite fermentation culture medium comprises the following components: 30-100g/L of glucose, 5-30g/L of corn steep liquor, 2-15g/L of yeast extract powder, 2-10g/L of corn steep liquor hydrolysate, 3-15g/L of ammonium sulfate, 2-10g/L of sodium glutamate, 1.0-4.0g/L of magnesium sulfate, 1.0-4.0g/L of dipotassium phosphate, 0.5-2g/L of potassium dihydrogen phosphate, 0.5-2g/L of calcium chloride, 0.02-0.1g/L, VB 10.01.01-0.05 g/L of biotin, 0.001-0.005g/L of manganese sulfate, 0.001-0.005g/L of ferrous sulfate and 0.2-0.5mL/L of defoaming agent; controlling fermentation conditions: pH6.6-7.2, temperature 30-37 deg.C, tank pressure 0.05-0.1MPa, ventilation 10-30L/min, stirring at 200rpm, circularly adjusting tank pressure, ventilation and stirring in sequence to maintain dissolved oxygen at 30-40%, controlling the concentration of base sugar at 10-15g/L by adding sugar, and culturing for 60-80h to obtain fermentation liquid containing inosine and guanosine; the feeding sugar is a culture mode which is prepared by 50-80g/L glucose and supplements a carbon source in a culture medium in a feeding mode in the culture process. The concentration of the base sugar is the concentration of the glucose in the process culture medium, and the content of the glucose is detected by an SBA-50 biosensor analyzer; in the step, two strains of bacillus amyloliquefaciens are inoculated into the same fermentation tank for composite culture, and due to different glycoside-producing metabolic modes of different strains, respective metabolic products of inosine and guanosine are gradually accumulated in the culture process.

During compound culture, the inoculation amounts of the two strains are in accordance with the inoculation ratio and the inoculation OD₆₀₀Determining products, wherein the ratio of the products is 1: about 1. OD₆₀₀In fermentation liquor capable of indirectly reactingThe bacteria content, the inoculation OD is difficult to control due to the process₆₀₀And therefore, the inoculation ratio needs to be adjusted to ensure that the number of the inoculated strains is basically the same.

S5, culturing escherichia coli: scraping a ring from a slant culture medium of escherichia coli, inoculating the ring into a shake flask, wherein the liquid culture medium in the shake flask is 30-100ml and comprises the following components: 2-10g/L of tryptone, 1-5g/L of yeast extract, 2-10g/L of sodium chloride and 7.0-7.2 of pH; the culture conditions are that the rotating speed is 100-300rpm and the temperature is 32-35 ℃; when OD is reached₆₀₀When the culture medium is 10-15, inoculating the shake flask into a fermentation tank for culture, wherein the culture medium comprises the following components: 20-100g/L of glucose, 2-10g/L of yeast extract powder, 5-30g/L of corn steep liquor, 1-5g/L of ammonium sulfate, 1.0-4g/L of dipotassium phosphate, 0.1-0.6g/L of magnesium sulfate, 0.02-0.1g/L, VB 10.01, 10.01-0.05 g/L of biotin and 0.2-0.5mL/L of defoaming agent; controlling fermentation conditions: pH6.6-7.2, temperature 28-35 deg.C, tank pressure 0.05-0.1MPa, ventilation 10-20L/min, stirring at 200rpm, and circulating and adjusting tank pressure, ventilation and stirring sequentially to maintain dissolved oxygen at 40-50%; when OD is reached₆₀₀When the protein was 20 to 30, the expression of acid phosphorylase was induced by addition of IPTG, OD₆₀₀Finishing the fermentation at 80-120 to obtain an escherichia coli fermentation liquid; IPTG, known as isopropyl-beta-D-thiogalactopyranoside, is a powerful inducer that can induce the expression of enzymes/proteins. In this patent, the expression of acid phosphorylase can be induced.

Preparation of IPTG stock: dissolving 2g IPTG in 10ml distilled water, filtering with 0.22 μm filter membrane for sterilization, subpackaging to 1 ml/part, and storing at-20 deg.C for use;

in this step, the E.coli is an engineering bacterium which can produce nucleoside phosphorylase after IPTG induction.

S6, transformation: s4, inoculating fermentation liquor containing inosine and guanosine obtained in the composite culture into a fermentation tank, and adjusting the pH value to 3.5-5.5; adding polyphosphate in a fermentation tank according to 1-3 times of the total weight of inosine and guanosine, finally inoculating 10-20 vol% of the polyphosphate into an S5. escherichia coli fermentation liquid in the fermentation tank, and converting at 28-37 ℃ for 10-15h to convert inosine and guanosine into inosinic acid and guanylic acid to obtain a conversion liquid containing I + G; in the step, nucleoside phosphorylase produced by the engineering bacteria of escherichia coli can transfer phosphate groups on polyphosphate to inosine and guanosine to form inosinic acid and guanylic acid.

And (4) obtaining a conversion solution containing I + G after conversion, and obtaining an I + G finished product through subsequent further separation and extraction.

Further, in the S6. transformation, the pH of a fermentation broth containing inosine and guanosine was adjusted with hydrochloric acid or sulfuric acid.

Further, in the S6. conversion, the polyphosphate is any one or combination of sodium pyrophosphate, sodium tripolyphosphate and sodium hexametaphosphate.

The invention has the advantages that:

1. the invention replaces the traditional process of producing the flavor-developing nucleotide by a chemical phosphorylation method, simplifies the conventional I + G production process and also solves the following problems in the existing fermentation process: scientific researchers obtain a production strain capable of accumulating a large amount of certain flavor nucleotide or nucleoside by means of mutation breeding or engineering bacteria transformation and the like, then obtain a single nucleoside or nucleotide finished product with higher purity by optimizing a series of fermentation processes, conversion conditions and extraction processes, and finally mix the two flavor nucleotides according to a certain proportion to obtain a conversion solution containing I + G. In the production process, IMP and GMP are subjected to various processes such as fermentation process optimization, extraction and separation of single finished products, nucleoside phosphorylation, finished product mixing and the like. The whole process is relatively long and complex, and the investment in resources, equipment and the like is large, so that the improvement of the international competitiveness of the I + G product is not facilitated.

2. The invention utilizes the characteristic that the main metabolic pathways of IMP and GMP are basically the same, the produced strains are Bacillus amyloliquefaciens mutant strains, two strains capable of producing inosine and guanosine are simultaneously cultured in a mixed culture mode, and the purpose of simultaneously accumulating two nucleosides in fermentation liquor can be realized by matching with a proper fermentation and conversion control process, and the corresponding nucleotides can be obtained by further converting nucleoside enzyme by escherichia coli. The production method has the advantages of simplifying the conventional I + G production process, reducing the investment of working procedures in the separate production process, saving a large amount of labor and resource cost, creating possibility for developing a new I + G production process and bringing considerable economic benefit.

Description of the drawings:

in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a liquid phase peak diagram of a conversion solution obtained in example 3;

FIG. 2 is a crystal structure diagram (x60 times) of the conversion solution obtained in example 3.

The specific implementation mode is as follows:

the technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples, two seed solutions of Bacillus amyloliquefaciens strains and an Escherichia coli fermentation broth were prepared;

the seed liquid of two bacillus amyloliquefaciens strains is prepared by respectively inoculating two bacillus amyloliquefaciens strains B.amyloliquefaciens G10 and B.amyloliquefaciens8311 to a slant culture medium, wherein the slant culture medium comprises the following components: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride, 20g/L of agar powder and 7.0-7.2 of pH; culturing in an incubator at 35-37 deg.C for 25 hr; then scraping a loopful of bacteria on two slant culture mediums and respectively inoculating the loopful of bacteria in two shake flasks, wherein the liquid culture medium in the shake flasks is 50ml and comprises the following components: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 7.0-7.2 of pH; culturing at 35-37 deg.C at 200rpm for 25 hr, and measuring OD₆₀₀Respectively inoculating two first-stage seed solutions of bacillus amyloliquefaciens into two second-stage seed tanks according to the inoculation amount of 10 vol% for culture, and inoculating two second-stage seed tanksThe culture medium used by the grade seeds comprises: 20g/L of glucose, 10g/L of yeast extract powder, 10g/L of corn steep liquor, 5g/L of peptone, 5g/L of sodium glutamate, 2.5g/L of magnesium sulfate, 2.0g/L of urea, 1.0g/L of dipotassium hydrogen phosphate and 0.3mL/L of defoaming agent; the secondary seeding tank culture conditions are as follows: the pot pressure is 0.08MPa, the ventilation volume is 12L/min, the culture temperature is 35-37 ℃, the rotation speed is 300rpm, and the OD is measured after 15h of culture₆₀₀Two seed solutions of bacillus amyloliquefaciens strains were obtained for use. Wherein the defoaming agent is a Dow DF-103 polyether defoaming agent.

The preparation method of the escherichia coli fermentation liquid comprises the following steps: inoculating an escherichia coli strain to a slant culture medium, wherein the slant culture medium comprises the following components: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride, 20g/L of agar powder and 7.0-7.2 of pH; culturing in an incubator at 35-37 deg.C for 25 hr; scraping a ring from a slant culture medium of escherichia coli, inoculating the ring into a shake flask, wherein 50ml of liquid culture medium is arranged in the shake flask, and the liquid culture medium comprises the following components: 5g/L of tryptone, 3g/L of yeast extract, 5g/L of sodium chloride and 7.0-7.2 of pH; the culture conditions are that the rotating speed is 200rpm and the temperature is 32-35 ℃; when OD is reached₆₀₀When 13, inoculating the shake flask into a fermentation tank for culture, wherein the culture medium comprises the following components: 20g/L of glucose, 10g/L of yeast extract powder, 30g/L of corn steep liquor, 5g/L of ammonium sulfate, 4g/L of dipotassium phosphate, 0.6g/L of magnesium sulfate, 0.1g/L of biotin, 10.05g/L of VBs, 0.3mL/L of antifoaming agents and the balance of water. Controlling fermentation conditions: pH 7.0, temperature 35 deg.C, tank pressure 0.05-0.1MPa, aeration 10-20L/min, stirring at 300rpm, and circulating and adjusting tank pressure, aeration, and stirring sequentially to maintain dissolved oxygen at 45%. When OD is reached₆₀₀Adding IPTG (isopropyl thiogalactoside) to 25, cooling to 30 ℃ and carrying out induced expression of acid phosphorylase to OD (OD)₆₀₀The fermentation was terminated at 80, 100, 120, and 150 to obtain OD₆₀₀The four kinds of colibacillus fermentation liquid, 80, 100, 120 and 150, are used.

Experimental example 1 Bacillus amyloliquefaciens strain B. amyloliquefaciens G10 fermented alone

The seed solution of Bacillus amyloliquefaciens strain B. amyloliquefaciens G10 was inoculated into a fermenter at an inoculum size of 10 vol%. The fermentation medium comprises the following components: 100g/L of glucose, 30g/L of corn steep liquor, 15g/L of yeast extract powder, 10g/L of corn steep liquor hydrolysate, 15g/L of ammonium sulfate,10g/L of sodium glutamate, 4.0g/L of magnesium sulfate, 4g/L of dipotassium phosphate, 2g/L of monopotassium phosphate, 2g/L of calcium chloride, 0.1g/L of biotin, 10.05g/L of VBI, 0.005g/L of manganese sulfate, 0.005g/L of ferrous sulfate, 0.3mL/L of defoaming agent and the balance of water. Controlling fermentation conditions: pH 6.8, temperature 37 deg.C, tank pressure 0.05-0.1MPa, ventilation amount 20L/min, stirring at 500rpm, circularly adjusting tank pressure, ventilation amount, and stirring to maintain dissolved oxygen at 35%, and controlling the concentration of the sugar at 12g/L by adding sugar. Adjusting pH of the fermentation broth to 4.2 with hydrochloric acid after 80h fermentation, adding sodium pyrophosphate in an amount of 2 times of the fermentation broth, and inoculating OD in an amount of 15%₆₀₀The fermentation broth of escherichia coli (120) was inoculated into a fermenter and transformed at 30 ℃ for 15 hours to obtain a transformed solution.

The experimental example was repeated 3 times, and after the fermentation was completed, the contents of IMP and GMP in the fermentation broth were measured by HPLC, and the fermentation indexes are shown in table 1 below.

EXAMPLE 2 Bacillus amyloliquefaciens strain B. amyloliquefaciens8311 fermented alone

Inoculating the seed solution of bacillus amyloliquefaciens strain B. amyloliquefaciens8311 into a fermentation tank according to the inoculation amount of 10%. The fermentation medium comprises the following components: 100g/L of glucose, 30g/L of corn steep liquor, 15g/L of yeast extract powder, 10g/L of corn steep liquor hydrolysate, 15g/L of ammonium sulfate, 10g/L of sodium glutamate, 4.0g/L of magnesium sulfate, 4g/L of dipotassium hydrogen phosphate, 2g/L of potassium dihydrogen phosphate, 2g/L of calcium chloride, 0.1g/L of biotin, 10.05g/L of VBE, 0.005g/L of manganese sulfate, 0.005g/L of ferrous sulfate, 0.3mL/L of antifoaming agent and the balance of water. Controlling fermentation conditions: pH 6.8, temperature 37 ℃, tank pressure 0.05-0.1MPa, ventilation 10-30L/min, stirring 200-800rpm, circularly adjusting tank pressure, ventilation and stirring in sequence to maintain dissolved oxygen at 35%, and controlling the concentration of the base sugar at 12g/L by adding sugar in the process. Adjusting pH of the fermentation broth to 4.2 with hydrochloric acid after 80h fermentation, adding sodium pyrophosphate in an amount of 2 times of inosine and guanosine, and inoculating OD in an amount of 15%₆₀₀The fermentation broth of escherichia coli (120) was inoculated into a fermenter and transformed at 30 ℃ for 15 hours to obtain a transformed solution.

The experimental example was repeated 3 times, and after the fermentation was completed, the contents of IMP and GMP in the fermentation broth were measured by HPLC, and the fermentation indexes are shown in table 1 below.

Example 3 Complex fermentation of Bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and Bacillus amyloliquefaciens strain B.amyloliquefaciens8311

The seed solutions of bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and bacillus amyloliquefaciens strain B.amyloliquefaciens8311 were inoculated into the same fermentation tank according to the inoculum size of 10%, and the fermentation media and process control process of bacillus amyloliquefaciens and escherichia coli were the same as those of experimental example 1. Adjusting pH of the fermentation broth to 4.2 with hydrochloric acid after 80h fermentation, adding sodium pyrophosphate in an amount of 2 times of inosine and guanosine, and inoculating OD in an amount of 15%₆₀₀The fermentation broth of escherichia coli (120) was inoculated into a fermenter and transformed at 30 ℃ for 15 hours to obtain a transformed solution.

This example was repeated 3 times, and after the end of the fermentation, the contents of IMP and GMP in the fermentation broth were determined by HPLC, and the fermentation indices are shown in table 1 below. And (3) selecting the primary conversion solution to carry out liquid phase peak diagram and crystal structure detection, wherein the crystal detection means is to utilize an OLYMPUS CX-40 microscope to carry out amplification observation on the crystal structure, and the detection aims at comparing and distinguishing the crystal forms of IMP and GMP and the crystal structures. The results are shown in FIGS. 1 and 2.

Example 4 Complex fermentation of Bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and Bacillus amyloliquefaciens strain B.amyloliquefaciens8311

The seed solutions of bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and bacillus amyloliquefaciens strain B.amyloliquefaciens8311 were inoculated into the same fermentation tank according to the inoculum size of 15%, and the fermentation media and process control process of bacillus amyloliquefaciens and escherichia coli were the same as those of experimental example 1. Adjusting pH of the fermentation broth to 4.2 with hydrochloric acid after 80h fermentation, adding sodium tripolyphosphate according to 2 times of the ratio of inosine to guanosine, and finally inoculating OD according to 20% of inoculation ratio₆₀₀The fermentation broth of 80 E.coli was inoculated into a fermenter and transformed at 32 ℃ for 15 hours to obtain a transformed broth.

This example was repeated 3 times, and after the end of the fermentation, the contents of IMP and GMP in the fermentation broth were determined by HPLC, and the fermentation indices are shown in table 1 below.

Example 5 Complex fermentation of Bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and Bacillus amyloliquefaciens strain B.amyloliquefaciens8311

The seed solutions of bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and bacillus amyloliquefaciens strain B.amyloliquefaciens8311 were inoculated into the same fermentation tank according to the inoculum size of 5%, and the fermentation media and process control process of bacillus amyloliquefaciens and escherichia coli were the same as those of experimental example 1. Adjusting pH of the fermentation broth to 5.0 with sulfuric acid after 80h fermentation, adding sodium tripolyphosphate at a ratio of 1.5 times of inosine and guanosine, and inoculating OD at a ratio of 10%₆₀₀Coli was inoculated into a fermenter and transformed at 32 ℃ for 10 hours to obtain a transformed liquid.

This example was repeated 3 times, and after the end of the fermentation, the contents of IMP and GMP in the fermentation broth were determined by HPLC, and the fermentation indices are shown in table 1 below.

Example 6 Complex fermentation of Bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and Bacillus amyloliquefaciens strain B.amyloliquefaciens8311

The seed solutions of bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and bacillus amyloliquefaciens strain B.amyloliquefaciens8311 were inoculated into the same fermentation tank according to the inoculum size of 10%, and the fermentation media and process control process of bacillus amyloliquefaciens and escherichia coli were the same as those of experimental example 1. Adjusting pH of the fermentation broth to 4.5 with sulfuric acid after 80h fermentation, adding sodium tripolyphosphate according to 2 times of the ratio of inosine to guanosine, and finally inoculating OD according to 10% of inoculation ratio₆₀₀Coli was inoculated into a fermenter and transformed at 32 ℃ for 12 hours to obtain a transformed liquid.

This example was repeated 3 times, and after the end of the fermentation, the contents of IMP and GMP in the fermentation broth were determined by HPLC, and the fermentation indices are shown in table 1 below.

Example 7 Complex fermentation of Bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and Bacillus amyloliquefaciens strain B.amyloliquefaciens8311

The seed solutions of bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and bacillus amyloliquefaciens strain B.amyloliquefaciens8311 were inoculated into the same fermentation tank according to the inoculum size of 10%, and the fermentation media and process control process of bacillus amyloliquefaciens and escherichia coli were the same as those of experimental example 1. After 70h of fermentation, adjusting the pH of the fermentation liquor to 3.5 with sulfuric acid, and adding inosineAdding sodium tripolyphosphate at a ratio of 1 time of guanosine, and inoculating with OD at a ratio of 10%₆₀₀Coli was inoculated into a fermenter and transformed at 28 ℃ for 12 hours to obtain a transformed liquid.

This example was repeated 3 times, and after the end of the fermentation, the contents of IMP and GMP in the fermentation broth were determined by HPLC, and the fermentation indices are shown in table 1 below.

Example 8 Complex fermentation of Bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and Bacillus amyloliquefaciens strain B.amyloliquefaciens8311

The seed solutions of bacillus amyloliquefaciens strain B.amyloliquefaciens G10 and bacillus amyloliquefaciens strain B.amyloliquefaciens8311 were inoculated into the same fermentation tank according to the inoculum size of 10%, and the fermentation media and process control process of bacillus amyloliquefaciens and escherichia coli were the same as those of experimental example 1. Adjusting the pH of the fermentation broth to 4.5 with sulfuric acid after the fermentation is finished for 75h, adding sodium tripolyphosphate according to 3 times of the ratio of inosine to guanosine, and finally inoculating the OD according to 15 percent of inoculation ratio₆₀₀Coli was inoculated into a fermentor and transformed at 30 ℃ for 15h to obtain a transformed solution.

This example was repeated 3 times, and after the end of the fermentation, the contents of IMP and GMP in the fermentation broth were determined by HPLC, and the fermentation indices are shown in table 1 below.

TABLE 1 Table for measuring contents of flavor-producing nucleotides in the obtained conversion solutions of examples 1 to 8

The liquid phase peak profile of figure 1 was analyzed and tabulated below, and in contrast to the IMP + GMP standard, it was determined that the two peaks represented IMP and GMP, respectively.

As can be seen from fig. 2, the IMP and GMP prepared in example 3 have uniform crystal structures, with a regular crystal morphology of IMP and a more irregular crystal morphology of GMP.

As can be seen from table 1 above, by controlling the inoculation ratio and fermentation/transformation process of bacillus amyloliquefaciens strain b.amyloliquefaciens G10 and bacillus amyloliquefaciens strain b.amyloliquefaciens8311, the ratio and content of IMP and GMP are greatly different. Among them, it is understood from examples 1 and 2 that the transformed product obtained by culturing Bacillus amyloliquefaciens strain B. amyloliquefaciens G10 alone has substantially no GMP; when bacillus amyloliquefaciens strain B. amyloliquefaciens8311 is independently cultured, IMP is basically not contained in an obtained conversion product; from examples 3-8, bacillus amyloliquefaciens strains B.amyloliquefaciens G10 and B.amyloliquefaciens8311 are subjected to composite culture and transformation by adopting the process required by the patent, the obtained transformed product contains IMP and GMP, the proportion of the IMP and the GMP meets the requirement of GB 1886.171-2016 food additive 5-flavor nucleotide disodium on the proportion of the IMP and the GMP, and the IMP and the GMP can meet the requirement of GB 1886.171-2016 food additive 5-flavor nucleotide disodium in the obtained product after subsequent separation and extraction.

Description of the drawings:

HPLC determination refers to a method for determining flavor-developing nucleotide by liquid chromatography, is a high performance liquid chromatography detection method, and is a chromatographic process taking 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.) larger than 100 μm. 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-efficiency liquid phase has small granularity (5-10 mu m), fast mass transfer and high column efficiency.

The invention adopts an Agilent Technologies1200 to detect the content of the flavor-developing nucleotide accumulated in the fermentation liquor, and the specific method is as follows:

a chromatographic column: XDB-C18 column (5 μm, 4.6X 250mm)

Mobile phase A: 2.7218g of potassium dihydrogen phosphate were weighed, 800mL of ultrapure water was added, and stirring was carried out until all crystals were completely dissolved. Transferring to a 1000mL volumetric flask for constant volume, adjusting the pH value of the solution to 4.5 by using phosphoric acid, and filtering for later use.

Mobile phase B: acetonitrile (chromatographically pure) water 99.5:0.5(V/V)

Column temperature: 40 deg.C

Flow rate: 1ml/min

A detector: ultraviolet detector (λ 254nm)

Sample introduction amount: 2 μ L.

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.