Coupling fermentation process of threonine and isoleucine as well as compound amino acid product and application of compound amino acid product

Abstract

The invention provides a coupling fermentation process of threonine and isoleucine and application of the threonine and isoleucine to production of a compound amino acid product, and belongs to the technical field of biological fermention.The coupling fermentation process comprises the following steps that threonine production strains and isoleucine production strains are mixed according to different modes and inoculated to a coupling fermentation culture medium, and then coupling fermentation is conducted; the inoculation proportion during mixed inoculation is that the volume ratio x of the threonine production bacterial liquid to the isoleucine production bacterial liquid is greater than or equal to 0 and greater than or equal to 10, and the biomass ratio of the threonine production bacterial liquid to the isoleucine production bacterial liquid is 0.8-1.2. According to the technical scheme provided by the invention, threonine and isoleucine can be simultaneously produced through coupled fermentation, so that the production cost is reduced, and the production efficiency is improved; meanwhile, the ratio of threonine to isoleucine in the compound amino acid product can be adjusted by regulating and controlling the coupling inoculation mode and proportion, so that the requirements of different markets and application scenes are met.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation, and in particular to a coupled fermentation process of threonine and isoleucine and a composite amino acid product and application thereof. Background Art

[0002] Threonine and isoleucine are essential amino acids for humans and animals, and are widely used in food, medicine, feed additives and other fields. As important amino acids for feeding, threonine and isoleucine have the functions of promoting growth and enhancing immunity, improving feed-to-meat ratio and improving meat quality, which can improve the nutritional value and utilization rate of feed, accelerate the growth rate of livestock, and reduce breeding costs, and have a wide market demand.

[0003] The current production methods of threonine and isoleucine mainly rely on microbial fermentation, but still follow the traditional single product fermentation production process. In order to ensure output and benefits, the synthesis efficiency has to be reduced. However, the feed application field has relatively low requirements for product purity. Since the existing fermentation does not fully consider the characteristics of feed applications and market demand, there are problems such as high cost and low efficiency, and the synthetic potential of the production strains cannot be fully utilized.

[0004] At present, the microorganisms used to synthesize threonine and isoleucine in industrial production are mainly Escherichia coli and Corynebacterium glutamicum. Different amino acid producing bacteria of the same category are very similar in growth and metabolism, and there are significant differences only in the metabolic pathways related to the synthesis of the target amino acid; and threonine is the direct precursor of isoleucine in the metabolic pathways of microorganisms. Therefore, the similarity of threonine and isoleucine producing bacteria of the same category, and the correlation of the two amino acids in their biosynthetic pathways provide a basis for the coupled fermentation production of threonine and isoleucine. However, the fermentation processes in current industrial production are all single product fermentation, threonine participates in the reaction as a precursor raw material of isoleucine, and there is only one amino acid product in the product.

[0005] The inventors of the present application disclosed in a Chinese patent with publication number CN114410701B and patent name "Genetically engineered bacteria with high yield of L-isoleucine and method for producing L-isoleucine by fermentation" that the efficient production of isoleucine under the condition of adding the precursor substance threonine is achieved by genetically engineered strains. However, the threonine used therein is derived from the finished threonine product produced by fermentation production of industrial strains and preliminary purification, which has a high cost and only isoleucine is produced in the product. Summary of the invention

[0006] In view of this, the present invention provides a coupled fermentation process of threonine and isoleucine and a composite amino acid product and application thereof, so as to realize coupled fermentation to generate threonine and isoleucine, reduce production costs and improve production efficiency.

[0007] To achieve the above object, the present invention provides a coupled fermentation process of threonine and isoleucine, comprising the following steps: The threonine producing strain and the isoleucine producing strain are mixed and inoculated into a coupled fermentation medium, and then coupled fermentation is performed; The inoculation ratio during mixed inoculation is that the volume ratio of the threonine production bacterial solution to the isoleucine production bacterial solution is x, 10≥x>0, and the biomass ratio of the threonine production bacterial solution to the isoleucine production bacterial solution is 0.8-1.2; The coupled fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt and a growth factor, and the pH of the coupled fermentation medium is 6.5-7.5.

[0008] Optionally, the mixed inoculation also includes mixed inoculation of threonine and isoleucine production strains under one or more combinations of the same or different fermentation stages, time periods and inoculation ratios; the fermentation stages include a seed shake flask stage, a seed tank stage and a fermentation tank stage; at least one of the seed shake flask stage, the seed tank stage and the fermentation tank stage uses a coupled fermentation medium.

[0009] Optionally, the time period includes an initial growth period, an initial logarithmic growth period and a mid-to-late logarithmic growth period; the initial growth period is 0 to 3 hours for the growth of threonine and isoleucine strains, the initial logarithmic growth period is 6 to 9 hours for the growth of threonine and isoleucine strains, and the mid-to-late logarithmic growth period is 8 to 12 hours for the growth of threonine and isoleucine strains.

[0010] Optionally, the nitrogen source includes one or more combinations of inorganic nitrogen sources and organic nitrogen sources; the inorganic nitrogen source includes one or more combinations of ammonia, soluble ammonium salts and amino acids; the organic nitrogen source includes one or more combinations of plant-based proteins and hydrolysates thereof, animal-based proteins and hydrolysates thereof, and microbial-based proteins and hydrolysates thereof; the soluble ammonium salts include one or more combinations of ammonium sulfate and ammonium chloride, and the amino acids include one or more combinations of glutamic acid and methionine; the plant-based protein and its hydrolysate include one or more combinations of soy peptone, soybean meal hydrolysate, corn steep liquor, corn steep liquor powder, corn steep liquor extract and malt extract; the animal-based protein and its hydrolysate include one or more combinations of beef extract powder, beef extract, fish meal, casein hydrolysate and acid-hydrolyzed casein; the microbial-based protein and its hydrolysate include one or more combinations of yeast powder, yeast extract powder, yeast extract, yeast paste, yeast broken wall material and yeast hydrolysate.

[0011] Optionally, the inorganic salt includes one or more of soluble sodium salt, soluble magnesium salt, soluble calcium salt and soluble salt of trace metal elements; the trace metal elements include zinc, manganese, copper, molybdenum, iron and cobalt; the growth factor includes one or more of ammonium ferric citrate, choline chloride and vitamins; the vitamin includes VB 1 , biotin, VB 3 ,VB 6 One or a combination of two or more.

[0012] Optionally, the carbon source includes one or more of starch hydrolysate, cellulose hydrolysate, glucose, and sucrose; the hydrolysate of starch and cellulase includes one or more of starch hydrolysate, cassava hydrolysate, and non-food carbon source hydrolysate; the non-food carbon source includes one or more of straw and corn cobs; and the pH of the coupled fermentation medium is adjusted using one or more of ammonia water, sodium hydroxide, potassium hydroxide, sulfuric acid, and phosphoric acid.

[0013] Optionally, the amino acid fermentation production strain used in the coupled fermentation process is a wild or genetically engineered bacterial species, including Escherichia coli, Bacillus, Brevibacterium, yeast, Corynebacterium glutamicum or Streptomyces.

[0014] Optionally, the bacterial species is Escherichia coli or Corynebacterium glutamicum.

[0015] In order to achieve the above object, the present invention also provides a composite amino acid product prepared by the coupled fermentation process of threonine and isoleucine, wherein the composite amino acid product contains threonine and isoleucine in different proportions.

[0016] The present invention provides an application of a composite amino acid product prepared by the coupled fermentation process of threonine and isoleucine in food or feed.

[0017] The above technical solution of the present invention includes at least the following beneficial effects: The coupled fermentation process of threonine and isoleucine provided by the present invention makes the fermentation production process of the two highly compatible, greatly enhances the utilization of carbon source metabolic flow and energy in the fermentation system, and improves the fermentation production efficiency of the target amino acid; the amino acid products finally obtained do not need to be separated one by one, and can be used to prepare composite amino acid products for feeding. Therefore, the co-production of threonine and isoleucine by coupled fermentation can significantly reduce its production cost and improve production efficiency; at the same time, it can also effectively reduce energy consumption and wastewater and waste gas emissions, achieve the purpose of energy saving and emission reduction, and has great production potential and market prospects.

[0018] The coupled fermentation process of threonine and isoleucine provided by the present invention prepares a composite amino acid product containing both threonine and isoleucine. Isoleucine and threonine are common essential amino acids, but different types of animals have different requirements for isoleucine and threonine. In addition, animals of the same type but at different growth stages also have different requirements for isoleucine and threonine. Therefore, the technical solution provided by the present invention can regulate the ratio of threonine to isoleucine in the composite amino acid product according to different mixed inoculation methods and ratios. The application of composite amino acid products with different ratios can meet the needs of different markets and application scenarios, and can also reduce the cost of reprocessing when the composite amino acids are used. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described in combination with the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0020] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0021] As used herein and unless otherwise specified, the term "about" or "approximately" refers to a measurable value such as an amount, a time period, etc., and includes a variation of ±20%, more preferably ±10%, even better ±5%, and still more preferably ±1% from the given value, as long as such variation is suitable for practicing the disclosed method. The coupled fermentation process of threonine and isoleucine in the present invention refers to the process in which the product obtained by the same batch fermentation contains both threonine and isoleucine.

[0022] The amino acid fermentation production strain used in the coupled fermentation process of the present invention is a wild or genetically engineered strain, including Escherichia coli (E.coli), Bacillus (Bacillus), Brevibacterium (Brevibacterium), Yeast (Yeast), Corynebacterium glutamicum (Corynebacterium glutamicum) or Streptomyces (Streptomyces). The biomass OD600 refers to the absorbance of a certain solution at a wavelength of 600nm. OD600 is a standard method for tracking the growth of microorganisms in liquid cultures; the culture solution without bacterial solution is used as a blank solution, and then the bacterial culture solution after quantitative culture is obtained. The OD600 value can be detected using a commercial instrument. The amino acids mentioned in the present invention, such as threonine and isoleucine, are all L-amino acids unless otherwise specified.

[0023] The method and application of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0024] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0025] Determination of amino acid concentration: amino acid standards were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). Take 1 mL of fermentation broth, centrifuge at 10000 r / min for 5 min to remove the bacteria, filter the obtained filtrate through a filter membrane with a pore size of 0.22 μm, and dilute the sample filtrate to an appropriate multiple. The amino acid concentration in the sample is determined using an OPA-derived high-performance liquid chromatography column HPLC method. (See https: / / www.agilent.com / library / applications / 5990-4547EN.pdf) The high-performance liquid chromatograph is Shimadzu Nexera LC-40, and the chromatographic column is Agilent ZORBAXEclipse Plus C18, 4.6×250mm 5μm. The detector is a DAD diode array detector with a detection wavelength of 338nm and a reference wavelength of 390nm. The mobile phase composition, ratio change, flow rate and column temperature are all set according to the above method.

[0026] Example 1 Activation screening and rejuvenation of bacterial strains The threonine production strain (Escherichia coli or Corynebacterium glutamicum) and the isoleucine production strain (Escherichia coli or Corynebacterium glutamicum) were inoculated onto activation medium agar plates by streaking method for activation; the medium composition was glucose 1g / L, peptone 5g / L, NaCl 1g / L, yeast extract powder 5g / L, KH 2 PO 4 1g / L, agar powder 20g / L, pH=6.5-7.5.

[0027] In particular, 40 g / L isoleucine was added when activating the threonine-producing strain, and 80 g / L threonine was added when activating the isoleucine-producing strain, so as to screen strains that can tolerate high concentrations of coupled culture products.

[0028] During activation, the activation plate was incubated at 37°C for 12-16 hours, and single colonies with clear edges and stable morphology were selected and inoculated into the activation medium. The medium composition was glucose 1g / L, tryptone 5g / L, NaCl 1g / L, yeast extract powder 5g / L, KH 2 PO 4 1g / L, pH = 6.5-7.5. After culturing at 220rpm and 37℃ for 8-12 hours, the strain was stored in a 20% glycerol tube at -80℃ for later use.

[0029] Example 2 Medium adaptation and coupled fermentation experiments in shake flask system (1) Preparation of shake flask-related culture medium Seed culture medium: peptone 10 g / L, yeast extract powder 5 g / L, KH 2 PO 4 1g / L, pH=6.5-7.5.

[0030] Isoleucine shake flask fermentation medium: glucose 20 g / L, threonine 20 g / L, yeast extract 1 g / L, (NH 4 ) 2 SO 4 8 g / L, KH 2 PO 4 4 g / L, MgSO 4 2 g / L, citric acid 2 g / L, betaine 2 g / L, vitamin B 1 0.02 g / L, Vitamin B 3 0.2 g / L, pH=6.5-7.5. When used in fermentation experiments with threonine-producing bacteria, no threonine is added.

[0031] Threonine shake flask fermentation medium: glucose 40 g / L, corn syrup 3 g / L, (NH 4 )2 SO 4 10 g / L, KH 2 PO 4 2g / L, MgSO 4 1 g / L, choline chloride 0.2 g / L, FeSO 4 0.01 g / L, MnSO 4 0.01 g / L, Vitamin B 1 0.005 g / L, Vitamin B 3 0.005 g / L, Vitamin B 6 0.005 g / L, biotin 0.002 g / L, pH = 6.5-7.5. When used for isoleucine production bacteria fermentation, add 20 g / L threonine.

[0032] (2) Basic process of shake flask experiment Inoculate 1 mL of activated glycerol-frozen bacteria (or activated non-frozen bacterial suspension) into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium, and culture at 220 rpm and 37 °C for 10 hours to obtain shake flask seeds. Transfer the shake flask seeds to a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium at a ratio of 10% (v:v) (5 mL), and culture at 220 rpm and 37 °C for 24 hours. Take samples at different time periods to measure OD and product content.

[0033] (3) Preliminary adaptation experiment of threonine and isoleucine producing bacteria in different fermentation media The shake flask seeds of threonine or isoleucine producing bacteria (Escherichia coli or Corynebacterium glutamicum) were inoculated into threonine shake flask fermentation medium (T-medium) and isoleucine shake flask fermentation medium (I-medium) respectively. The two types of culture media can meet the fermentation needs of different strains by adding threonine or not. The two types of culture media were cultured at 220rpm and 37℃. After 12 hours, the pH was adjusted to 6.8-7.2 with ammonia water every 2 hours. After 24 hours of culture, samples were taken to measure the biomass (OD600 value) and amino acid yield. The fermentation results are shown in Tables 1 and 2. Whether in terms of biomass or product content, the effects of the two types of threonine and isoleucine producing bacteria (Escherichia coli and Corynebacterium glutamicum) in T-medium are better than those in I-medium, indicating that T-medium can be used as the initial basic coupling fermentation medium to adapt to the growth and metabolism of threonine and isoleucine producing bacteria, and the corresponding target amino acid yields in Escherichia coli are higher than those in Corynebacterium glutamicum.

[0034] Table 1. Results of 24-hour fermentation of different strains in different shake flask fermentation media (Escherichia coli)

[0035] Table 2. Results of 24-hour fermentation of different strains in different shake flask fermentation media (Corynebacterium glutamicum)

[0036] (4) Shake flask coupled fermentation of threonine and isoleucine producing bacteria Based on the threonine shake flask fermentation medium (T-medium), the shake flask seeds of threonine and isoleucine producing bacteria were inoculated into the fermentation shake flasks at different mixing ratios (1:1, 1:2, 2:1, v:v) for coupled fermentation experiments. At the same time, the threonine producing bacteria alone fermentation (1:0) and isoleucine alone fermentation (0:1, T-medium added with 5 g / L threonine) were also set up as control groups. The shake flasks were cultured at 220rpm and 37℃. After 12 hours, the pH was adjusted to 6.8-7.2 every 2 hours with ammonia water. After 24 hours of culture, samples were taken to measure the OD600 value and amino acid yield. The fermentation results are shown in Tables 3 and 4. Both types of threonine and isoleucine producing bacteria (Escherichia coli and Corynebacterium glutamicum) showed good growth compatibility and metabolic synergy during coupled fermentation. In addition, isoleucine producing bacteria can directly use threonine synthesized by threonine producing bacteria to produce isoleucine, which illustrates the feasibility of coupled fermentation production of threonine and isoleucine. Considering the growth rate of the strain and the amino acid yield, the subsequent examples are mainly based on Escherichia coli. Table 3. Results of coupled fermentation of threonine and isoleucine producing bacteria (Escherichia coli) in T-medium

[0037] Table 4. Results of coupled fermentation of threonine and isoleucine producing bacteria (Corynebacterium glutamicum) in T-medium

[0038] Example 3 Adaptation optimization of coupled fermentation medium in shake flask system and fermenter (1) Evaluation and comparison of organic nitrogen sources in shake flask system Organic nitrogen sources play a vital role in bacterial growth and metabolism. In order to evaluate the potential effects of different organic nitrogen sources in the original threonine and isoleucine shake flask fermentation medium on coupled fermentation, organic nitrogen sources derived from corn syrup, including corn syrup from different manufacturers, corn syrup dry powder, corn syrup paste, and organic nitrogen sources derived from yeast, including different types of yeast extract powder, yeast autolyzed powder, yeast powder, yeast cell wall and yeast hydrolyzate, were systematically compared and preliminarily evaluated. A nitrogen source evaluation medium was designed based on the threonine shake flask fermentation medium (T-medium). The medium composition was: glucose 40 g / L, organic nitrogen source to be tested 3 g / L, (NH 4 )2 SO 4 10 g / L, KH 2 PO 4 2 g / L, MgSO 4 1 g / L, choline chloride 0.2 g / L, FeSO4 0.01 g / L, MnSO 4 0.01 g / L, Vitamin B 1 0.005 g / L, Vitamin B 3 0.005 g / L, Vitamin B 6 0.005 g / L, biotin 0.002 g / L, pH = 6.5-7.5. The cultured threonine and isoleucine shake flask seeds were mixed in a 1:1 ratio, inoculated with a 10% (v:v) inoculation amount into the nitrogen source evaluation medium, cultured at 220 rpm and 37 °C, and adjusted to pH 6.8-7.2 with ammonia water every 2 hours after 12 hours, and sampled for OD600 value and amino acid yield (threonine + isoleucine) at 24 hours. The evaluation results are shown in Table 5. Considering the growth and production of the strains and the cost of raw materials, it was finally determined that yeast extract powder and corn steep liquor were added to the coupled fermentation medium to maintain the growth and metabolism of the bacteria.

[0039] Table 5. Comparative results of evaluation of different organic nitrogen sources in shake flask system

[0040] (2) Optimization of coupled fermentation medium in a 5-liter fermenter system Based on the fermentation medium of threonine (T-medium) and the evaluation results of organic nitrogen sources in (1), a basic coupled fermentation medium was designed. The medium composition was as follows: glucose 40 g / L, yeast extract 5 g / L, corn steep liquor 10 g / L, (NH 4 ) 2 SO 4 10g / L, KH 2 PO 4 8g / L, MgSO 4 4 g / L, choline chloride 0..01 g / L, FeSO 4 0.01 g / L, MnSO 4 0.01 g / L, Vitamin B 1 0.001 g / L, Vitamin B 3 0.001 g / L, Vitamin B 60.001 g / L, biotin 0.001 g / L, pH = 6.5-7.5. The organic nitrogen source, inorganic salts and nutritional growth factors were optimized by referring to the isoleucine shake flask fermentation medium to meet the dynamic needs of growth and metabolic synthesis of different strains.

[0041] As described in Example 2, the cultured threonine and isoleucine shake flask seeds were mixed in a 1:1 ratio and inoculated into a 5L fermenter containing 2L of basic coupled fermentation medium at a 10% (v:v) inoculation amount. The pH of the fermenter was adjusted to 6.5-7.5 by ammonia water, the temperature was constant at 37°C, the ventilation was 1-3 L / min, the dissolved oxygen was controlled at about 15-30%, and the glucose concentration in the tank was maintained at 0.5-2.0 g / L by adding 50-70% glucose solution. During the process, samples were taken regularly to measure the OD600 value and amino acid yield. The optimization results are shown in Table 6. Based on the above system optimization results, a coupled fermentation medium for the joint production of threonine and isoleucine in a fermenter was designed. The medium contains a carbon source, a nitrogen source, an inorganic salt and a nutrient growth factor, and the preferred composition is: glucose 20-40 g / L, corn steep liquor 10-30 g / L, yeast powder 1-5 g / L, (NH 4 ) 2 SO 4 2-8 g / L, KH 2 PO 4 2-8 g / L, MgSO 4 1-4 g / L, FeSO 4 0.01-0.5 g / L, MnSO 4 0.01-0.5 g / L, choline chloride 0.01-0.5 g / L, vitamin B 1 0.001-0.05 g / L, vitamin B 3 0.001-0.05 g / L, vitamin B 6 0.001-0.05 g / L, biotin 0.001-0.05 g / L, pH=6.5-7.5.

[0042] Table 6. Single factor system optimization results of basic coupled fermentation medium in 5-liter fermenter

[0043] Example 4 Coupling fermentation of threonine and isoleucine producing bacteria in fermenter (1) Preparation of relevant culture media Shake flask seed culture medium: peptone 10 g / L, yeast extract 5 g / L, KH 2 PO 41g / L, pH=6.5-7.5.

[0044] The isoleucine seed tank culture medium contains a carbon source, a nitrogen source, an inorganic salt and a nutrient growth factor, and the preferred composition is: glucose 20-40 g / L, threonine 10-30 g / L, yeast powder 1-5 g / L, (NH 4 )2SO 4 2-8 g / L, KH 2 PO 4 2-8 g / L, MgSO 4 1-4 g / L, citric acid 1-5 g / L, betaine 2-8 g / L, vitamin B 1 0.01-0.05 g / L, vitamin B 3 0.1-0.5g / L, trace metal salt mother solution 2 mL / L, pH=6.5-7.5.

[0045] The threonine seed tank culture medium contains a carbon source, a nitrogen source, an inorganic salt and a nutrient growth factor, and the preferred composition is: glucose 20-40 g / L, corn syrup 10-30 g / L, yeast powder 1-5 g / L, (NH 4 ) 2 SO 4 2-8 g / L, KH 2 PO 4 2-8 g / L, MgSO 4 1-4 g / L, FeSO 4 0.01-0.5 g / L, MnSO 4 0.01-0.5 g / L, vitamin B 1 0.001-0.05 g / L, vitamin B 3 0.001-0.05 g / L, vitamin B 6 0.001-0.05 g / L, biotin 0.001-0.05 g / L, trace metal salt mother solution 2mL / L, pH=6.5-7.5.

[0046] Trace metal mother liquor formula: zinc sulfate 1 g / L, manganese sulfate 1 g / L, copper sulfate 0.5 g / L, boric acid 0.15 g / L, ammonium molybdate 0.1 g / L, ferrous sulfate 1 g / L, cobalt chloride 1.5 g / L.

[0047] The coupled fermentation medium contains a carbon source, a nitrogen source, an inorganic salt and a nutrient growth factor, and the preferred composition is: glucose 20-40 g / L, corn syrup 10-30 g / L, yeast powder 1-5 g / L, (NH 4 ) 2 SO 42-8 g / L, KH 2 PO 4 2-8 g / L, MgSO 4 1-4 g / L, FeSO 4 0.01-0.5 g / L, MnSO 4 0.01-0.5 g / L, choline chloride 0.01-0.5 g / L, vitamin B 1 0.001-0.05 g / L, vitamin B 3 0.001-0.05 g / L, vitamin B 6 0.001-0.05 g / L, biotin 0.001-0.05 g / L, pH=6.5-7.5.

[0048] (2) Basic process of coupled fermentation The activated strain or its glycerol cryopreserved tube was inoculated into the shake flask seed medium. After the shake flask seeds were cultured at 220rpm and 37℃ for 8-10 hours, they were transferred to the seed tank or fermentation tank at a 1-10% (v:v) inoculation rate. The pH of the seed tank was maintained at 6.5-7.5 by adjusting with ammonia water, the temperature was constant at 37℃, the ventilation was 1-3 L / min, and the dissolved oxygen was controlled at more than 30%. After 8-12 hours of culture, the inoculation rate was transferred to a 5L fermentation tank containing 2L coupled fermentation medium at a 1-10% (v:v) inoculation rate. The pH of the fermentation tank was maintained at 6.5-7.5 by adjusting with ammonia water, the temperature was constant at 37℃, the ventilation was 1-3 L / min, and the dissolved oxygen was controlled at about 15-30%. The glucose concentration in the tank was maintained at 0.5-2.0 g / L by adding 50-70% glucose solution. During the process, samples were taken regularly to measure the OD600 value and amino acid yield.

[0049] Threonine-producing bacteria and isoleucine-producing bacteria can be mixed and inoculated at different proportions in the shake flask stage, seed tank stage and fermentation tank stage at the same stage or at different stages, and start coupled fermentation. For details, please refer to the subsequent implementation cases.

[0050] Example 5 Mixed inoculation and coupled fermentation of different shake flask seeds in fermenter After the shake flask seeds of threonine and isoleucine producing bacteria were cultured for 8-12 hours, they were mixed in different proportions (v:v) and inoculated into the fermenter for coupled fermentation. At the same time, the shake flask seeds of threonine producing bacteria and isoleucine producing bacteria were fermented separately in the coupled fermentation medium as a control (when the isoleucine producing bacteria were fermented separately, 35 g / L threonine was added to the coupled fermentation medium). The 24-hour fermentation results are shown in Table 7. The threonine produced during the coupled fermentation can fully meet the synthesis requirements of isoleucine. When the inoculation ratio is greater than 10, threonine is mainly produced; when the inoculation ratio is less than 1, isoleucine is mainly produced; when the inoculation ratio is in the range of 1-10, both threonine and isoleucine can be effectively synthesized. Threonine producing bacteria and isoleucine producing bacteria show good growth and metabolic synergy during coupled fermentation.

[0051] Table 7. Results of coupled fermentation with different ratios of seed mixture inoculation in shake flasks

[0052] Example 6 Mixed inoculation and coupled fermentation of seeds from different seed tanks in a fermentation tank (1) Inoculation and coupled fermentation of seeds in seed tanks at different time stages with different mixing ratios After the shake flask seeds of threonine and isoleucine producing bacteria were cultured for 8-12 hours, they were transferred to their respective seed tanks at an inoculation rate of 1-10% (v:v). After culturing to the early logarithmic growth stage (6-9 hours) and the middle logarithmic growth stage (8-12 hours), they were mixed and inoculated into the fermentation tank at different ratios (v:v) for coupled fermentation. The 24-hour fermentation results are shown in Table 8.

[0053] Table 8. Coupled fermentation results of two mixed ratios of inoculation in seed tanks at different time periods

[0054] (2) Inoculation of seeds in seed tanks at different mixing ratios in the middle logarithmic growth period and coupled fermentation After the shake flask seeds of threonine and isoleucine producing bacteria were cultured for 8-12 hours, they were transferred to their respective seed tanks at an inoculation rate of 1-10% (v:v), and then mixed and inoculated into the fermentation tank at different ratios (v:v) for coupled fermentation after 8-12 hours of culture. The 24-hour fermentation results are shown in Table 9. At different inoculation ratios, the biomass and yield of the seed tank seed mixed inoculation coupled fermentation were better than those of the corresponding shake flask seed mixed inoculation coupled fermentation. Both threonine and isoleucine can be effectively synthesized, which is consistent with the change trend of the shake flask seed mixed inoculation coupled fermentation.

[0055] Table 9. Coupling fermentation results of seed tanks with different ratios of mixed inoculation

[0056] Example 7: Mixed inoculation and coupled fermentation of isoleucine shake flask seeds and threonine seed tank seeds in a fermenter The seed tank seeds of threonine-producing bacteria and the shake flask seeds of isoleucine-producing bacteria in the middle logarithmic growth period were mixed in different proportions (v:v) and inoculated into the fermenter for coupled fermentation. The 24-hour fermentation results are shown in Table 10. Due to the large differences in the composition of the shake flask seed medium and the coupled fermentation medium, and the large differences in the OD of the seed tank seeds and the shake flask seeds, the biomass and isoleucine yields under the two mixed inoculation ratios were compared with the corresponding seed tank seed mixed inoculation coupled fermentation.

[0057] Table 10. Results of coupled fermentation with mixed inoculation of seed tank seeds and shake flask seeds at different ratios

[0058] Example 8 The seeds from the isoleucine seed tank are mixed and inoculated into the threonine fermentation tank for coupled fermentation After the threonine seed tank seeds were transferred to the fermentation tank filled with the coupled fermentation medium at an inoculation rate of 1-10% (v:v) for fermentation for 8-12 hours, the isoleucine seed tank seeds in the middle of logarithmic growth (8-12 hours) were inoculated at different ratios (v:v) for coupled fermentation. The 24-hour fermentation results are shown in Table 11. Compared with the results of the coupled fermentation of the seed tank seeds mixed inoculation at the corresponding ratio, the biomass was lower and the proportion of isoleucine production in the total output decreased. It should be that the nutrients in the threonine fermentation tank were consumed in large quantities by the threonine production bacteria, resulting in the slower growth of the isoleucine production bacteria, thereby reducing its biomass and yield. Table 11. Coupling fermentation results of different ratios of seed tank seeds and fermentation tank mixed inoculation

[0059] The above are preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A coupled fermentation process of threonine and isoleucine, characterized in that: The steps include: The threonine producing strain and the isoleucine producing strain are mixed and inoculated into a coupled fermentation medium, and then coupled fermentation is performed; The inoculation ratio during mixed inoculation is that the volume ratio of the threonine production bacterial solution to the isoleucine production bacterial solution is x, 10≥x>0, and the biomass ratio of the threonine production bacterial solution to the isoleucine production bacterial solution is 0.8-1.2; The coupled fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt and a growth factor, and the pH of the coupled fermentation medium is 6.5-7.

5.

2. The coupled fermentation process of threonine and isoleucine according to claim 1, characterized in that: The mixed inoculation comprises mixing and inoculating the production strains of threonine and isoleucine in one or more combinations of the same or different fermentation stages, time periods and inoculation ratios; the fermentation stages comprise a seed shake flask stage, a seed tank stage and a fermentation tank stage; and at least one of the seed tank stage and the fermentation tank stage uses a coupled fermentation medium.

3. The coupled fermentation process of threonine and isoleucine according to claim 2, characterized in that: The time period includes an initial growth period, an initial logarithmic growth period and a mid-to-late logarithmic growth period; the initial growth period is 0 to 3 hours for the growth of threonine and isoleucine strains, the initial logarithmic growth period is 6 to 9 hours for the growth of threonine and isoleucine strains, and the mid-to-late logarithmic growth period is 8 to 12 hours for the growth of threonine and isoleucine strains.

4. The coupled fermentation process of threonine and isoleucine according to claim 1, characterized in that: The nitrogen source includes one or more combinations of inorganic nitrogen sources and organic nitrogen sources; the inorganic nitrogen source includes one or more combinations of ammonia, soluble ammonium salts and amino acids; the organic nitrogen source includes one or more combinations of plant-based proteins and hydrolysates thereof, animal-based proteins and hydrolysates thereof, and microbial-based proteins and hydrolysates thereof; the soluble ammonium salts include one or more combinations of ammonium sulfate and ammonium chloride, and the amino acids include one or more combinations of glutamic acid and methionine; the plant-based proteins and hydrolysates thereof include one or more combinations of soy peptone, soybean meal hydrolysate, corn steep liquor, corn steep liquor dry powder, corn steep liquor extract and malt extract; the animal-based proteins and hydrolysates thereof include one or more combinations of beef extract powder, beef extract, fish meal, casein hydrolysate and acid-hydrolyzed casein; the microbial-based proteins and hydrolysates thereof include one or more combinations of yeast powder, yeast extract powder, yeast extract, yeast paste, yeast wall-broken material and yeast hydrolysate.

5. The coupled fermentation process of threonine and isoleucine according to claim 1, characterized in that: The inorganic salts include one or a combination of two or more of soluble sodium salts, soluble magnesium salts, soluble calcium salts and soluble salts of trace metal elements; the trace metal elements include zinc, manganese, copper, molybdenum, iron and cobalt; the growth factors include one or a combination of two or more of ammonium ferric citrate, choline chloride and vitamins; the vitamins include one or a combination of two or more of VB1, biotin, VB3 and VB6.

6. The coupled fermentation process of threonine and isoleucine according to claim 1, characterized in that: The carbon source includes one or more of starch hydrolysate, cellulose hydrolysate, glucose, and sucrose; the hydrolysate of starch and cellulase includes one or more of starch hydrolysate, cassava hydrolysate, and non-grain carbon source hydrolysate; the non-grain carbon source includes one or more of straw and corn cob; and the pH of the coupled fermentation medium is adjusted by using one or more of ammonia water, sodium hydroxide, potassium hydroxide, sulfuric acid, and phosphoric acid.

7. The coupled fermentation process of threonine and isoleucine according to claim 1, characterized in that: The amino acid fermentation production strain used in the coupled fermentation process is a wild or genetically engineered strain, which includes Escherichia coli, Bacillus, Brevibacterium, yeast, Corynebacterium glutamicum or Streptomyces.

8. The coupled fermentation process of threonine and isoleucine according to claim 7, characterized in that: The bacterial species is Escherichia coli or Corynebacterium glutamicum.

9. A composite amino acid product prepared by the coupled fermentation process of threonine and isoleucine according to any one of claims 1 to 8, characterized in that: The composite amino acid product contains threonine and isoleucine in different proportions.

10. Use of the composite amino acid product prepared by the coupled fermentation process of threonine and isoleucine as claimed in claim 9 in food or feed.

Images