Method for producing L-leucine and L-isoleucine through fermentation

Abstract

The embodiment of the invention provides a fermentation method of L-leucine and L-isoleucine. According to the method, at least one L-leucine or L-isoleucine producing strain is used for fermentation culture; according to the method, relatively cheap molasses with rich carbon and nitrogen sources is selected as materials, corn steep liquor with more impurities is abandoned, and the molasses is converted into a nutrient substance containing more carbon and nitrogen sources capable of being utilized by strains through enzymolysis; in addition, in order to control the ratio of carbon and nitrogen sources in the fermentation liquor, a more accurate material supplementing process is selected, so that the fermentation requirements of strains can be guaranteed, and the situation that the liquid nitrogen source of the fermentation liquor is too high and too low can be effectively avoided, and the purposes of increasing the yield and the sugar acid conversion rate and reducing heteroacid byproducts are achieved.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a method for producing L-leucine and L-isoleucine by fermentation, the method comprising the use of Corynebacterium glutamicum in the fermentation of L-leucine and L-isoleucine. Background Art

[0002] Branched-chain amino acids (BCAAs, including leucine, isoleucine, and valine), as important ingredients in the field of sports nutrition and health, have developed rapidly in recent years, and their application scenarios have continued to expand, but their market and technology also face some challenges. As the global market continues to expand, the global BCAAs market size will exceed US$1 billion in 2023, with a compound annual growth rate (CAGR) of approximately 8%-10%, mainly driven by sports nutrition, health foods, and medical applications. BCAAs, as anti-fatigue and recovery-promoting supplements, are widely used by fitness people and athletes, especially in strength training and endurance sports; in addition, studies have shown the potential of BCAAs in improving insulin resistance (diabetes) and adjuvant treatment of liver disease; BCAAs beverages, functional snacks and other products are gradually recognized by the general public and used in daily exercise.

[0003] The fermentation strains of BCAAs mainly include Corynebacterium glutamicum, Brevibacterium flavum and Escherichia coli. Traditional strains have low acid production, high impurity acid, and low efficiency, and are gradually replaced by metabolic engineering strains. With the continuous improvement of strains, the process also needs to be continuously optimized. However, the matching degree between the materials and processes used in the fermentation process of BCAAs is not perfect, which leads to the phenomenon that most excellent strains have low acid production due to the lack of matching fermentation processes.

[0004] Therefore, it is necessary to provide a fermentation method of recombinant bacteria to increase the yield of branched-chain amino acids, reduce impurity acids, and improve the sugar-acid conversion ratio. Summary of the invention

[0005] One or more embodiments of the present specification provide a fermentation method for L-leucine and L-isoleucine. The method uses at least one L-leucine or L-isoleucine production bacteria for fermentation culture; the method selects relatively cheap molasses as a material, which has a rich carbon and nitrogen source, discards corn pulp with more impurities, and converts molasses into a nutrient containing more carbon and nitrogen sources that can be used by the strain through enzymatic hydrolysis; in addition, in order to control the carbon and nitrogen source ratio in the fermentation liquid, a more precise feeding process is selected, which can not only guarantee the fermentation needs of the strain, but also effectively avoid excessive and low nitrogen sources in the fermentation liquid, thereby achieving the purpose of increasing yield, sugar-acid conversion rate, and reducing miscellaneous acid by-products.

[0006] One of one or more embodiments of the present specification provides a method for producing L-leucine by fermentation, the method comprising: using Corynebacterium glutamicum to ferment and produce L-leucine, the Corynebacterium glutamicum comprising at least one of IBBH-15, IBCLQ-257 and IBCLQ-257e; during the L-leucine fermentation process, increasing the production of L-leucine by regulating the supply of nutrients; the nutrients comprising molasses enzymatic hydrolysate.

[0007] In some embodiments, the molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses for enzymatic hydrolysis; the molasses includes at least one of beet molasses and sucrose molasses.

[0008] In some embodiments, the molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses and performing enzymatic hydrolysis for 2-6 hours at pH 4.5-6.0 and temperature 45-60°C.

[0009] In some embodiments, the production of L-leucine is increased by regulating the supply of nutrients during the L-leucine fermentation process, including: during the L-leucine fermentation process, feed sugar and the molasses enzymatic hydrolyzate are added in a ratio of 6:1-10:1 to control residual sugar.

[0010] In some embodiments, when the feed sugar and the molasses enzymatic hydrolysate are added, their concentrations are kept consistent.

[0011] In some embodiments, the method further comprises: during the L-leucine fermentation process, after the residual sugar drops to 2 wt %, starting to feed feed sugar and molasses enzymatic hydrolysate to control the residual sugar at 1-3 wt %.

[0012] In some embodiments, the method further comprises: in the fermentation culture feeding stage, in order to control the residual sugar at 1.5-2.5wt%, starting to supplement the glucose solution with a concentration of 40-50wt% and the molasses hydrolyzate with a concentration of 40-50wt%, the flow rate ratio of the glucose solution and the molasses hydrolyzate is controlled to be 6:1-10:1, and the proportion of the molasses hydrolyzate to the fermentation medium is 2.5-3.5wt%.

[0013] In some embodiments, during the L-leucine fermentation process, the seed culture medium includes glucose with an initial concentration of 2.5-3.5 wt % and molasses enzymatic hydrolyzate with an initial concentration of 1-2 wt %.

[0014] In some embodiments, during the L-leucine fermentation process, the fermentation medium includes glucose with an initial concentration of 3.5-4.5wt% and molasses hydrolyzate with a concentration of 0.5-1.1wt%, wherein the molasses hydrolyzate is selected from at least one of beet molasses hydrolyzate and sucrose molasses hydrolyzate, and the molasses hydrolyzate is hydrolyzed by at least one of sucrase or protease.

[0015] In some embodiments, the method further includes: during the L-leucine fermentation process, the initial pH is 6.7, and the rotation speed and ventilation are alternately adjusted to maintain the dissolved oxygen at 5-10%; after the bacterial OD562 grows to 20, the pH is increased to 6.9, and after the bacterial OD562 grows to 30, the pH is increased to 7.1; after the bacterial OD562 grows to 40, the pH is increased to 7.2.

[0016] One of one or more embodiments of the present specification provides a method for producing L-isoleucine by fermentation, the method comprising: using Corynebacterium glutamicum to ferment and produce L-isoleucine, wherein the Corynebacterium glutamicum includes at least one of BCIL-253, IBCL-1 (IBCL-01) and IBCIL-253k; during the L-isoleucine fermentation process, increasing the production of L-isoleucine by regulating the supply of nutrients; the nutrients include molasses enzymatic hydrolysate.

[0017] In some embodiments, the molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses for enzymatic hydrolysis; the molasses includes at least one of beet molasses and sucrose molasses.

[0018] In some embodiments, the molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses and performing enzymatic hydrolysis for 2-6 hours at pH 4.5-6.0 and temperature 45-60°C.

[0019] In some embodiments, the production of L-isoleucine is increased by regulating the supply of nutrients during the L-isoleucine fermentation process, including: during the L-isoleucine fermentation process, feed sugar and the molasses enzymatic hydrolyzate are added in a ratio of 4:1-7:1 to control residual sugar.

[0020] In some embodiments, when the feed sugar and the molasses enzymatic hydrolysate are added, their concentrations are kept consistent.

[0021] In some embodiments, the method further comprises: during the L-isoleucine fermentation process, when the residual sugar drops to 5.5-6.5wt%, starting to feed inorganic salt feed solution to control the osmotic pressure of the fermentation liquid at 690-710mosm / L; when the residual sugar is lower than the residual sugar and drops to 1.5-2.5wt%, starting to feed feed sugar and molasses enzymatic hydrolyzate to control the residual sugar at 1.5-2.5wt%.

[0022] In some embodiments, the method further comprises: when the residual sugar drops to 1.5-2.5wt%, starting to feed supplementary liquid A and liquid B, controlling the flow rate ratio of the liquid A and the liquid B to be 4:1-7:1, the proportion of the molasses hydrolyzate to the fermentation medium is 3.5-4.5wt%, and continuing to feed inorganic salt feed liquid to control the osmotic pressure of the fermentation liquid at 890-910mosm / L; the liquid A comprises a glucose solution with a concentration of 40-50wt%, a potassium dihydrogen phosphate solution with a concentration of 0.1wt% and a magnesium sulfate solution with a concentration of 0.05wt%, and the liquid B comprises a molasses hydrolyzate with a concentration of 40-50wt%.

[0023] In some embodiments, during the L-isoleucine fermentation process, the seed culture medium includes glucose with an initial concentration of 4.5-5.5 wt % and molasses enzymatic hydrolyzate with an initial concentration of 2.5-3.5 wt %.

[0024] In some embodiments, during the L-isoleucine fermentation process, the fermentation medium includes glucose with an initial concentration of 9.5-10.5wt% and molasses hydrolyzate with a concentration of 1.5-2.5wt%, wherein the molasses hydrolyzate is selected from at least one of beet molasses hydrolyzate and sucrose molasses hydrolyzate, and the molasses hydrolyzate is hydrolyzed by at least one of sucrase or protease.

[0025] In some embodiments, the initial fermentation temperature during the L-isoleucine fermentation process is 30° C. and the pH is 6.8. During the fermentation process, the ventilation and rotation speed are continuously adjusted to control the dissolved oxygen at 8-12%. The OD562 of the bacteria in the fermented liquid grows to 38-42, and the pH is adjusted to 6.9-7.1. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] This specification will be further described in the form of exemplary embodiments, which will be described in detail by the accompanying drawings. These embodiments are not restrictive, and in these embodiments, the same number represents the same structure, wherein:

[0027] Figure 1 is an exemplary flow chart of the method for producing L-leucine by fermentation according to some embodiments of the present specification;

[0028] Figure 2 is an exemplary flow chart of the method for producing L-isoleucine by fermentation according to some embodiments of the present specification. DETAILED DESCRIPTION

[0029] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

[0030] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0031] Branched-chain amino acids have become one of the core raw materials for sports medicine, clinical nutrition and functional food development due to their unique role in promoting protein synthesis, regulating energy metabolism and supporting immune function. However, the relevant fermentation production technology is difficult to meet the market's requirements for large-scale, low-cost and high-purity preparation of branched-chain amino acids. In the industrial fermentation process, there are still problems such as low branched-chain amino acid yield, low sugar-acid ratio conversion rate and more impurities.

[0032] In view of this, one of the embodiments of the present specification provides a fermentation method of recombinant bacteria, comprising: inoculating at least one recombinant strain into a seed culture medium for cultivation to obtain a seed liquid; inoculating the seed liquid into a fermentation medium for fermentation culture; during the fermentation culture, increasing the production of branched-chain amino acids by regulating at least one of the amount of inducer added, dissolved oxygen level, pH and nutrient supply.

[0033] Figure 1 is an exemplary flow chart of the fermentation method for producing L-leucine according to some embodiments of this specification. Figure 1 As shown, the method for producing L-leucine by fermentation may include the following steps.

[0034] Step S11, using Corynebacterium glutamicum to ferment and produce L-leucine, wherein the Corynebacterium glutamicum includes at least one of IBBH-15, IBCLQ-257 and IBCLQ-257e.

[0035] Corynebacterium glutamicum is originally a Gram-positive bacterium isolated from soil, and is widely used in the industrialized production of amino acids because of its high-yield glutamic acid characteristic. Corynebacterium glutamicum has important value in the fields of food, medicine, feed additives due to its stable metabolic regulation characteristics, such as for preparing nutritional supplements or pharmaceutical raw materials, etc. Exemplarily, Corynebacterium glutamicum can be used for amino acid production, for example, by metabolic engineering, Corynebacterium glutamicum is optimized for efficient production of branched-chain amino acids (such as L-leucine, L-isoleucine, L-valine, etc.).

[0036] In some embodiments, Corynebacterium glutamicum IBBH-15, IBCLQ-257 and IBCLQ-257e can be obtained as follows:

[0037] Corynebacterium glutamicum IBBH-15 is a mutant strain of Corynebacterium glutamicum, deposited in the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with a deposit number of CGMCC No.15720 and a deposit date of May 2, 2018.

[0038] The non-coding sRNA s25 was expressed in the Corynebacterium glutamicum mutant IBBH-15 to obtain strain IBBH-pXMJ19-s257, denoted as IBCLQ-257. For non-coding sRNA s25, please refer to the Chinese published patent application (publication number CN118086306A).

[0039] The non-coding sRNA s25 was expressed by the Ptac promoter at the pta site of IBBH-15, and the resulting strain IBBH-s257e was designated as IBCLQ-257e.

[0040] The specific method of fermentation production can be found in the relevant description of the embodiments below.

[0041] Step S12, during the L-leucine fermentation process, increasing the production of L-leucine by regulating the supply of nutrients; the nutrients include molasses enzymatic hydrolyzate.

[0042] Nutrients are compounds required for microbial growth and product synthesis, including carbon sources, nitrogen sources, inorganic salts, growth factors, etc., which provide energy, structural materials and elements required for metabolic regulation for the bacteria. For example, nutrients can be glucose, molasses, molasses hydrolysate, ammonium sulfate, corn syrup, potassium dihydrogen phosphate, magnesium sulfate, biotin, vitamin B1, and some other substances, such as yeast extract, peptone, urea, sodium chloride, nucleotides, etc.

[0043] Molasses hydrolysate is the product of molasses after enzymatic hydrolysis. By adding specific enzymes (such as sucrase, cellulase, protease, etc.), the complex macromolecules in molasses (such as sucrose, polysaccharides, proteins) are decomposed into small molecules (such as monosaccharides, oligosaccharides, free amino acids) that are easier for strains to use.

[0044] Molasses enzymatic hydrolysate has the following effects:

[0045] Improve the utilization rate, convert the sucrose in molasses that cannot be directly utilized into glucose and fructose (through sucrase hydrolysis), and improve the effectiveness of carbon sources;

[0046] Release nitrogen source: decompose the protein in molasses into free amino acids, replenishing the nitrogen source required for fermentation;

[0047] Reduce interference from impurities: Degrade macromolecular substances such as colloids or pigments that may inhibit bacterial growth.

[0048] In some embodiments, the molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses for enzymatic hydrolysis; the molasses includes at least one of beet molasses and sucrose molasses.

[0049] For example, the molasses enzymatic hydrolysate can be obtained by adding sucrase and / or protease to molasses, and performing enzymatic hydrolysis at pH 4.5-6.0 and temperature 45-60° C. for 2-6 hours, for example, 2 hours, 3 hours, 4 hours, 4.5 hours or 6 hours.

[0050] In some embodiments, the method of increasing the production of L-leucine by regulating the supply of nutrients during the L-leucine fermentation process includes: during the L-leucine fermentation process, feeding feed sugar and the molasses enzymatic hydrolyzate at a ratio of 6:1-10:1 to control residual sugar. For example, the feeding ratio may be 6:1, 7:1, 8:1, 9:1 or 10:1.

[0051] In some embodiments, when the feed sugar and the molasses enzymatic hydrolysate are added, their concentrations are kept consistent.

[0052] In some embodiments, during the L-leucine fermentation process, after the residual sugar drops to 2 wt %, feed sugar and molasses enzymatic hydrolysate are started to be added to control the residual sugar at 1-3 wt %.

[0053] In some embodiments, the method further comprises: in the fermentation culture feeding stage, in order to control the residual sugar at 1.5-2.5wt%, starting to supplement the glucose solution with a concentration of 40-50wt% and the molasses hydrolyzate with a concentration of 40-50wt%, the flow rate ratio of the glucose solution and the molasses hydrolyzate is controlled to be 6:1-10:1, and the proportion of the molasses hydrolyzate to the fermentation medium is 2.5-3.5wt%.

[0054] In some embodiments, during the L-leucine fermentation process, the seed culture medium includes glucose with an initial concentration of 2.5-3.5 wt % and molasses enzymatic hydrolyzate with an initial concentration of 1-2 wt %.

[0055] In some embodiments, during the L-leucine fermentation process, the fermentation medium includes glucose with an initial concentration of 3.5-4.5wt% and molasses hydrolyzate with a concentration of 0.5-1.1wt%, wherein the molasses hydrolyzate is selected from at least one of beet molasses hydrolyzate and sucrose molasses hydrolyzate, and the molasses hydrolyzate is hydrolyzed by at least one of sucrase or protease.

[0015] In some embodiments, during the L-leucine fermentation process, the initial pH is 6.7, and the rotation speed and ventilation are alternately adjusted to maintain the dissolved oxygen at 5-10%; after the bacterial OD562 grows to 20, the pH is increased to 6.9, and after the bacterial OD562 grows to 30, the pH is increased to 7.1; after the bacterial OD562 grows to 40, the pH is increased to 7.2.

[0032] Figure 2 is an exemplary flow chart of the fermentation method for producing L-isoleucine according to some embodiments of this specification. Figure 2 As shown, the method for producing L-isoleucine by fermentation may include the following steps.

[0056] Step S21, using Corynebacterium glutamicum to ferment and produce L-isoleucine, wherein the Corynebacterium glutamicum includes at least one of BCIL-253, IBCL-1 and IBCIL-253k.

[0057] In some embodiments, the Corynebacterium glutamicum IBCL-1, BCIL-253 and IBCIL-253k can be obtained as follows:

[0058] Corynebacterium glutamicum IBCL-1 (Corynebacterium glutamicum), deposited on May 30, 2022, with a deposit number of CCTCC NO:M2022764.

[0059] The non-coding sRNA s25 was expressed in Corynebacterium glutamicum IBCL-1 to obtain strain IBCL-pXMJ19-s253, which was designated as IBCIL-253. For non-coding sRNA s25, please refer to the Chinese published patent application (publication number CN118086306A).

[0060] The non-coding sRNA s25 was expressed by the Ptac promoter at the pta site of Corynebacterium glutamicum IBCL-1, and the strain IBCL-s253k was obtained, which was recorded as IBCIL-253k.

[0061] The specific method of fermentation production can be found in the relevant description of the embodiments below.

[0062] Step S22, during the L-isoleucine fermentation process, increasing the production of L-isoleucine by regulating the supply of nutrients; the nutrients include molasses enzymatic hydrolyzate.

[0063] For detailed information on nutrients and molasses hydrolysate, see Figure 1 The relevant description will not be repeated here.

[0064] In some embodiments, the molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses for enzymatic hydrolysis; the molasses includes at least one of beet molasses and sucrose molasses.

[0065] For example, the molasses enzymatic hydrolysate can be obtained by adding sucrase and / or protease to molasses and performing enzymatic hydrolysis at pH 4.5-6.0 and temperature 45-60° C. for 2-6 hours.

[0066] In some embodiments, the production of L-isoleucine is increased by regulating the supply of nutrients during the L-isoleucine fermentation process, including: during the L-isoleucine fermentation process, feed sugar and the molasses enzymatic hydrolyzate are added in a ratio of 4:1-7:1 to control residual sugar.

[0067] In some embodiments, when the feed sugar and the molasses enzymatic hydrolysate are added, their concentrations are kept consistent.

[0068] In some embodiments, during the L-isoleucine fermentation process, when the residual sugar drops to 5.5-6.5wt%, inorganic salt feed solution is started to be added to control the osmotic pressure of the fermentation liquid at 690-710mosm / L; when the residual sugar is lower than the residual sugar and drops to 1.5-2.5wt%, feed sugar and molasses enzymatic hydrolyzate are started to be added to control the residual sugar at 1.5-2.5wt%.

[0069] In some embodiments, when the residual sugar drops to 1.5-2.5wt%, liquid A and liquid B are added, and the flow rate ratio of the liquid A and the liquid B is controlled to be 4:1-7:1, the ratio of the molasses hydrolyzate to the fermentation medium is 3.5-4.5wt%, and inorganic salt feed liquid is continued to be added to control the osmotic pressure of the fermentation liquid at 890-910mosm / L; the liquid A includes a glucose solution with a concentration of 40-50wt%, a potassium dihydrogen phosphate solution with a concentration of 0.1wt% and a magnesium sulfate solution with a concentration of 0.05wt%, and the liquid B includes a molasses hydrolyzate with a concentration of 40-50wt%.

[0070] In some embodiments, during the L-isoleucine fermentation process, the seed culture medium includes glucose with an initial concentration of 4.5-5.5 wt % and molasses enzymatic hydrolyzate with an initial concentration of 2.5-3.5 wt %.

[0071] In some embodiments, during the L-isoleucine fermentation process, the fermentation medium includes glucose with an initial concentration of 9.5-10.5wt% and molasses hydrolyzate with a concentration of 1.5-2.5wt%, wherein the molasses hydrolyzate is selected from at least one of beet molasses hydrolyzate and sucrose molasses hydrolyzate, and the molasses hydrolyzate is hydrolyzed by at least one of sucrase or protease.

[0072] In some embodiments, the initial fermentation temperature during the fermentation of L-isoleucine is 30° C. and the pH is 6.8. During the fermentation process, the ventilation and rotation speed are continuously adjusted to control the dissolved oxygen at 8-12%. The OD562 of the bacteria in the fermented liquid grows to about 40, for example, 38-42, and the pH is adjusted to about 7.0, for example, 6.9-7.1.

[0073] The above fermentation method will be described in detail below through a number of examples and comparative examples. It should be noted that the reaction conditions, reaction materials and the amount of reaction materials in the examples are only for illustration and do not limit the protection scope of this specification. The comparative examples are the control groups of the examples.

[0074] In this specification, unless otherwise specified, percentages and percentage contents are all by mass. Unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from commercial channels. At the same time, in this specification, unless otherwise specified, leucine refers to L-leucine, and isoleucine refers to L-isoleucine.

[0075] Embodiment 1 Preferred embodiment of leucine fermentation

[0076] 1. The composition of the seed culture medium is as follows:

[0077] Primary seed culture medium: 3wt% glucose, 1.5wt% enzymatic beet molasses, 0.5wt% ammonium sulfate, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate, 1wt% calcium carbonate, pH 6.7-7.2; divided into 500ml / 5000ml; sterilization conditions 121℃ / 20min;

[0078] Secondary seed tank culture medium: glucose 3wt%, enzymatic beet molasses 1.5wt%, ammonium sulfate 0.5wt%, potassium dihydrogen phosphate 0.1wt%, magnesium sulfate 0.05wt%, pH 6.8-7.2, sterilization conditions 121°C / 20min.

[0079] 2. The fermentation medium is as follows:

[0080] Standard fermentation tank fermentation initial medium: glucose 4wt%, beet molasses hydrolysate 0.8wt%, ammonium sulfate 0.4wt%, potassium dihydrogen phosphate 0.11wt%, magnesium sulfate 0.05wt%, ferrous sulfate 0.001wt%, manganese sulfate 0.001wt%, biotin 0.00001wt%, vitamin B1 0.00003wt%, L-methionine 0.01wt%, L-isoleucine 0.01wt%; natural pH, 121℃*20min;

[0081] Feed solution: Solution A: 40-50wt% glucose; Solution B: 40-50wt% beet molasses enzymatic hydrolysate; when used, the concentrations of the two need to be kept consistent.

[0082] 3. Fermentation tank process (500L):

[0083] IBCLQ-257, IBCLQ-257e, and IBBH-15 were inoculated into the primary seed culture medium of a shake flask, respectively, and cultured at 30°C, 85 rpm, and reciprocated for 16 h to obtain 500 mL of the shake flask seed solution. The maturity indicator of the primary seed solution was OD 562 8-12;

[0084] The seed solution in the shake flask was added to the medium of the secondary seed tank of 50L at a ratio of 0.2% (V / V), 30℃, pH 6.8, dissolved oxygen not less than 10%, and cultured for 16 hours. The maturity indicator of the secondary seed solution was OD 562 is 8-10, and the secondary seed solution is obtained;

[0085] 10% of the secondary seed solution was inoculated into a 500L fermenter, the fermentation volume was 300L, the fermentation temperature was 30°C, pH 6.7, the speed was 200RPM, the ventilation volume was 60LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were adjusted alternately to keep the dissolved oxygen at 5-10%; the bacterial OD 562 After the growth reached 20, the pH increased to 6.9, and the feed was started when the residual sugar dropped to 2wt%. At this time, the flow rate ratio of feed A and feed B was 8 / 1; the bacterial OD 562 After the cell OD reached 30, the pH was raised to 7.1 and 1 mM lactose was added once (no addition is required for IBBH-15 strain); 562 After increasing to 40, the pH value was raised to 7.2; the residual sugar was controlled at 2wt%, and the feed sugar was removed from the tank after it was exhausted.

[0086] Table 1 Main results of Example 1 strain L-Leucine yield wt% Sugar acid conversion rate % Secondary acid wt% IBCLQ-257 6.65 33.25 0.058 IBCLQ-257e 6.34 31.70 0.076 IBBH-15 5.57 27.85 0.15

[0087] As shown in Example 1, the core of the present invention is to obtain beet molasses hydrolyzate by enzymolysis of beet molasses using sucrase and papain, and then in the leucine fermentation process, residual sugar is controlled by adding glucose solution and beet molasses hydrolyzate in a certain ratio. This method can not only control the residual sugar, but also control the total amount of nitrogen source by adding an organic nitrogen source, thereby preventing the bacteria from growing too fast. After the enzymolysis, sucrose in the beet molasses is completely hydrolyzed into glucose and fructose, which are easier to be used by the bacteria, and papain can enzymolyze large molecular proteins so that the nitrogen source is fully utilized, thereby achieving the purpose of fully utilizing the carbon and nitrogen sources of the molasses.

[0088] In the fermentation process, acid production refers to the actual concentration of L-leucine in the fermentation broth. The calculation formula for the sugar-acid conversion rate is as follows:

[0089] Sugar-acid conversion rate = (actual L-leucine concentration × actual fermentation volume) / (actual total glucose consumption × 100%)

[0090] Meanwhile, heteroacid refers to the sum of valine, lysine, alanine, and glutamate in the fermentation broth, and these two parameters are used together to evaluate the efficiency of the fermentation process and the product composition.

[0091] In summary, Corynebacterium glutamicum IBCLQ-257, IBCLQ-257e, and IBBH-15 have obvious advantages in L-leucine production, sugar-acid conversion rate, and miscellaneous acids in the process of Example 1.

[0092] Example 2 Confirmation of the initial addition amount range

[0093] 1. The seed formula and culture process are consistent with those in Example 1.

[0094] 2. The fermentation medium is as follows:

[0095] Standard fermentation tank fermentation initial medium: glucose 4wt%, beet molasses hydrolysate 0.5-1.1wt%, ammonium sulfate 0.4wt%, potassium dihydrogen phosphate 0.11wt%, magnesium sulfate 0.05wt%, ferrous sulfate 0.001wt%, manganese sulfate 0.001wt%, biotin 0.00001wt%, vitamin B1 0.00003wt%, L-methionine 0.01wt%, L-isoleucine 0.01wt%; natural pH, 121℃*20min;

[0096] Feed solution: Solution A: 40-50wt% glucose; Solution B: 40-50wt% beet molasses enzymatic hydrolysate; when used, the concentrations of the two need to be kept consistent.

[0097] 3. Fermentation tank process (500L):

[0098] IBCLQ-257, IBCLQ-257e, and IBBH-15 were inoculated into the primary seed culture medium of a shake flask, respectively, and cultured at 30°C, 85 rpm, and reciprocated for 16 h to obtain 500 mL of the shake flask seed solution. The maturity indicator of the primary seed solution was OD 562 8-12;

[0099] The seed solution in the shake flask was inoculated into the medium of the secondary seed tank of 50L at a ratio of 0.2% (V / V), 30℃, pH 6.8, dissolved oxygen not less than 10%, and cultured for 16 hours. The maturity indicator of the secondary seed solution was OD 562 8-10, to obtain the secondary seed liquid; 10% of the secondary seed liquid was inoculated into a 500L fermenter, the initial fermentation liquid volume was 60%, the fermentation temperature was 30°C, the pH was 6.7, the speed was 200RPM, the ventilation volume was 60LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 5-10%; the bacterial OD 562 After the growth reached 20, the pH increased to 6.9, and the feed was started when the residual sugar dropped to 2wt%. At this time, the flow rate ratio of feed A and feed B was 8 / 1; the bacterial OD 562 After the cell OD reached 30, the pH was raised to 7.1 and 1 mM lactose was added once (no addition was required for IBBH-15 strain); 562 After increasing to 40, the pH value was raised to 7.2; the residual sugar was controlled at 2wt%, and the feed sugar was removed from the tank after it was exhausted.

[0100] Table 2 Main results of Example 2

[0101] Feed A / B is the volume ratio of the two feed liquids. Since the concentrations of feed sugar and feed molasses are basically the same, it can also be regarded as the mass ratio of the two when they are added.

[0102] As shown in Example 2, this example optimizes the initial concentration of beet molasses hydrolysate and verifies that under different initial concentration conditions, the results show that the initial addition amount is within the range of 0.5-1.1 wt%.

[0103] Example 3 Determining the ratio of glucose and molasses during feeding

[0104] 1. The seed formula and culture process are consistent with those in Example 1.

[0105] 2. The fermentation medium is as follows:

[0106] Standard fermentation tank fermentation initial medium: glucose 4wt%, molasses hydrolyzate 0.8wt%, ammonium sulfate 0.4wt%, potassium dihydrogen phosphate 0.11wt%, magnesium sulfate 0.05wt%, ferrous sulfate 0.001wt%, manganese sulfate 0.001wt%, biotin 0.00001wt%, vitamin B1 0.00003wt%, L-methionine 0.01wt%, L-isoleucine 0.01wt%; natural pH, 121℃*20min;

[0107] Feed solution: Solution A: 40-50wt% glucose; Solution B: 40-50wt% beet molasses enzymatic hydrolysate; when used, the concentrations of the two need to be kept consistent.

[0108] 3. Fermentation tank process (500L):

[0109] IBCLQ-257, IBCLQ-257e, and IBBH-15 were inoculated into the primary seed culture medium of a shake flask, respectively, and cultured at 30°C, 85 rpm, and reciprocated for 16 h to obtain 500 mL of the shake flask seed solution. The maturity indicator of the primary seed solution was OD 562 For 8-12.

[0110] The seed solution in the shake flask was added to the medium of the secondary seed tank of 50L at a ratio of 0.2% (V / V), 30℃, pH 6.8, dissolved oxygen not less than 10%, and cultured for 16 hours. The maturity indicator of the secondary seed solution was OD 562 8-10, to obtain the secondary seed liquid; 10% of the secondary seed liquid was inoculated into a 500L fermenter, the initial fermentation liquid volume was 60%, the fermentation temperature was 30°C, the pH was 6.7, the speed was 200RPM, the ventilation volume was 60LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 5-10%; the bacterial OD 562 After the growth reached 20, the pH increased to 6.9, and the feed was started when the residual sugar dropped to 2wt%. At this time, the flow rate ratio of feed A and feed B was 6 / 1 to 10 / 1; the cell OD 562 After the cell OD reached 30, the pH was raised to 7.1 and 1 mM lactose was added once (no addition is required for IBBH-15 strain); 562 After increasing to 40, the pH is increased to 7.2; if the molasses is exhausted first, continue to add feed sugar; if the feed sugar is exhausted first, stop adding beet molasses; control the residual sugar to 2wt%, and remove from the tank after the feed sugar is exhausted.

[0111] Table 3 Main results of Example 3

[0112] As shown in Example 3, this example optimizes the AB feeding ratio. The results show that the ideal effect can be achieved within the range of 6 / 1 to 10 / 1, which also shows that the ratio between sugar and molasses during feeding needs to be strictly controlled within the range.

[0113] Example 4: Determination of sugarcane enzymatic hydrolysate can also be used in the current process

[0114] 1. The seed formula and culture process are consistent with those in Example 1.

[0115] 2. The fermentation medium is as follows:

[0116] Standard fermentation tank fermentation initial medium: glucose 4wt%, (beet / sugar cane) molasses hydrolysate 0.8wt%, ammonium sulfate 0.4wt%, potassium dihydrogen phosphate 0.11wt%, magnesium sulfate 0.05wt%, ferrous sulfate 0.001wt%, manganese sulfate 0.001wt%, biotin 0.00001wt%, vitamin B1 0.00003wt%, L-methionine 0.01wt%, L-isoleucine 0.01wt%; natural pH, 121℃*20min;

[0117] Feed solution: Solution A: 40-50wt% glucose; Solution B: 40-50wt% beet molasses enzymatic hydrolysate; when used, the concentrations of the two need to be kept consistent.

[0118] 3. Fermentation tank process (500L):

[0119] IBCLQ-257, IBCLQ-257e, and IBBH-15 were inoculated into the primary seed culture medium of a shake flask, respectively, and cultured at 30°C, 85 rpm, and reciprocated for 16 h to obtain 500 mL of the shake flask seed solution. The maturity indicator of the primary seed solution was OD 562 For 8-12.

[0120] The seed solution in the shake flask was added to the medium of the secondary seed tank of 50L at a ratio of 0.2% (V / V), 30℃, pH 6.8, dissolved oxygen not less than 10%, and cultured for 16 hours. The maturity indicator of the secondary seed solution was OD 562 8-10, to obtain the secondary seed liquid; 10% of the secondary seed liquid was inoculated into a 500L fermenter, the initial fermentation liquid volume was 60%, the fermentation temperature was 30°C, the pH was 6.7, the speed was 200RPM, the ventilation volume was 60LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 5-10%; the bacterial OD 562 After the growth reached 20, the pH increased to 6.9, and the feed was started when the residual sugar dropped to 2wt%. At this time, the flow rate ratio of feed A and feed B was 8 / 1; the cell OD 562After the cell OD reached 30, the pH was raised to 7.1 and 1 mM lactose was added once (no addition is required for IBBH-15 strain); 562 After increasing to 40, the pH value was raised to 7.2; the residual sugar was controlled at 2wt%, and the feed sugar was removed from the tank after it was exhausted.

[0121] Table 4 Main results of Example 4

[0122] As shown in Example 4, in this example, sugarcane molasses and beet molasses enzymatic hydrolysate are used together. Sugarcane molasses enzymatic hydrolysate can be used instead of beet molasses, but the effect is slightly different from that of beet molasses.

[0123] This example demonstrates that the enzymatic hydrolysis method and the feeding method are suitable for leucine fermentation from sugarcane molasses.

[0124] Example 5 confirmed that partially enzymatically hydrolyzed molasses can also be used for fermentation

[0125] 1. The seed formula and culture process are consistent with those in Example 1.

[0126] 2. The fermentation medium is as follows:

[0127] The initial culture medium of standard fermenter fermentation: 4wt% glucose, 0.7wt% partially enzymatically hydrolyzed molasses hydrolysate, 0.4wt% ammonium sulfate, 0.11wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate, 0.001wt% ferrous sulfate, 0.001wt% manganese sulfate, 0.00001wt% biotin, 0.00003wt% vitamin B1, 0.01wt% L-methionine, 0.01wt% L-isoleucine; natural pH, 121°C*20min.

[0128] Feed solution: Solution A: 40-50wt% glucose; Solution B: 40-50wt% beet molasses enzymatic hydrolysate; when used, the concentrations of the two need to be kept consistent.

[0129] 3. Fermentation tank process (500L):

[0130] IBCLQ-257, IBCLQ-257e, and IBBH-15 were inoculated into the primary seed culture medium of a shake flask, respectively, and cultured at 30°C, 85 rpm, and reciprocated for 16 h to obtain 500 mL of the shake flask seed solution. The maturity indicator of the primary seed solution was OD 562 For 8-12.

[0131] The seed solution in the shake flask was inoculated into the medium of the secondary seed tank of 50L at a ratio of 0.2% (V / V), 30℃, pH 6.8, dissolved oxygen not less than 10%, and cultured for 16 hours. The maturity indicator of the secondary seed solution was OD 562 8-10, to obtain the secondary seed liquid; 10% of the secondary seed liquid was inoculated into a 500L fermenter, the initial fermentation liquid volume was 60%, the fermentation temperature was 30°C, the pH was 6.7, the speed was 200RPM, the ventilation volume was 60LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 5-10%; the bacterial OD 562 After the growth reached 20, the pH increased to 6.9, and the feed was started when the residual sugar dropped to 2wt%. At this time, the flow rate ratio of feed A and feed B was 8 / 1; the bacterial OD 562 After the cell OD reached 30, the pH was raised to 7.1 and 1 mM lactose was added once (no addition was required for IBBH-15 strain); 562 After increasing to 40, the pH value was raised to 7.2; the residual sugar was controlled at 2wt%, and the feed sugar was removed from the tank after it was exhausted.

[0132] Table 5 Main results of Example 5

[0133] As shown in Example 5, in this example, beet / sugarcane molasses was hydrolyzed using only sucrase or protease, and then the corresponding molasses was added through the feed stream. The results showed that the leucine yield and sugar-acid ratio of the partially hydrolyzed molasses decreased compared with Example 1, and the fermentation broth miscellaneous acids corresponding to the molasses that was not hydrolyzed by protease increased.

[0134] This example demonstrates that partially enzymatically hydrolyzed molasses can still improve the effect of leucine, but the improvement capacity is limited compared to Example 1.

[0135] Comparative Example 1: One-time addition of molasses process versus fed-batch process

[0136] 1. The seed formula and cultivation process are consistent with those in Example 1.

[0137] 2. The fermentation medium is as follows:

[0138] The initial culture medium of the standard fermentation tank was as follows: 4wt% glucose, 0.5-3.0wt% beet molasses hydrolysate, 0.4wt% ammonium sulfate, 0.11wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate, 0.001wt% ferrous sulfate, 0.001wt% manganese sulfate, 0.00001wt% biotin, 0.00003wt% vitamin B1, 0.01wt% L-methionine, and 0.01wt% L-isoleucine; natural pH, 121°C*20min.

[0139] Feed solution: Solution A 40-50wt% glucose.

[0140] 3. Fermentation tank process (500L):

[0141] IBCLQ-257, IBCLQ-257e, and IBBH-15 were inoculated into the primary seed culture medium of a shake flask, respectively, and cultured at 30°C, 85 rpm, and reciprocated for 16 h to obtain 500 mL of the shake flask seed solution. The maturity indicator of the primary seed solution was OD 562 For 8-12.

[0142] The seed solution in the shake flask was inoculated into the medium of the secondary seed tank of 50L at a ratio of 0.2% (V / V), 30℃, pH 6.8, dissolved oxygen not less than 10%, and cultured for 16 hours. The maturity indicator of the secondary seed solution was OD 562 8-10, to obtain the secondary seed liquid; 10% of the secondary seed liquid was inoculated into a 500L fermenter, the initial fermentation liquid volume was 60%, the fermentation temperature was 30°C, the pH was 6.7, the speed was 200RPM, the ventilation volume was 60LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 5-10%; the bacterial OD 562 After the growth reached 20, the pH value was raised to 6.9, and the sugar was added when the residual sugar dropped to 2wt%, and the residual sugar was controlled at 2wt%; 562 After the cell OD reached 30, the pH was raised to 7.1 and 1 mM lactose was added once (no addition was required for IBBH-15 strain); 562 After increasing to 40, the pH increased to 7.2.

[0143] Table 6 Main results of comparative example 1

[0144] The above-mentioned one-time added molasses concentration is the total amount of added molasses divided by the initial fermentation volume.

[0145] As shown in Comparative Example 1, a one-time addition of molasses will lead to a decrease in acid production and sugar-acid conversion rate. Compared with the feed-batch process, a one-time addition of molasses will result in too much or too little organic nitrogen source in the fermentation liquid, which will lead to abnormal acid production by the bacteria.

[0146] This comparative example proves that the fed-batch process of molasses has obvious advantages over the one-time addition.

[0147] Comparative Example 2 confirmed that the initial addition amount should not be too high or too low

[0148] 1. The seed formula and cultivation process are consistent with those in Example 1.

[0149] 2. The fermentation medium is as follows:

[0150] The initial culture medium of standard fermentation tank fermentation was as follows: 4wt% glucose, 0.3-0.6wt% or 1.1-1.2wt% beet molasses enzymatic hydrolysis, 0.4wt% ammonium sulfate, 0.11wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate, 0.001wt% ferrous sulfate, 0.001wt% manganese sulfate, 0.00001wt% biotin, 0.00003wt% vitamin B1, 0.01wt% L-methionine, and 0.01wt% L-isoleucine; natural pH, 121°C*20min.

[0151] Feed solution: Solution A: 40-50wt% glucose; Solution B: 40-50wt% beet molasses enzymatic hydrolysate; when used, the concentrations of the two need to be kept consistent.

[0152] 3. Fermentation tank process (500L):

[0153] IBCLQ-257, IBCLQ-257e, and IBBH-15 were inoculated into the primary seed culture medium of a shake flask, respectively, and cultured at 30°C, 85 rpm, and reciprocated for 16 h to obtain 500 mL of the shake flask seed solution. The maturity indicator of the primary seed solution was OD 562 For 8-12.

[0154] The seed solution in the shake flask was inoculated into the medium of the secondary seed tank of 50L at a ratio of 0.2% (V / V), 30℃, pH 6.8, dissolved oxygen not less than 10%, and cultured for 16 hours. The maturity indicator of the secondary seed solution was OD 562 8-10, to obtain the secondary seed liquid; 10% of the secondary seed liquid was inoculated into a 500L fermenter, the initial fermentation liquid volume was 60%, the fermentation temperature was 30°C, the pH was 6.7, the speed was 200RPM, the ventilation volume was 60LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 5-10%; the bacterial OD 562 After the growth reached 20, the pH value was raised to 6.9, and the sugar was added when the residual sugar dropped to 2wt%, and the residual sugar was controlled at 2wt%; 562 After the cell OD reached 30, the pH was raised to 7.1 and 1 mM lactose was added once (no addition was required for IBBH-15 strain); 562 After increasing to 40, the pH increased to 7.2.

[0155] Table 7 Main results of comparative example 2

[0156] The above initial concentration is the actual concentration of molasses in the initial culture medium; the above total molasses concentration is the total amount of molasses divided by the volume of the tank below.

[0157] As shown in Comparative Example 2, too high or too low initial molasses, too high or too low molasses feeding rate, and too high or too low total molasses amount will cause acid production and sugar-acid conversion rate to decrease. Compared with Examples 1 and 2, this comparative example illustrates several extreme addition conditions of several molasses, indicating the importance of controlling the initial addition amount of molasses, controlling the feeding ratio rate, and controlling the total amount of molasses in the examples.

[0158] Comparative Example 3 confirmed that molasses must be enzymatically hydrolyzed to achieve the effect of increasing acid production

[0159] 1. The seed formula and culture process are consistent with those in Example 1.

[0160] 2. The fermentation medium is as follows:

[0161] The initial culture medium of standard fermenter fermentation: 4wt% glucose, 0.8wt% beet / sugarcane molasses stock solution, 0.4wt% ammonium sulfate, 0.11wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate, 0.001wt% ferrous sulfate, 0.001wt% manganese sulfate, 0.00001wt% biotin, 0.00003wt% vitamin B1, 0.01wt% L-methionine, 0.01wt% L-isoleucine; natural pH, 121℃*20min.

[0162] Feed solution: Solution A: 40-50wt% glucose; Solution B: 40-50wt% molasses enzymatic hydrolyzate; when used, the concentrations of the two need to be kept consistent.

[0163] 3. Fermentation tank process (500L):

[0164] IBCLQ-257, IBCLQ-257e, and IBBH-15 were inoculated into the primary seed culture medium of a shake flask, respectively, and cultured at 30°C, 85 rpm, and reciprocated for 16 h to obtain 500 mL of the shake flask seed solution. The maturity indicator of the primary seed solution was OD 562 For 8-12.

[0165] 10% of the secondary seed solution was inoculated into a 500L fermenter, the fermentation volume was 300L, the fermentation temperature was 30°C, pH 6.7, the speed was 200RPM, the ventilation volume was 60LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were adjusted alternately to keep the dissolved oxygen at 5-10%; the bacterial OD 562 After the growth reached 20, the pH increased to 6.9, and the feed was started when the residual sugar dropped to 2wt%. At this time, the flow rate ratio of feed A and feed B was 8 / 1; the bacterial OD 562 After the cell OD reached 30, the pH was raised to 7.1 and 1 mM lactose was added once (no addition is required for IBBH-15 strain); 562After increasing to 40, the pH value was raised to 7.2; the residual sugar was controlled at 2wt%, and the feed sugar was removed from the tank after it was exhausted.

[0166] Table 8 Main results of comparative example 3

[0167] As shown in Comparative Example 3, when the molasses that has not been enzymatically hydrolyzed is subjected to L-leucine fermentation, its acid production and sugar-acid conversion rate do not reach the effect of enzymatic hydrolysis molasses. This comparative example shows that enzymatic hydrolysis of molasses is beneficial to L-leucine fermentation. After enzymatic hydrolysis, the sucrose in the molasses is enzymatically hydrolyzed into glucose and fructose, and the crude protein is decomposed, which is more beneficial to the metabolism of the bacteria. The molasses that has not been enzymatically hydrolyzed cannot achieve this effect.

[0168] Comparative Example 4 confirms the process advantages provided by the embodiments of this specification

[0169] 1. The composition of the seed culture medium is as follows:

[0170] Primary seed culture medium: glucose 3wt%, corn slurry 4wt%, ammonium sulfate 0.5wt%, potassium dihydrogen phosphate 0.1wt%, magnesium sulfate 0.05wt%, calcium carbonate 1wt%, pH 6.7-7.2; packaged in 500 ml / 5000 ml; sterilization conditions 121°C / 20min.

[0171] Secondary seed tank culture medium: glucose 3wt%, corn slurry 4wt%, ammonium sulfate 0.5wt%, potassium dihydrogen phosphate 0.1wt%, magnesium sulfate 0.05wt%, pH 6.8-7.2, sterilization conditions 121°C / 20min.

[0172] 2. The fermentation medium is as follows:

[0173] The initial culture medium of standard fermenter fermentation: glucose 10wt%, corn slurry 1wt%, ammonium sulfate 0.5wt%, potassium dihydrogen phosphate 0.11wt%, magnesium sulfate 0.25wt%, ferrous sulfate 0.001wt%, manganese sulfate 0.001wt%, biotin 0.00001wt%, vitamin B1 0.00003wt%, L-methionine 0.01wt%, L-isoleucine 0.01wt%, natural pH, 121℃*20min.

[0174] Feed solution: Solution A, 40-50wt% glucose, 0.05wt% magnesium sulfate.

[0175] 3. Fermentation tank process (500L):

[0176] IBCLQ-257, IBCLQ-257e, and IBBH-15 were inoculated into the primary seed culture medium of a shake flask, respectively, and cultured at 30°C, 85 rpm, and reciprocated for 16 h to obtain 500 mL of the shake flask seed solution. The maturity indicator of the primary seed solution was OD 562 For 8-12.

[0177] 10% of the secondary seed solution was inoculated into a 500L fermenter, the fermentation volume was 300L, the fermentation temperature was 30°C, pH 6.7, the speed was 200RPM, the ventilation volume was 60LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were adjusted alternately to keep the dissolved oxygen at 5-10%; the bacterial OD 562 After the growth reached 20, the pH increased to 6.9, and the feed was started when the residual sugar dropped to 2wt%; 562 After the cell OD reached 30, the pH was raised to 7.1 and 1 mM lactose was added once (no addition is required for IBBH-15 strain); 562 After increasing to 40, the pH value was raised to 7.2; 1 mM was added after 48 h to control the residual sugar to 2-3 wt%, and the feed sugar was removed from the tank after it was exhausted.

[0178] Table 9 Main results of comparative example 4 strain L-Leucine yield wt% Sugar acid conversion rate % Miscellaneous acid wt% IBCLQ-257 6.01 27.8 0.12 IBCLQ-257e 5.76 26.6 0.15 IBBH-15 4.95 24.5 0.29

[0179] As shown in Comparative Example 4, this comparative example uses corn steep liquor for L-leucine fermentation. By comparing with Example 1, it can be seen that the use of enzymatic molasses for L-leucine fermentation has higher acid production and sugar-acid conversion rate, and less impurities. In addition, molasses has fewer impurities than corn steep liquor, lighter color, and is more conducive to subsequent separation and purification.

[0180] Example 6 Preferred embodiment, beet molasses enzymatic hydrolysate, fed-feed, optimal feed AB ratio 5 / 1

[0181] 1. The composition of the seed culture medium is as follows:

[0182] Primary seed culture medium: glucose 3wt%, corn steep liquor powder 1.0wt%, ammonium sulfate 0.5wt%, KH2PO4 0.1wt%, MgSO4·7H2O 0.05wt%, calcium carbonate 1.0wt%, pH 6.7-7.2; packaged in 500 ml / 5000 ml; sterilization condition 121°C / 20min.

[0183] Secondary seed tank culture medium: glucose 5wt%, beet molasses enzymatic hydrolyzate 3.0wt%, ammonium sulfate 0.5wt%, KH2PO4 0.1wt%, MgSO4·7H2O 0.05wt%, pH 6.8-7.2; sterilization condition 121°C / 20min.

[0184] 2. The fermentation medium is as follows:

[0185] The initial culture medium of the standard fermenter was as follows: 10 wt% glucose, 2 wt% beet molasses enzymatic hydrolysate, 1.0 wt% ammonium sulfate, 0.1 wt% potassium dihydrogen phosphate, and 0.1 wt% magnesium sulfate.

[0186] Feed solution: Solution A: 40-50wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate; Solution B: 40-50wt% beet molasses enzymatic hydrolyzate; Solution C: 10-12% ammonium sulfate solution; When used, the concentrations of glucose and molasses need to be kept consistent.

[0187] 3. Fermentation tank process (500L):

[0188] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 It is 0.6-0.7*25.

[0189] Step 2: The seed solution in the shake flask was inoculated into a 50L secondary seed tank medium at a ratio of 1% (V / V), and fermented at 30°C, 200rpm, aeration ratio of 0.3VVM, pH 6.8, and dissolved oxygen not less than 10%. After 16 hours of seed tank culture, the secondary maturity indicator: OD 562 It is 0.7-0.8*25; the secondary seed liquid is obtained.

[0190] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562 When the cell culture medium grows to about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (IBCL-1 strain does not need to be added); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding ammonium sulfate feed solution; (2) when the residual sugar content is lower than 2 wt%, the residual sugar content is controlled at about 2.0 wt% by adding feed solution, and the feed ratio of feed A and feed B is controlled to 5 / 1. At the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by adding inorganic salt feed solution, and the fermentation ends when the sugar is consumed.

[0191] Table 10 Main results of Example 6 strain L-Isoleucine wt% Sugar acid conversion rate % Secondary acid wt% IBCIL-253 7.25 41.9 0.06 IBCIL-253k 7.03 40.64 0.09 IBCL-1 6.58 38.0 0.12

[0192] As shown in Example 6, the core of the present invention is to obtain beet molasses hydrolyzate by enzymolysis of beet molasses using sucrase and protease, and then control the residual sugar by adding glucose solution and beet molasses hydrolyzate in a certain ratio during the isoleucine fermentation process. This method can not only control the residual sugar, but also prevent the bacteria from growing too fast by adding an organic nitrogen source. After the enzymolysis, the sucrose in the beet molasses is completely hydrolyzed into glucose and fructose, which are easier to be used by the bacteria, and the protease can enzymolyze large molecular proteins to make full use of the nitrogen source, thereby achieving the purpose of fully utilizing the carbon and nitrogen sources of the molasses.

[0193] In the fermentation process, acid production refers to the actual concentration of L-isoleucine in the fermentation broth.

[0194] The calculation formula of sugar-acid conversion rate is as follows:

[0195] Sugar-acid conversion rate = (actual L-isoleucine concentration × actual fermentation volume) / (actual total amount of glucose consumed × 100%).

[0196] Meanwhile, heteroacid refers to the sum of valine, lysine, alanine, and glutamate in the fermentation broth, and these two parameters are used together to evaluate the efficiency of the fermentation process and the product composition.

[0197] In summary, Corynebacterium glutamicum IBCIL-253, IBCIL-253k, and IBCL-1 have obvious advantages in L-isoleucine production, sugar-acid conversion rate, and miscellaneous acids in the process of Example 6.

[0198] Example 7 Confirming the initial addition amount range and determining the feed AB ratio

[0199] 1. The seed process is consistent with that in Example 6.

[0200] 2. The fermentation medium is as follows:

[0201] Standard fermentation tank fermentation initial culture medium: glucose 10wt%, beet molasses enzymatic hydrolyzate 1.5-2.5wt%, ammonium sulfate 1.0wt%, potassium dihydrogen phosphate 0.1wt%, magnesium sulfate 0.1wt%;

[0202] Feed solution: Solution A: 40-50wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate; Solution B: 40-50wt% beet molasses enzymatic hydrolyzate; Solution C: 10-12% ammonium sulfate solution; When used, the concentrations of glucose and molasses need to be kept consistent.

[0203] 3. Fermentation tank process (500L):

[0204] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 It is 0.6-0.7*25.

[0205] Step 2: The seed solution in the shake flask was inoculated into a 50L secondary seed tank medium at a ratio of 1% (V / V), and fermented at 30°C, 200rpm, aeration ratio of 0.3VVM, pH 6.8, and dissolved oxygen not less than 10%. After 16 hours of seed tank culture, the secondary maturity indicator: OD 562 It is 0.7-0.8*25; the secondary seed liquid is obtained.

[0206] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562 When the cell culture medium grows to about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (IBCL-1 strain does not need to be added); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding ammonium sulfate feed solution; (2) when the residual sugar content is lower than 2 wt%, the residual sugar content is controlled at about 2.0 wt% by adding feed solution, and the feed ratio of feed A and feed B is controlled to 4 / 1-7 / 1. At the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by continuing to add inorganic salt feed solution. The fermentation ends when the sugar is consumed.

[0207] Table 11 Main results of Example 7

[0208] As shown in Example 7, the initial addition amount of beet molasses hydrolysate is controlled at 1.5-2.5wt%, and the corresponding results are better when the flow addition A / B range is 4 / 1-7 / 1, which is slightly lower than that of Example 6. This example proves that the initial addition amount of molasses hydrolysate and the ratio of A and B during feeding are very important for the increase of acid production in L-isoleucine fermentation and the control of miscellaneous acids.

[0209] Example 8 Determining the suitability of molasses enzymatic hydrolysate process for beet molasses and sugarcane molasses

[0210] 1. The seed process is consistent with that in Example 6.

[0211] 2. The fermentation medium is as follows:

[0212] The initial culture medium of the standard fermentation tank was as follows: 10 wt % glucose, 2 wt % of a mixture of beet and sugarcane molasses enzymatic hydrolysates in a certain proportion, 1.0 wt % ammonium sulfate, 0.1 wt % potassium dihydrogen phosphate, and 0.1 wt % magnesium sulfate.

[0213] Feed solution: Solution A: 40-50wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate; Solution B: 40-50wt% beet and sugarcane molasses enzymatic hydrolyzate; Solution C: 10-12% ammonium sulfate solution; When used, the concentrations of glucose and molasses need to be kept consistent.

[0214] 3. Fermentation tank process (500L):

[0215] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 It is 0.6-0.7*25.

[0216] Step 2: The seed solution in the shake flask was inoculated into a 50L secondary seed tank medium at a ratio of 1% (V / V), and fermented at 30°C, 200rpm, aeration ratio of 0.3VVM, pH 6.8, and dissolved oxygen not less than 10%. After 16 hours of seed tank culture, the secondary maturity indicator: OD 562 It is 0.7-0.8*25; the secondary seed liquid is obtained.

[0217] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562 When the cell culture medium grows to about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (IBCL-1 strain does not need to be added); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding ammonium sulfate feed solution; (2) when the residual sugar content is lower than 2 wt%, the residual sugar content is controlled at about 2.0 wt% by adding feed solution, and the feed ratio of feed A and feed B is controlled to 5 / 1. At the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by adding inorganic salt feed solution, and the fermentation ends when the sugar is consumed.

[0218] Table 12 Main results of Example 8

[0219] As shown in Example 8, after the sugarcane molasses is hydrolyzed by sucrase and protease, it can be mixed with beet molasses hydrolysate in a certain ratio to achieve the desired effect. However, the sugarcane molasses hydrolysate alone is slightly lower than the beet molasses in Example 6.

[0220] This example demonstrates that sugarcane molasses hydrolysate can be used as an organic nitrogen source in the L-isoleucine process.

[0221] Example 9 confirmed that partially enzymatically hydrolyzed molasses can also be used for fermentation

[0222] 1. The seed process is consistent with that in Example 6.

[0223] 2. The fermentation medium is as follows:

[0224] Standard fermenter fermentation initial medium: glucose 10wt%, partially enzymatically hydrolyzed beet / sugarcane molasses 2wt%, ammonium sulfate 1.0wt%, potassium dihydrogen phosphate 0.1wt%, magnesium sulfate 0.1wt%.

[0225] Feed solution: Solution A: 40-50wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate; Solution B: 40-50wt% partially enzymatically hydrolyzed beet or sugarcane molasses; Solution C: 10-12% ammonium sulfate solution; When used, the concentrations of glucose and molasses need to be kept consistent.

[0226] 3. Fermentation tank process (500L):

[0227] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 It is 0.6-0.7*25.

[0228] Step 2: The seed solution in the shake flask was inoculated into a 50L secondary seed tank medium at a ratio of 1% (V / V), and fermented at 30°C, 200rpm, aeration ratio of 0.3VVM, pH 6.8, and dissolved oxygen not less than 10%. After 16 hours of seed tank culture, the secondary maturity indicator: OD 562 It is 0.7-0.8*25; the secondary seed liquid is obtained.

[0229] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562When the cell culture medium grows to about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (IBCL-1 strain does not need to be added); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding ammonium sulfate feed solution; (2) when the residual sugar content is lower than 2 wt%, the residual sugar content is controlled at about 2.0 wt% by adding feed solution, and the feed ratio of feed A and feed B is controlled to 5 / 1. At the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by adding inorganic salt feed solution, and the fermentation ends when the sugar is consumed.

[0230] Table 13 Main results of Example 9

[0231] As shown in Example 9, two types of molasses were hydrolyzed by sucrase and protease, respectively, and the hydrolyzates were obtained and then subjected to L-isoleucine fermentation verification. This example shows that partially hydrolyzed molasses can still be used in fermentation, but the result is lower than that of complete hydrolysis.

[0232] Example 10 determines the range of total amount of molasses used

[0233] 1. The seed process is consistent with that in Example 6.

[0234] 2. The fermentation medium is as follows:

[0235] The initial culture medium of the standard fermenter was as follows: 10 wt% glucose, 2 wt% beet molasses enzymatic hydrolysate, 1.0 wt% ammonium sulfate, 0.1 wt% potassium dihydrogen phosphate, and 0.1 wt% magnesium sulfate.

[0236] Feed solution: Solution A: 40-50wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate; Solution B: 40-50wt% beet molasses enzymatic hydrolyzate; Solution C: 10-12% ammonium sulfate solution; When used, the concentrations of glucose and molasses need to be kept consistent.

[0237] 3. Fermentation tank process (500L):

[0238] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 It is 0.6-0.7*25.

[0239] Step 2: The seed solution in the shake flask was inoculated into a 50L secondary seed tank medium at a ratio of 1% (V / V), and fermented at 30°C, 200rpm, aeration ratio of 0.3VVM, pH 6.8, and dissolved oxygen not less than 10%. After 16 hours of seed tank culture, the secondary maturity indicator: OD 562 is 0.7-0.8*25; obtain the secondary seed solution;

[0240] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562 When the cell size is about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (IBCL-1 strain does not need to be added); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding ammonium sulfate feed solution; (2) when the residual sugar content is lower than 2 wt%, the residual sugar content is controlled at about 2.0 wt% by adding feed solution, and the feed ratio of feed A and feed B is controlled to 4 / 1. At the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by adding inorganic salt feed solution, and the fermentation ends when the sugar is consumed.

[0241] Table 14 Main results of Example 10

[0242] The total molasses concentration is the total amount of molasses divided by the volume of the fermentation tank.

[0243] As shown in Example 10, the optimization verification of the total amount of molasses was carried out respectively, and the results showed that the ideal result could be achieved when the total concentration of molasses hydrolysate was 3.5-4.5wt%. This result shows that there is a certain requirement for the amount of molasses hydrolysate, which cannot be too low or too high.

[0244] Comparative Example 5: One-time addition of molasses process versus fed-batch process

[0245] 1. The seed process is consistent with that in Example 6.

[0246] 2. The fermentation medium is as follows:

[0247] The initial culture medium for standard fermentation tank fermentation was as follows: 10 wt% glucose, 2.5-4.5 wt% beet molasses enzymatic hydrolysate, 1.0 wt% ammonium sulfate, 0.1 wt% potassium dihydrogen phosphate, and 0.1 wt% magnesium sulfate.

[0248] Feed solution: Solution A: 40-50wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate; Solution C: 10-12% ammonium sulfate solution; when used, the concentrations of glucose and molasses need to be kept consistent.

[0249] 3. Fermentation tank process (500L):

[0250] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 It is 0.6-0.7*25.

[0251] Step 2: The seed solution in the shake flask was inoculated into a 50L secondary seed tank medium at a ratio of 1% (V / V), and fermented at 30°C, 200rpm, aeration ratio of 0.3VVM, pH 6.8, and dissolved oxygen not less than 10%. After 16 hours of seed tank culture, the secondary maturity indicator: OD 562 is 0.7-0.8*25; obtain the secondary seed solution;

[0252] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562 When the cell culture medium grows to about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (no addition is required for IBCL-1 strain); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding ammonium sulfate feed solution; (2) when the residual sugar content is lower than 2 wt%, the residual sugar content is controlled at about 2.0 wt% by adding feed solution, and at the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by continuing to add inorganic salt feed solution, and the fermentation ends when the sugar is consumed.

[0253] Table 15 Main results of comparative example 5

[0254] The concentration of the molasses hydrolysate added once is the amount of molasses hydrolysate added according to the initial fermentation volume.

[0255] As shown in Comparative Example 5, the acid production results of the comparative examples with the one-time addition of molasses are lower than those of the comparative examples. This is mainly because the one-time addition of too high an organic nitrogen source will cause the bacteria to grow too fast and waste more sugar for bacteria reproduction, which will lead to the acid production failing to achieve the expected effect. Therefore, the fed-addition method is more suitable for L-isoleucine fermentation.

[0256] Comparative Example 6 confirmed that the initial addition amount should not be too high or too low

[0257] 1. The seed process is consistent with that in Example 6.

[0258] 2. The fermentation medium is as follows:

[0259] The initial culture medium of the standard fermenter fermentation was as follows: 10 wt% glucose, 1 or 3 wt% beet molasses hydrolysate, 1.0 wt% ammonium sulfate, 0.1 wt% potassium dihydrogen phosphate, and 0.1 wt% magnesium sulfate.

[0260] Feed solution: Solution A: 40-50wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate; Solution B: 40-50wt% beet molasses enzymatic hydrolyzate; Solution C: 10-12% ammonium sulfate solution; When used, the concentrations of glucose and molasses need to be kept consistent.

[0261] 3. Fermentation tank process (500L):

[0262] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 It is 0.6-0.7*25.

[0263] Step 2: The seed solution in the shake flask was inoculated into a 50L secondary seed tank medium at a ratio of 1% (V / V), and fermented at 30°C, 200rpm, aeration ratio of 0.3VVM, pH 6.8, and dissolved oxygen not less than 10%. After 16 hours of seed tank culture, the secondary maturity indicator: OD 562 It is 0.7-0.8*25; the secondary seed liquid is obtained.

[0264] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562When the cell culture medium grows to about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (IBCL-1 strain does not need to be added); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding ammonium sulfate feed solution; (2) when the residual sugar content is lower than 2 wt%, the residual sugar content is controlled at about 2.0 wt% by adding feed solution, and the feed ratio of feed A and feed B is controlled to 5 / 1. At the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by adding inorganic salt feed solution, and the fermentation ends when the sugar is consumed.

[0265] Table 16 Main results of comparative example 6

[0266] As shown in Comparative Example 6, verifications were conducted with different initial addition amounts of molasses hydrolysate. This comparative example shows that the initial addition amount is particularly important during the addition of molasses hydrolysate, and as described in the example, it needs to be controlled within the range of 1.5-2.5 wt %.

[0267] Comparative Example 7 confirmed that the ratio of feed AB should not be too high or too low

[0268] 1. The seed process is consistent with that in Example 6.

[0269] 2. The fermentation medium is as follows:

[0270] Standard fermentation tank fermentation initial medium: glucose 10wt%, beet molasses enzymatic hydrolysate 1.8-2.2wt%, ammonium sulfate 1.0wt%, potassium dihydrogen phosphate 0.1wt%, magnesium sulfate 0.1wt%;

[0271] Feed solution: Solution A: 40-50wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate; Solution B: 40-50wt% beet molasses enzymatic hydrolyzate; Solution C: 10-12% ammonium sulfate solution; When used, the concentrations of glucose and molasses need to be kept consistent.

[0272] 3. Fermentation tank process (500L):

[0273] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 It is 0.6-0.7*25.

[0274] Step 2: The seed solution in the shake flask was inoculated into a 50L secondary seed tank medium at a ratio of 1% (V / V), and fermented at 30°C, 200rpm, aeration ratio of 0.3VVM, pH 6.8, and dissolved oxygen not less than 10%. After 16 hours of seed tank culture, the secondary maturity indicator: OD 562 It is 0.7-0.8*25; the secondary seed liquid is obtained.

[0275] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562 When the cell size is about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (no addition is required for IBCL-1 strain); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding ammonium sulfate feed solution; (2) when the residual sugar content is lower than 2 wt%, the residual sugar content is controlled at about 2.0 wt% by adding feed solution, and the feed ratio of feed A and feed B is controlled to 3 / 1 or 8 / 1. At the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by continuing to add inorganic salt feed solution, and the fermentation ends when the sugar is consumed.

[0276] Table 17 Main results of comparative example 7

[0277] As shown in Comparative Example 7, the ratio of feed A and feed B was verified respectively, and the results showed that the ratio of feed AB needed to be in the range of 4 / 1-7 / 1 of Example 7, and too high or too low would not meet the demand.

[0278] Comparative Example 8 confirmed that molasses must be enzymatically hydrolyzed to achieve the effect of increasing acid production

[0279] 1. The seed process is consistent with that in Example 6.

[0280] 2. The fermentation medium is as follows:

[0281] Standard fermenter fermentation initial medium: glucose 10wt%, beet / cane molasses 2wt%, ammonium sulfate 1.0wt%, potassium dihydrogen phosphate 0.1wt%, magnesium sulfate 0.1wt%.

[0282] Feed solution: Solution A: 40-50wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate; Solution B: 40-50wt% beet / sugarcane molasses; Solution C: 10-12% ammonium sulfate solution; When used, the concentrations of glucose and molasses need to be kept consistent.

[0283] 3. Fermentation tank process (500L):

[0284] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 It is 0.6-0.7*25.

[0285] Step 2: The seed solution in the shake flask was inoculated into a 50L secondary seed tank medium at a ratio of 1% (V / V), and fermented at 30°C, 200rpm, aeration ratio of 0.3VVM, pH 6.8, and dissolved oxygen not less than 10%. After 16 hours of seed tank culture, the secondary maturity indicator: OD 562 It is 0.7-0.8*25; the secondary seed liquid is obtained.

[0286] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562 When the cell culture medium grows to about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (IBCL-1 strain does not need to be added); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding ammonium sulfate feed solution; (2) when the residual sugar content is lower than 2 wt%, the residual sugar content is controlled at about 2.0 wt% by adding feed solution, and the feed ratio of feed A and feed B is controlled to 5 / 1. At the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by adding inorganic salt feed solution, and the fermentation ends when the sugar is consumed.

[0287] Table 18 Main results of comparative example 8

[0288] As shown in Comparative Example 8, the L-isoleucine fermentation verification was carried out using unhydrolyzed molasses, and the result was not ideal, and the fermentation effect of enzymatically hydrolyzed molasses was not achieved. Therefore, it can be concluded from this comparative example that enzymatic hydrolysis of molasses is more conducive to L-isoleucine fermentation.

[0289] Comparative Example 9 Confirms the advantages of the new process provided by the embodiments of this specification

[0290] 1. The composition of the seed culture medium is as follows:

[0291] Primary seed culture medium: glucose 3wt%, corn steep liquor powder 1.0wt%, ammonium sulfate 0.5wt%, KH2PO4 0.1wt%, MgSO4·7H2O 0.05wt%, calcium carbonate 1.0wt%, pH 6.7-7.2; packaged in 500 ml / 5000 ml; sterilization condition 121°C / 20min.

[0292] Secondary seed tank culture medium: glucose 5wt%, corn steep liquor dry powder 1.0wt%, ammonium sulfate 0.5wt%, KH2PO4 0.1wt%, MgSO4·7H2O 0.05wt%, pH 6.8-7.2; sterilization condition 121°C / 20min.

[0293] 2. The fermentation medium is as follows:

[0294] The initial culture medium of the standard fermenter was as follows: 12 wt % glucose, 0.8 wt % corn steep liquor powder, 0.5 wt % ammonium sulfate, 0.1 wt % potassium dihydrogen phosphate, and 0.05 wt % magnesium sulfate.

[0295] Feed sugar solution: 40wt% glucose, 0.1wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate.

[0296] Inorganic salt feed solution: ammonium sulfate 10wt%.

[0297] Fermentation waste liquid: The separated waste liquid obtained by fermentation separation is rich in a large amount of ammonium sulfate, about 10-12wt%.

[0298] 3. Fermentation tank process:

[0299] Step 1: Inoculate the one-ring L-isoleucine producing bacteria IBCIL-253, IBCIL-253k, and IBCL-1 into the primary seed culture medium of the shake flask respectively, and culture at 30°C, 90rpm, and shake for 16 hours to obtain 500mL of the shake flask seed solution. The primary maturity indicator is: OD 562 0.6-0.7*25;

[0300] Step 2: The seed solution in the shake flask was added to the medium of the secondary seed tank of 50L at a ratio of 1% (V / V), and fermented at 30℃, 90rpm, pH 6.8, and dissolved oxygen not less than 10%. After 16h of seed tank culture, the secondary maturity indicator: OD 562 It is 0.7-0.8*25; the secondary seed liquid is obtained.

[0301] Step 3: (1) 10% of the secondary seed liquid was inoculated into the initial fermentation medium of the standard fermentation tank in a 500L fermentation tank. The initial fermentation temperature was 30°C and the pH was 6.8. During the fermentation process, the ventilation and speed were continuously adjusted to control the dissolved oxygen at about 10%. The OD of the strain in the fermentation liquid was 562When the cell culture medium grows to about 40, the pH is adjusted to about 7.0, and 1 mM lactose is added at one time (IBCL-1 strain does not need to be added); when the residual sugar content of the fermentation broth drops to 6 wt%, the osmotic pressure of the fermentation broth is controlled at about 700 mosm / L by adding inorganic salt feed solution; (2) when the residual sugar content is lower than 1 wt%, the residual sugar content of the fermentation broth is controlled at about 1.0 wt% by adding feed sugar solution, and at the same time, the osmotic pressure of the fermentation broth is controlled at about 900 mosm / L by continuing to add inorganic salt feed solution, and the fermentation ends when the sugar is consumed.

[0302] Table 19 Main results of comparative example 9 strain L-Isoleucine wt% Sugar acid conversion rate % Secondary acid wt% IBCIL-253 66.4 38.38 0.10 IBCIL-253k 65.3 37.75 0.18 IBCL-1 61.7 36.29 0.21

[0303] As shown in Comparative Example 9, corn steep liquor was used for L-isoleucine fermentation. By comparing with Example 6, it can be seen that the use of enzymatic molasses for L-isoleucine fermentation has higher acid production and sugar-acid conversion rate and less impurities. In addition, molasses has fewer impurities than corn steep liquor, lighter color, and is more conducive to subsequent separation and purification.

[0304] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0305] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0306] Similarly, it should be noted that in order to simplify the description disclosed in this specification and thus help understand one or more embodiments of the invention, in the above description of the embodiments of this specification, multiple features are sometimes combined into one embodiment, figure or description thereof. However, this disclosure method does not mean that the features required by the subject matter of this specification are more than the features mentioned in the claims. In fact, the features of the embodiments are less than all the features of the single embodiment disclosed above.

[0307] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0308] Each patent, patent application, patent application publication, and other materials, such as articles, books, specifications, publications, documents, etc., cited in this specification are hereby incorporated by reference in their entirety. Except for application history documents that are inconsistent with or conflicting with the contents of this specification, documents that limit the broadest scope of the claims of this specification (currently or later attached to this specification) are also excluded. It should be noted that if the descriptions, definitions, and / or use of terms in the materials attached to this specification are inconsistent or conflicting with the contents described in this specification, the descriptions, definitions, and / or use of terms in this specification shall prevail.

[0309] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A method for producing L-leucine by fermentation, characterized in that: The method comprises: L-leucine is produced by fermentation using Corynebacterium glutamicum, wherein the Corynebacterium glutamicum includes at least one of IBBH-15, IBCLQ-257 and IBCLQ-257e; In the L-leucine fermentation process, the production of L-leucine is increased by regulating the supply of nutrients; the nutrients include molasses enzymatic hydrolysate.

2. The method according to claim 1, characterized in that The molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses for enzymatic hydrolysis; the molasses includes at least one of beet molasses and sucrose molasses.

3. The method according to claim 2, characterized in that The molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses and performing enzymatic hydrolysis for 2-6 hours at a pH of 4.5-6.0 and a temperature of 45-60°C.

4. The method according to claim 1, characterized in that: In the L-leucine fermentation process, the production of L-leucine is increased by regulating the supply of nutrients, including: During the L-leucine fermentation process, feed sugar and the molasses enzymatic hydrolyzate are added in a ratio of 6:1-10:1 to control residual sugar.

5. The method according to claim 4, characterized in that When the feed sugar and the molasses enzymatic hydrolysate are added, the concentrations of the two are kept consistent.

6. The method according to claim 4, characterized in that The method further comprises: In the L-leucine fermentation process, after the residual sugar drops to 2 wt%, feed sugar and molasses enzymatic hydrolysate are started to be added to control the residual sugar to be 1-3 wt%.

7. The method according to claim 4, characterized in that The method further comprises: During the fermentation culture feeding stage, in order to control the residual sugar at 1.5-2.5wt%, a glucose solution with a concentration of 40-50wt% and a molasses hydrolyzate with a concentration of 40-50wt% are added to supplement the fermentation medium, and the flow rate ratio of the glucose solution and the molasses hydrolyzate is controlled to be 6:1-10:1, and the proportion of the molasses hydrolyzate to the fermentation medium is 2.5-3.5wt%.

8. The method according to claim 1, characterized in that In the L-leucine fermentation process, the seed culture medium includes glucose with an initial concentration of 2.5-3.5 wt% and molasses enzymatic hydrolyzate with an initial concentration of 1-2 wt%.

9. The method according to claim 1, characterized in that: In the L-leucine fermentation process, the fermentation medium includes glucose with an initial concentration of 3.5-4.5wt% and molasses hydrolyzate with a concentration of 0.5-1.1wt%, wherein the molasses hydrolyzate is selected from at least one of beet molasses hydrolyzate and sucrose molasses hydrolyzate, and the molasses hydrolyzate is hydrolyzed by at least one of sucrase or protease.

10. The method according to claim 1, characterized in that The method further comprises: During the L-leucine fermentation process, the initial pH was 6.7, and the rotation speed and ventilation were alternately adjusted to maintain the dissolved oxygen at 5-10%; after the bacterial OD562 increased to 20, the pH increased to 6.9, after the bacterial OD562 increased to 30, the pH increased to 7.1; after the bacterial OD562 increased to 40, the pH increased to 7.

2.

11. A method for producing L-isoleucine by fermentation, characterized in that: The method comprises: L-isoleucine is fermented and produced by Corynebacterium glutamicum, wherein the Corynebacterium glutamicum comprises at least one of BCIL-253, IBCL-1 and IBCIL-253k; In the L-isoleucine fermentation process, the production of L-isoleucine is increased by regulating the supply of nutrients; the nutrients include molasses enzymatic hydrolysate.

12. The method according to claim 11, characterized in that The molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses for enzymatic hydrolysis; the molasses includes at least one of beet molasses and sucrose molasses.

13. The method according to claim 12, characterized in that The molasses enzymatic hydrolyzate is obtained by adding sucrase and / or protease to molasses and performing enzymatic hydrolysis for 2-6 hours at a pH of 4.5-6.0 and a temperature of 45-60°C.

14. The method according to claim 11, characterized in that In the L-isoleucine fermentation process, the production of L-isoleucine is increased by regulating the supply of nutrients, including: During the L-isoleucine fermentation process, feed sugar and the molasses enzymatic hydrolyzate are added in a ratio of 4:1-7:1 to control residual sugar.

15. The method according to claim 14, characterized in that When the feed sugar and the molasses enzymatic hydrolysate are added, the concentrations of the two are kept consistent.

16. The method according to claim 14, characterized in that The method further comprises: During the L-isoleucine fermentation process, When the residual sugar content drops to 5.5-6.5 wt%, inorganic salt feed solution is added to control the osmotic pressure of the fermentation liquid at 690-710 mosm / L; When the residual sugar level drops below 1.5-2.5 wt%, feed sugar and molasses enzymatic hydrolysate are added to control the residual sugar level at 1.5-2.5 wt%.

17. The method according to claim 14, characterized in that The method further comprises: When the residual sugar drops to 1.5-2.5wt%, start to feed liquid A and liquid B, control the flow rate ratio of liquid A to liquid B to be 4:1-7:1, the ratio of molasses enzymatic hydrolyzate to fermentation medium to be 3.5-4.5wt%, and continue to feed inorganic salt feed liquid to control the osmotic pressure of the fermentation liquid to be 890-910mosm / L; The liquid A comprises a glucose solution with a concentration of 40-50wt%, a potassium dihydrogen phosphate solution with a concentration of 0.1wt% and a magnesium sulfate solution with a concentration of 0.05wt%, and the liquid B comprises a molasses enzymatic hydrolyzate with a concentration of 40-50wt%.

18. The method according to claim 11, characterized in that In the L-isoleucine fermentation process, the seed culture medium includes glucose with an initial concentration of 4.5-5.5 wt% and molasses enzymatic hydrolyzate with an initial concentration of 2.5-3.5 wt%.

19. The method according to claim 11, characterized in that In the L-isoleucine fermentation process, the fermentation medium includes glucose with an initial concentration of 9.5-10.5wt% and molasses hydrolyzate with a concentration of 1.5-2.5wt%, wherein the molasses hydrolyzate is selected from at least one of beet molasses hydrolyzate and sucrose molasses hydrolyzate, and the molasses hydrolyzate is hydrolyzed by at least one of sucrase or protease.

20. The method according to claim 1, characterized in that During the fermentation of L-isoleucine, the initial fermentation temperature is 30° C. and the pH is 6.

8. During the fermentation, the ventilation and the rotation speed are continuously adjusted to control the dissolved oxygen at 8-12%. The OD562 of the bacteria in the fermentation liquid grows to 38-42, and the pH is adjusted to 6.9-7.1.

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