Method for producing L-alanine

Abstract

The invention discloses a method for producing L-alanine. The method comprises the following steps: inoculating a seed solution of a strain into a fermentation culture medium for fermentation; the fermentation culture medium is prepared from p-aminobenzoic acid, riboflavin, pyridoxine, ferrous sulfate, yeast powder, peptone, K2HPO4 and KH2PO4; supplementing a carbon source and a nitrogen source in the fermentation process; and / or pyruvic acid accounting for 0.2 wt%-0.8 wt% of the fermentation liquor is added. In some embodiments, when the concentration of the L-alanine is not increased any more, the tank is stopped, the maximum concentration of the L-alanine can reach 121 g / L, and the conversion rate can reach 98%; the yield and the purity of the L-alanine can be remarkably improved, and the method has important industrial application value.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation and bioengineering, and in particular to a method for producing L-alanine. Background Art

[0002] Amino acids, as the basic unit of proteins, the basic substance of life, have important physiological functions. With the continuous expansion of the application scope of amino acid products in the fields of medicine, food and feed additives, the demand for amino acids in various terminal application markets has also increased dramatically. Therefore, as one of the pillars of the entire fermentation industry, the amino acid manufacturing industry is receiving increasing attention.

[0003] L-Alanine is an important natural amino acid, widely used in the fields of medicine, food, feed, etc. Traditional L-amino acid manufacturing is mainly achieved through chemical synthesis or aerobic fermentation. Compared with chemical synthesis, microbial fermentation can realize the direct production of amino acids using renewable resources as raw materials, reducing the dependence on petroleum-based raw materials, and solving the problems of high pollution and high energy consumption of chemical synthesis. Aerobic fermentation has the characteristics of fast growth and high yield, but a large amount of carbon source used for cell growth in aerobic fermentation is prone to cause problems such as low sugar-acid conversion rate and high energy consumption. The traditional L-alanine production method has problems such as low yield and high cost. Some technologies have been used to transform strains used to produce L-alanine, such as fermentation production by high-yield genetically engineered bacteria. However, there is a lack of effective fermentation production methods that improve the production method itself in the art.

[0004] Therefore, developing a new, efficient and low-cost method for producing L-alanine has important practical significance. Summary of the invention

[0005] Based on this, at least a method for producing L-alanine is provided.

[0006] In a first aspect of the present application, a method for producing L-alanine is provided, the method comprising: inoculating a seed liquid of a bacterial species into a fermentation medium for fermentation; wherein the fermentation medium comprises 0.03 g / L~0.1 g / L p-aminobenzoic acid, 0.02 g / L~0.08 g / L riboflavin, 0.01 g / L~0.06 g / L pyridoxine, 0.6 g / L~1.82 g / L ferrous sulfate, 15 g / L~35 g / L yeast powder, 41 g / L~75 g / L peptone, 0.6 g / L~2.8 g / L K2HPO4 and 1.8 g / L~2.4 g / L KH2PO4; during the fermentation process, one or more of the following adjustments shown in A1)~A2) are performed: A1) supplementing a carbon source and a nitrogen source to maintain the carbon-nitrogen ratio of the fermentation liquid at (20-76):1; A2) adding 0.2 wt%~0.8 wt% of the fermentation liquid wt% of pyruvate; optionally, the pyruvate is added in the late fermentation period.

[0007] In some embodiments, adjusting A2) is performed after adjusting A1).

[0008] In some embodiments, the OD of the fermentation broth is 600 When the value reaches 30-50, adjust A1), and when the OD of the fermentation liquid 600 When the value reaches 70~90, adjust A2).

[0009] In some embodiments, the additional carbon source meets one or more of the following conditions B1) to B3):

[0010] B1) the carbon source accounts for 10 wt% to 30 wt% of the fermentation broth;

[0011] B2) the carbon source comprises at least one of glucose, starch hydrolyzed sugars and lignocellulose hydrolyzed sugars; and,

[0012] B3) The carbon source is added at a rate of 0.04 wt% to 0.06 wt% of the carbon source per minute; "wt%" is the mass percentage of the added carbon source in the fermentation liquid.

[0013] In some embodiments, the supplemental nitrogen source meets one or more of the following conditions C1) to C2):

[0014] C1) the nitrogen source accounts for 10 wt% to 30 wt% of the fermentation broth;

[0015] C2) The nitrogen source comprises at least one of ammonium sulfate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate and ammonium chloride.

[0016] The addition method of the nitrogen source, such as the flow rate, can be adjusted according to the carbon-nitrogen ratio in the fermentation liquid and the addition conditions of the carbon source.

[0017] In some embodiments, the method also includes seed culture; the seed culture includes primary seed culture, secondary seed culture and tertiary seed culture; the seed culture medium used for primary seed culture, secondary seed culture and tertiary seed culture each independently contains 2g / L~8g / L calcium chloride, 9g / L~20g / L yeast powder, 14g / L~40g / L casein peptone, 6g / L~27g / L magnesium sulfate, 3g / L~9g / L ammonium sulfate hydrochloride, 3g / L~8g / L riboflavin and 5g / L~20g / L anhydrous glucose.

[0018] In some embodiments, the seed culture meets one or more of the conditions shown in the following D1) to D4):

[0019] D1) OD of the first-stage seed solution obtained from the first-stage seed culture 600 The value is 0.8~1.4;

[0020] D2) OD of the secondary seed solution obtained from the secondary seed culture 600 The value is 4~8;

[0021] D3) OD of the third-level seed solution obtained by third-level seed culture 600 The value is 10~20;

[0022] D4) The inoculation amount of the first-stage seed solution, the second-stage seed solution and the third-stage seed solution is independently 2 wt% to 8 wt%; "wt%" refers to the mass percentage of the seed solution to the seed culture medium or fermentation medium to be inoculated.

[0023] In some embodiments, the bacterial species includes at least one of Corynebacterium glutamicum and Escherichia coli.

[0024] In some embodiments, the fermentation is anaerobic fermentation.

[0025] In some embodiments, the conditions of the anaerobic fermentation are:

[0026] The temperature is 26℃~35℃, and the pH of the fermentation liquid is 6~7.5.

[0027] This application has at least the following advantages:

[0028] By controlling the carbon-nitrogen ratio in the fermentation broth, the synthesis environment of L-alanine is optimized; by adding the precursor pyruvic acid, the synthesis efficiency of L-alanine is improved. Through an embodiment of the present application, an L-alanine yield of up to 121 g / L is achieved, and the conversion rate can reach 98%. It can be seen that the method of the present application can improve the yield and purity of L-alanine and has important industrial application value. DETAILED DESCRIPTION

[0029] In order to facilitate understanding of the present application, the present application will be described in more detail below. The preferred embodiments of the present application are given below. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0031] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other situations where "one or more" are used in other ways to mean "one or more" are also understood in the same way unless otherwise specified.

[0032] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0033] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0034] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0035] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0036] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0037] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0038] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0039] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0040] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0041] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0042] The contributions of this application include at least a. designing an optimized culture medium for seed culture and expansion culture of microorganisms; b. supplementing the bacteria with appropriate nutrients for continuous growth of the bacteria by adjusting the ingredients and methods of feed during expansion culture; c. during the growth of the bacteria, adjusting the growth environment of the bacteria through process control strategies to achieve the best growth conditions for the bacteria, such as high-speed growth of the bacteria in the early stage and sufficient synthesis of L-alanine in the later stage, so as to achieve high-concentration synthesis of L-alanine. In short, this application provides an efficient fermentation process for producing L-alanine, which at least promotes the synthesis of L-alanine by adding precursor pyruvate to the fermentation system (for example, in the later stage of fermentation) and accurately controlling the carbon-nitrogen ratio, thereby increasing the yield of L-alanine.

[0043] In a first aspect of the present application, a method for producing L-alanine is provided, the method comprising: inoculating a seed liquid of a bacterial species into a fermentation medium for fermentation; the fermentation medium comprises p-aminobenzoic acid, riboflavin, pyridoxine, ferrous sulfate, yeast powder, peptone, K2HPO4 and KH2PO4; supplementing a carbon source and a nitrogen source during the fermentation process; and / or, adding pyruvic acid accounting for 0.2 wt% to 0.8 wt% of the fermentation liquid.

[0044] In some embodiments, the method comprises:

[0045] The seed liquid of the strain is inoculated into a fermentation medium for fermentation; wherein the fermentation medium contains 0.03 g / L to 0.1 g / L p-aminobenzoic acid, 0.02 g / L to 0.08 g / L riboflavin, 0.01 g / L to 0.06 g / L pyridoxine, 0.6 g / L to 1.82 g / L ferrous sulfate, 15 g / L to 35 g / L yeast powder, 41 g / L to 75 g / L peptone, 0.6 g / L to 2.8 g / L K2HPO4 and 1.8 g / L to 2.4 g / L KH2PO4;

[0046] During the fermentation process, one or more of the following adjustments are made;

[0047] Add carbon source and nitrogen source to maintain the carbon-nitrogen ratio of the fermentation liquid at (20-76):1;

[0048] Add 0.2 wt% to 0.8 wt% pyruvic acid to the fermentation broth.

[0049] In some embodiments, the concentration of p-aminobenzoic acid in the fermentation medium is 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, or a range or value between any two values.

[0050] In some embodiments, the concentration of riboflavin in the fermentation medium is 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, or a range or value between any two values.

[0051] In some embodiments, the concentration of pyridoxine in the fermentation medium is 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, or a range or value between any two values.

[0052] In some embodiments, the concentration of ferrous sulfate in the fermentation medium is 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.82 g / L, or a range or value between any two values.

[0053] In some embodiments, the concentration of yeast powder in the fermentation medium is 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, or a range or value between any two values.

[0054] In some embodiments, the concentration of peptone in the fermentation medium is 41 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, or a range or value between any two values.

[0055] In some embodiments, the concentration of K2HPO4 in the fermentation medium is 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 2 g / L, 2.2 g / L, 2.4 g / L, 2.6 g / L, 2.8 g / L, or a range or value between any two values.

[0056] In some embodiments, the concentration of KH2PO4 in the fermentation medium is 1.8 g / L, 1.9 g / L, 2 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, or a range or value between any two values.

[0057] In some embodiments, the formula of the fermentation medium is 0.05 g / L p-aminobenzoic acid, 0.04 g / L riboflavin, 0.04 g / L pyridoxine, 1.2 g / L ferrous sulfate, 20 g / L yeast powder, 50 g / L peptone, 0.8 g / L K2HPO4, and 2 g / L KH2PO4.

[0058] In some embodiments, the formula of the fermentation medium is 0.03 g / L p-aminobenzoic acid, 0.02 g / L riboflavin, 0.01 g / L pyridoxine, 0.6 g / L ferrous sulfate, 15 g / L yeast powder, 41 g / L peptone, 0.6 g / L K2HPO4, and 1.8 g / L KH2PO4.

[0059] In some embodiments, the formula of the fermentation medium is 0.1 g / L p-aminobenzoic acid, 0.08 g / L riboflavin, 0.06 g / L pyridoxine, 1.82 g / L ferrous sulfate, 35 g / L yeast powder, 75 g / L peptone, 2.8 g / L K2HPO4, and 2.4 g / L KH2PO4.

[0060] Unless otherwise specified, the terms "fermentation broth", "fermentation effluent", "bioreactor flow" and the like in this application may be used interchangeably, meaning a liquid phase retaining microorganisms, feed materials (such as fermentation medium, etc.) and fermentation products, which may be contained in one or more bioreactors (such as fermenter, etc.). It should be understood that the fermentation broth is not limited to a certain time point, such as only existing at the end of fermentation, but exists continuously. After the start of fermentation, at any time point during the fermentation process, the above-mentioned liquid phase in the bioreactor can be referred to as fermentation broth. Exemplarily, the fermentation broth in the mass percentage of the carbon source in the fermentation broth mentioned when the carbon source is added is the fermentation broth at the moment of adding the carbon source.

[0061] In some embodiments, the pyruvate is added in the late fermentation period. Without wishing to be bound by any theory, it has been found that late addition of the precursor pyruvate can more efficiently synthesize L-alanine.

[0062] In some embodiments, before adding pyruvic acid, a carbon source and a nitrogen source are added to the fermentation broth. Without wishing to be bound by any theory, the inventors unexpectedly discovered that the addition of a carbon source and a nitrogen source and the addition of pyruvic acid in the culture process can at least achieve the effect of high-speed growth of the bacteria in the early stage and sufficient synthesis of L-alanine in the later stage, thereby achieving high-quality and / or high-concentration synthesis of L-alanine.

[0063] In some embodiments, the OD of the fermentation broth is 600 When the OD value of the fermentation liquid reaches 30 to 50, a carbon source and a nitrogen source are added. In some embodiments, when the OD value of the fermentation liquid is added, 600 The value reaches 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or a range or value between any two values.

[0064] In some embodiments, the OD of the fermentation broth is 600 Pyruvate is added when the OD value of the fermentation broth reaches 70 to 90. In some embodiments, 600 The value reaches 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or a range or value between any two values.

[0065] Unless otherwise specified, the term "OD" in this application is the abbreviation of optical density. The energy difference before and after light passes through the object is the energy absorbed by the object. At a specific wavelength, there is a quantitative relationship between the concentration of the same object and the absorbed energy. 600 " is the optical density value measured when the wavelength is set to 600nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate bacterial cell density. Detecting OD values ​​to indicate bacterial concentration is a common method used in this field. The OD value and bacterial concentration are linearly related.

[0066] According to common sense in the art, the amount of carbon source added can be determined according to the fermentation conditions. In some embodiments, the amount of the added carbon source accounts for 10 wt% to 30 wt% of the fermentation liquid, for example, 10 wt%, 12 wt%, 14 wt%, 16 wt%, 18 wt%, 20 wt%, 22 wt%, 24 wt%, 26 wt%, 28 wt%, 30 wt%, or a range or value between any two values.

[0067] The carbon source in the present application may be a conventional carbon source in the fermentation field, for example, glucose, starch hydrolyzed sugar or lignocellulose hydrolyzed sugar.

[0068] In some embodiments, the carbon source comprises one or more of glucose, starch hydrolyzed sugars, and lignocellulose hydrolyzed sugars.

[0069] In some embodiments, the carbon source is added at a rate of 0.04 wt% to 0.06 wt% of the carbon source per minute, for example 0.05 wt%; "wt%" is the mass percentage of the added carbon source in the fermentation liquid.

[0070] As mentioned above, the amount of carbon source and nitrogen source added can be as follows: maintaining the carbon-nitrogen ratio of the fermentation broth at (20-76):1.

[0071] Accordingly, the method of supplementing the nitrogen source, such as the amount of supplementation and the rate of supplementation, etc., can be understood.

[0072] In some embodiments, the added nitrogen source accounts for 10 wt% to 30 wt% of the fermentation broth, for example, it can be 10wt%, 12wt%, 14wt%, 16wt%, 18wt%, 20wt%, 22wt%, 24wt%, 26wt%, 28wt%, 30wt%, or a range or value between any two values.

[0073] The nitrogen source in the present application may be a conventional nitrogen source in the art, for example, may include at least one of ammonium sulfate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate and ammonium chloride.

[0074] The flow acceleration of the nitrogen source can be adjusted according to the carbon-nitrogen ratio in the fermentation liquid and the flow addition of the carbon source.

[0075] In some embodiments, the method further comprises seed culture; the seed culture comprises primary seed culture, secondary seed culture and tertiary seed culture.

[0076] In some embodiments, the seed culture medium used for primary seed culture, secondary seed culture and tertiary seed culture each independently contains 2g / L~8g / L calcium chloride, 9g / L~20g / L yeast powder, 14g / L~40g / L casein peptone, 6g / L~27g / L magnesium sulfate, 3g / L~9g / L ammonium sulfate hydrochloride, 3g / L~8g / L riboflavin and 5g / L~20g / L anhydrous glucose.

[0077] In the seed culture medium, the concentration of calcium chloride can optionally be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, or a range or value between any two values.

[0078] In the seed culture medium, the concentration of yeast powder can be optionally 9g / L, 10g / L, 11g / L, 12g / L, 13g / L, 14g / L, 15g / L, 16g / L, 17g / L, 18g / L, 19g / L, 20g / L, or a range or value between any two values.

[0079] In the seed culture medium, the concentration of casein peptone can be optionally 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, or a range or value between any two values.

[0080] In the seed culture medium, the concentration of magnesium sulfate can be optionally 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, or a range or value between any two values.

[0081] In the seed culture medium, the concentration of ammonium sulfate hydrochloride can optionally be 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, or a range or value between any two values.

[0082] In the seed culture medium, the concentration of riboflavin can optionally be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, or a range or value between any two values.

[0083] In the seed culture medium, the concentration of anhydrous glucose can be optionally 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, or a range or value between any two values.

[0084] In one embodiment, the formula of the seed culture medium is: 8 g / L calcium chloride, 9 g / L yeast powder, 14 g / L casein peptone, 6 g / L magnesium sulfate, 3 g / L ammonium sulfate hydrochloride, 3 g / L riboflavin, and 5 g / L anhydrous glucose.

[0085] In another embodiment, the formula of the seed culture medium is: 5 g / L calcium chloride, 12 g / L yeast powder, 22 g / L casein peptone, 15 g / L magnesium sulfate, 5 g / L ammonium sulfate hydrochloride, 5 g / L riboflavin, and 8 g / L anhydrous glucose.

[0086] In another embodiment, the formula of the seed culture medium is: 8 g / L calcium chloride, 20 g / L yeast powder, 40 g / L casein peptone, 27 g / L magnesium sulfate, 9 g / L ammonium sulfate hydrochloride, 8 g / L riboflavin, and 20 g / L anhydrous glucose.

[0087] In some embodiments, the OD of the primary seed solution obtained by primary seed culture is 600 The value is 0.8-1.4; for example, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or a range or value between any two values.

[0088] In some embodiments, the OD of the secondary seed solution obtained by the secondary seed culture is 600 The value is 4 to 8; for example, 4, 5, 6, 7, 8, or a range or value between any two values.

[0089] In some embodiments, the OD of the third seed solution obtained by the third seed culture is 600 The value is 10-20; for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range or value between any two values.

[0090] Optionally in the present application, the inoculation amount of the first-level seed liquid, the second-level seed liquid and the third-level seed liquid is independently 2 wt%~8 wt%; "wt%" is the mass percentage of the seed liquid to the seed culture medium or fermentation medium to be inoculated.

[0091] The strains that can be used in the present application include, but are not limited to, Corynebacterium glutamicum, Escherichia coli and other strains that can be used to produce L-alanine.

[0092] In some embodiments, the strain used in the method is Escherichia coli purchased from the American Type Culture Collection (ATCC9637).

[0093] In some embodiments, the strain used in the method is Corynebacterium glutamicum purchased from the American Type Culture Collection (ATCC13032).

[0094] In some embodiments, two strains as described above are used simultaneously in the method.

[0095] The fermentation in the present application may be anaerobic fermentation.

[0096] In some embodiments, the conditions of the anaerobic fermentation are:

[0097] The temperature is 26℃~35℃, and the pH of the fermentation liquid is 6~7.5.

[0098] Some examples are provided below.

[0099] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0100] L-alanine content detection method: Waters e2695 high performance liquid chromatography, Yilite C18 chromatographic column (4.6mm×250mm), mobile phase methanol-sodium hydrogen phosphate mixture (V (0.05mol / L sodium hydrogen phosphate with pH 6.5): V (methanol) = 1:9), flow rate 0.8mL / min, column temperature 30℃, injection volume 5μL, detection wavelength 215nm.

[0101] Bacterial OD detection method: dilute the sample to a range of OD600 between 0.2-0.8, and detect it using a spectrophotometer at a wavelength of 600 nm.

[0102] Conversion rate (%) calculation: the ratio of the mass of L-alanine obtained by fermentation to the mass of glucose consumed.

[0103] Escherichia coli was purchased from the American Type Culture Collection (ATCC9637), and Corynebacterium glutamicum was purchased from the American Type Culture Collection (ATCC13032).

[0104] Unless otherwise specified below, the "%" of inoculation amount refers to the mass percentage of seed liquid to the seed culture medium or fermentation medium to be inoculated.

[0105] Example 1

[0106] 1. Select Corynebacterium glutamicum as the strain, prepare the first-level seed culture medium, the second-level seed culture medium, and the third-level seed culture medium, with the formula: calcium chloride 8g / L, yeast powder 9g / L, casein peptone 14g / L, magnesium sulfate 6g / L, ammonium sulfate hydrochloride 3g / L, riboflavin 3g / L, anhydrous glucose 5g / L.

[0107] 2. Sterilize the culture medium at 121℃ for 15 minutes.

[0108] 3. Primary seed culture: The strain stored at low temperature is activated and cultured. The primary seeds are grown for 12 hours and the OD grows to 0.8.

[0109] 4. Secondary seed expansion: According to the inoculation amount of 2%, the activated strain seeds in step (3) were inoculated into the secondary seed culture medium for expansion culture. The secondary seeds were grown for 10 hours and the OD grew to 4.

[0110] 5. Tertiary seed culture: At a 3% inoculation rate, the activated strain seeds in step (4) were inoculated into the tertiary seed culture medium for expansion culture. The tertiary seeds were grown for 8 h and the OD grew to 10.

[0111] 6. Prepare fermentation medium with the following formula: p-aminobenzoic acid 0.03 g / L, riboflavin 0.02 g / L, pyridoxine 0.01 g / L, ferrous sulfate 0.6 g / L, yeast powder 15 g / L, peptone 41 g / L, K2HPO4 0.6 g / L, KH2PO4 1.8 g / L.

[0112] 7. The seed liquid cultured in step 5 was inoculated into the fermentation medium at 2 wt %, and the temperature was controlled at 26° C. and pH 6. The whole process was anaerobic fermentation to carry out L-alanine fermentation.

[0113] 8. When the OD of the fermentation bacteria reaches 40, add a certain amount of carbon source and nitrogen source to the bacteria.

[0114] 9. The carbon source added was 10% glucose by mass and the nitrogen source was 10% ammonium sulfate by mass. The carbon source was added at a rate of 0.04% of the total mass of the fermentation system per minute. The most suitable carbon-nitrogen ratio was 20:1 using the carbon and nitrogen sources, and the nitrogen source flow acceleration was adjusted according to the carbon-nitrogen ratio.

[0115] 10. After the OD of the fermentation strain reaches 80, add 0.2% by mass of pyruvic acid as a precursor to promote the synthesis of L-alanine.

[0116] 11. During the fermentation process, the L-alanine concentration should be tested every hour. When the L-alanine concentration does not increase for three consecutive hours, the fermentation can be stopped.

[0117] 12. After testing and calculation, the maximum concentration of L-alanine is 121g / L and the conversion rate reaches 98%.

[0118] Example 2

[0119] 1. Select Corynebacterium glutamicum as the strain, prepare the first-level seed culture medium, the second-level seed culture medium, and the third-level seed culture medium, the formula is: calcium chloride 5g / L, yeast powder 12g / L, casein peptone 22g / L, magnesium sulfate 15g / L, ammonium sulfate hydrochloride 5g / L, riboflavin 5g / L, anhydrous glucose 8g / L.

[0120] 2. Sterilize the culture medium at 121℃ for 15 minutes.

[0121] 3. Primary seed culture: The strain stored at low temperature is activated and cultured. The primary seeds are grown for 14 hours and the OD grows to 1.2.

[0122] 4. Secondary seed expansion: According to the inoculation amount of 2%, the activated strain seeds in step (3) were inoculated into the secondary seed culture medium for expansion. The secondary seeds were grown for 12 hours and the OD grew to 6.

[0123] 5. Tertiary seed culture: At a 3% inoculation rate, inoculate the activated strain seeds in step (4) into the tertiary seed culture medium for expansion culture. Grow the tertiary seeds for 10 h until the OD reaches 15.

[0124] 6. Prepare fermentation medium with the following formula: p-aminobenzoic acid 0.05 g / L, riboflavin 0.04 g / L, pyridoxine 0.04 g / L, ferrous sulfate 1.2 g / L, yeast powder 20 g / L, peptone 50 g / L, K2HPO4 0.8 g / L, KH2PO4 2 g / L.

[0125] 7. The seed liquid cultured in step 5 was inoculated into the fermentation medium at 5 wt %, and the temperature was controlled at 30°C and pH 6.5. The whole process was anaerobic fermentation to carry out L-alanine fermentation.

[0126] 8. When the OD of the fermentation bacteria reaches 40, add a certain amount of carbon source and nitrogen source to the bacteria.

[0127] 9. The carbon source added was 10% starch hydrolyzate by mass, and the nitrogen source was 10% diammonium phosphate by mass. The carbon source was added at a rate of 0.05% of the total mass of the fermentation system per minute. The most suitable carbon-nitrogen ratio was 40:1 using the carbon and nitrogen sources, and the nitrogen source flow acceleration was adjusted according to the carbon-nitrogen ratio.

[0128] 10. After the OD of the fermentation strain reaches 80, add 0.4% by mass of pyruvic acid as a precursor to promote the synthesis of L-alanine.

[0129] 11. During the fermentation process, the L-alanine concentration should be tested every hour. When the L-alanine concentration does not increase for three consecutive hours, the fermentation can be stopped.

[0130] 12. After testing and calculation, the maximum concentration of L-alanine is 118g / L and the conversion rate reaches 97%.

[0131] Example 3

[0132] 1. Select Corynebacterium glutamicum as the strain, prepare the first-level seed culture medium, the second-level seed culture medium, and the third-level seed culture medium, the formula is: calcium chloride 8g / L, yeast powder 20g / L, casein peptone 40g / L, magnesium sulfate 27g / L, ammonium sulfate hydrochloride 9g / L, riboflavin 8g / L, anhydrous glucose 20g / L.

[0133] 2. Sterilize the culture medium at 121℃ for 15 minutes.

[0134] 3. Primary seed culture: The strain stored at low temperature is activated and cultured. The primary seeds are grown for 12 hours and the OD grows to 1.4.

[0135] 4. Secondary seed expansion: According to the inoculation amount of 2%, the activated strain seeds in step (3) were inoculated into the secondary seed culture medium for expansion. The secondary seeds were grown for 14 hours and the OD grew to 8.

[0136] 5. Tertiary seed culture: At a 3% inoculation rate, the activated strain seeds in step (4) were inoculated into the tertiary seed culture medium for expansion culture. The tertiary seeds were grown for 14 h and the OD grew to 18.

[0137] 6. Prepare fermentation medium with the following formula: p-aminobenzoic acid 0.1 g / L, riboflavin 0.08 g / L, pyridoxine 0.06 g / L, ferrous sulfate 1.82 g / L, yeast powder 35 g / L, peptone 75 g / L, K2HPO4 2.8 g / L, KH2PO4 2.4 g / L.

[0138] 7. The seed liquid cultured in step 5 was inoculated into the fermentation medium at 8 wt %, and the temperature was controlled at 35°C and pH 7.5. The whole process was anaerobic fermentation to carry out L-alanine fermentation.

[0139] 8. When the OD of the fermentation bacteria reaches 40, add a certain amount of carbon source and nitrogen source to the bacteria.

[0140] 9. The carbon source added was 10% by weight of lignocellulose hydrolyzed sugar and the nitrogen source was 10% by weight of ammonium chloride. The carbon source was added at a rate of 0.06% of the total mass of the fermentation system per minute. The most suitable carbon-nitrogen ratio was 76:1 using the carbon source and nitrogen source, and the nitrogen source flow acceleration was adjusted according to the carbon-nitrogen ratio.

[0141] 10. After the OD of the fermentation strain reaches 80, add 0.8% by mass of pyruvic acid as a precursor to promote the synthesis of L-alanine.

[0142] 11. During the fermentation process, the L-alanine concentration should be tested every hour. When the L-alanine concentration does not increase for three consecutive hours, the fermentation can be stopped.

[0143] 12. After testing and calculation, the maximum concentration of L-alanine is 115g / L and the conversion rate reaches 96%.

[0144] Example 4

[0145] Fermentation was performed by referring to the steps basically the same as those in Example 1, except that in step 1, Escherichia coli was selected as the strain, and a primary seed culture medium, a secondary seed culture medium, and a tertiary seed culture medium were prepared with the following formula: 5 g / L calcium chloride, 12 g / L yeast powder, 22 g / L casein peptone, 15 g / L magnesium sulfate, 5 g / L ammonium sulfate hydrochloride, 5 g / L riboflavin, and 8 g / L anhydrous glucose.

[0146] Step 12. After testing and calculation, the maximum concentration of L-alanine is 102 g / L and the conversion rate reaches 92%.

[0147] Comparative Example 1

[0148] Fermentation was performed with reference to the same steps as in Example 1, except that in steps 8-9, the carbon source was 10% by mass glucose and the nitrogen source was 10% by mass ammonium sulfate. The carbon source was fed at a rate of 0.04% of the total mass of the fermentation system per minute, and the most suitable carbon-nitrogen ratio was 5:1 using the carbon source and the nitrogen source, and the nitrogen source flow acceleration was adjusted according to the carbon-nitrogen ratio.

[0149] Step 12. After testing and calculation, the maximum concentration of L-alanine is only 26g / L, and the conversion rate is only 18%

[0150] Comparative Example 2

[0151] The fermentation was carried out according to the same steps as in Example 1, except that in step 10, pyruvic acid was not added as a precursor, but water of the same mass was added.

[0152] Step 12. After testing and calculation, the maximum concentration of L-alanine is only 18g / L, and the conversion rate is only 12%.

[0153] Comparative Example 3

[0154] Fermentation was performed by referring to the same steps as in Example 1, except that in step 10, an equal mass of maleic acid was added as a precursor.

[0155] Step 12. After testing and calculation, the maximum concentration of L-alanine is only 22g / L, and the conversion rate is only 17%.

[0156] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0157] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for producing L-alanine, characterized in that The method comprises: Inoculating the seed liquid of the strain into a fermentation medium for fermentation; wherein the fermentation medium comprises 0.03 g / L to 0.1 g / L p-aminobenzoic acid, 0.02 g / L to 0.08 g / L riboflavin, 0.01 g / L to 0.06 g / L pyridoxine, 0.6 g / L to 1.82 g / L ferrous sulfate, 15 g / L to 35 g / L yeast powder, 41 g / L to 75 g / L peptone, 0.6 g / L to 2.8 g / L K2HPO4 and 1.8 g / L to 2.4 g / L KH2PO4; During the fermentation process, one or more of the following adjustments A1) to A2) are performed; A1) Add carbon source and nitrogen source to maintain the carbon-nitrogen ratio of the fermentation liquid at (20-76):1; A2) Add 0.2 wt%~0.8 wt% pyruvate to the fermentation broth in the late fermentation period.

2. The method according to claim 1, characterized in that Adjustment A2) is performed after adjustment A1).

3. The method according to claim 1 or 2, characterized in that OD of fermentation broth 600 When the value reaches 30-50, adjust A1), and when the OD of the fermentation liquid 600 When the value reaches 70~90, adjust A2).

4. The method according to any one of claims 1 to 3, characterized in that The additional carbon source meets one or more of the following conditions B1) to B3): B1) the carbon source accounts for 10 wt% to 30 wt% of the fermentation broth; B2) the carbon source comprises at least one of glucose, starch hydrolyzed sugars and lignocellulose hydrolyzed sugars; and, B3) The carbon source is added at a rate of 0.04 wt% to 0.06 wt% of the carbon source per minute; "wt%" is the mass percentage of the added carbon source in the fermentation liquid.

5. The method according to any one of claims 1 to 4, characterized in that The additional nitrogen source meets one or more of the following conditions C1) to C2): C1) the nitrogen source accounts for 10 wt% to 30 wt% of the fermentation broth; C2) The nitrogen source comprises at least one of ammonium sulfate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate and ammonium chloride.

6. The method according to any one of claims 1 to 5, characterized in that The method further comprises seed cultivation; the seed cultivation comprises primary seed cultivation, secondary seed cultivation and tertiary seed cultivation; The seed culture medium used for primary seed culture, secondary seed culture and tertiary seed culture each independently contains 2g / L~8g / L calcium chloride, 9g / L~20g / L yeast powder, 14g / L~40g / L casein peptone, 6g / L~27g / L magnesium sulfate, 3g / L~9g / L ammonium sulfate hydrochloride, 3g / L~8g / L riboflavin and 5g / L~20g / L anhydrous glucose.

7. The method according to claim 6, characterized in that The seed culture meets one or more of the following conditions D1) to D4): D1) OD of the first-stage seed solution obtained from the first-stage seed culture 600 The value is 0.8~1.4; D2) OD of the secondary seed solution obtained from the secondary seed culture 600 The value is 4~8; D3) OD of the third-level seed solution obtained by third-level seed culture 600 The value is 10~20; D4) The inoculation amount of the first-level seed solution, the second-level seed solution and the third-level seed solution is independently 2 wt% to 8 wt%; "wt%" is the mass percentage of the seed solution to the seed culture medium or fermentation medium to be inoculated.

8. The method according to any one of claims 1 to 7, characterized in that The bacterial species includes at least one of Corynebacterium glutamicum and Escherichia coli.

9. The method according to any one of claims 1 to 8, characterized in that The fermentation is anaerobic fermentation.

10. The method according to claim 9, characterized in that The conditions of the anaerobic fermentation are: The temperature is 26℃~35℃, and the pH of the fermentation liquid is 6~7.5.