A method for fermentative production of l-selenomethionine
The fermentation method using Corynebacterium glutamicum OMK-88 to prepare L-selenomethionine solves the problems of low production efficiency and insufficient safety in existing technologies, and achieves efficient, stable and safe production of L-selenomethionine.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-20
- Publication Date
- 2026-05-15
AI Technical Summary
Existing technologies are insufficient for the efficient production of L-selenomethionine, which has low toxicity and a stable structure, and other selenium-based food products have safety and stability issues.
L-homoserine and sodium methylselenool were fermented in a fermentation medium using Corynebacterium glutamicum OMK-88. Highly efficient transforming strains were obtained through multiple rounds of chemical mutagenesis screening to prepare L-selenomethionine.
It has achieved efficient production of L-selenomethionine with a concentration of over 70 g/L, exhibiting high bioavailability and food safety, avoiding the toxicity risks of inorganic selenium, and ensuring structural stability and consistent content.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of L-selenomethionine production technology, specifically relating to a strain of Corynebacterium glutamicum and a method for producing L-selenomethionine by fermentation using this strain. Background Technology
[0002] The molecular formula of selenomethionine is C5H. 11 NO2Se is a white or off-white crystalline powder with an optical rotation of +17.0° to +19.5°. In nature, selenium exists primarily in the organic form of plant-derived organoselenium compounds. Compared to inorganic selenium, L-selenomethionine has advantages such as lower toxicity and higher bioavailability (Environment Research, 2005, 98(1): 46-54). Currently, many European and American countries strongly advocate the use of organic selenium; for example, Sweden requires the use of organic selenium in piglet feed, and Japan has prohibited the use of inorganic selenium in feed. L-selenomethionine is stable and has significant efficacy (Current Protein and Peptide Science, 2014, 15(6): 598-607), possessing high practical value and broad application prospects.
[0003] The World Health Organization has recognized selenium as an essential trace element for both animals and humans. Currently, selenium-rich foods mainly include selenium-enriched yeast, selenized carrageenan, selenoproteins, selenium-enriched edible fungi powder, L-selenomethionine, and L-selenomethylselenocysteine. L-selenomethionine contains approximately 40% selenium in its structure and is the main naturally occurring form of selenium. Summary of the Invention Invention Overview
[0005] To address the aforementioned problems, the first aspect of this invention provides a strain of Corynebacterium glutamicum OMK-88, accession number: CCTCC NO: M 20222075, accession date: December 23, 2022. It has been biologically deposited by the China Center for Type Culture Collection (CCTCC, Wuhan University).
[0006] In a second aspect, the present invention proposes the application of Corynebacterium glutamicum OMK-88, as described in the first aspect, in the preparation of L-selenomethionine, comprising fermenting the Corynebacterium glutamicum in a fermentation medium containing a reaction substrate to obtain L-selenomethionine.
[0007] In some embodiments, in the application of the second aspect, Corynebacterium glutamicum OMK-88, in the preparation of L-selenomethionine, the fermentation substrate is any one or more of L-homoserine, sodium methylselenool, or methylselenool, and more specifically, the L-homoserine concentration is 50-70 g / L, and the sodium methylselenool or methylselenool concentration is 50-70 g / L.
[0008] In some embodiments, in the second aspect of the Corynebacterium glutamicum OMK-88 application, the fermentation medium includes one or more of the following: a carbon source, a nitrogen source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, ammonium sulfate, and trace elements.
[0009] The carbon source includes glucose.
[0010] The nitrogen source includes one or more of yeast extract and peptone.
[0011] In some embodiments, the second aspect of the Corynebacterium glutamicum OMK-88 application includes the steps of:
[0012] S1: Corynebacterium glutamicum cultured on agar plates.
[0013] S2: The mycelium from step S1 is expanded through primary or multiple stages of culture.
[0014] S3: Step S2 expands the culture of seed liquid for biotransformation.
[0015] In some embodiments, in the application of the second aspect, Corynebacterium glutamicum OMK-88, the volume ratio of the seed culture after one or more stages of cultivation to the biotransformation fermentation broth is 0.9:10-0.9:20, preferably 0.9:15, and particularly preferably 0.9:13, 0.9:12, 0.9:11, or 0.9:10.
[0016] In some embodiments, in the application of the second aspect, Corynebacterium glutamicum OMK-88, the pH of the plate culture medium is 7.4-7.6, preferably 7.4, 7.5, or 7.6; the rotation speed of the scaling-up step is 300-400 rpm, preferably 300, 350, or 400 rpm; the culture temperature is 36.5-37.5°C, preferably 36.5°C, 37°C, or 37.5°C; and the culture time is 13-16 h, preferably 14-16 h, and particularly preferably 15 h. The bioconversion step is carried out at a fermentation temperature of 37°C, a stirring speed of 400-500 rpm, and an aeration ratio of 1:0.1-1:0.4, preferably 1:0.1, 1:0.2, 1:0.3, or 1:0.4. The bioconversion time is 12-20 h, preferably 12 h-16 h, and particularly preferably 12 h or 16 h. The reaction substrates L-homoserine, sodium methylselenool, or methylselenool are added either all at once or in batches, with the latter referring to each addition.
[0017] In some embodiments, in the application of the second aspect, Corynebacterium glutamicum OMK-88, the fermentation medium comprises, based on the total volume of the fermentation medium, 2-5 g / L, preferably 3-5 g / L, particularly preferably 3-4 g / L; peptone 1-10 g / L, preferably 2 g / L-9 g / L, particularly preferably 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, or 9 g / L; and agar 10-30 g / L, preferably 15-2 g / L. 5 g / L, especially preferably 15 g / L, 20 g / L, or 25 g / L; potassium dihydrogen phosphate 1.0-2.5 g / L, preferably 1.2 g / L-2.0 g / L, especially preferably 1.2 g / L, 1.5 g / L, or 2.0 g / L; sodium chloride 0.5 g / L-10 g / L, preferably 0.8-5 g / L, especially preferably 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L; yeast powder 5 g / L. -20 g / L, preferably 10 g / L-15 g / L, especially preferably 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L; magnesium sulfate 0.1-0.3 g / L, preferably 0.2-0.3 g / L, especially preferably 0.2 g / L; ammonium sulfate 3-6 g / L, preferably 4-5 g / L, especially preferably 5 g / L; dipotassium hydrogen phosphate 2.0-2.6 g / L, preferably... The concentrations are selected as follows: 2.2-2.4 g / L, particularly preferably 2.4 g / L; manganese sulfate 0.0001-0.0003 g / L, preferably 0.0001 g / L; glucose 10-40 g / L, preferably 20-30 g / L, particularly preferably 20 g / L, 25 g / L, or 30 g / L; pH between 6.8 and 7.6, preferably 7. The pH can be adjusted using buffer salt solution, caustic soda, or water solvent.
[0018] The third aspect of the present invention also provides an organic selenium obtained by the preparation method of the second aspect.
[0019] All reagents used in this invention were purchased from the open and legal market and were not further purified.
[0020] Compared with the prior art, the present invention has the following main advantages:
[0021] (1) L-selenomethionine exhibits the advantage of low toxicity of organic selenium compared to inorganic selenium.
[0022] (2) L-Selenomethionine has advantages such as a well-defined structure, stable content, and a clear metabolic mechanism in the human body. The selenium content in products such as selenium-enriched yeast, selenized carrageenan, selenoproteins, and selenium-enriched edible fungi powder varies from batch to batch and is not fixed. Because the effective supplemental dose of selenium is close to the toxic dose, there are certain risks to the safety of consuming other selenium-based foods.
[0023] (3) L-selenomethionine and L-seleno-methylselenocysteine are the only two selenium-containing amino acids in nature and are known as the third generation of selenium supplements. Compared with other forms of selenium-rich foods, L-selenomethionine has higher food safety if used as the main source of selenium in animal food.
[0024] (4) L-Selenomethionine is mainly obtained by chemical synthesis. This invention develops a biological fermentation method, which has a high market acceptance.
[0025] (5) The concentration of L-selenomethionine in the fermentation broth of this invention is above 70 g / L, which has high efficiency.
[0026] Terminology definition:
[0027] Throughout this document, all figures disclosed herein, whether or not the words “approximately” or “about” are used, are approximate values. Based on the disclosed figures, each figure may vary by less than ±10% or by a difference that is considered reasonable by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.
[0028] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.
[0029] The term "wt%" refers to a percentage by mass.
[0030] The term "%vol" indicates a volume percentage.
[0031] The term "aeration ratio" refers to the volume of gas introduced per unit volume of fermentation broth per unit time (usually per minute). For example, a "1:0.4 aeration ratio" means that 1 L of fermentation broth is aerated with 0.4 L of gas per minute. In scale-up culture, the term "inoculum volume" refers to the percentage of the volume of the medium containing *Corynebacterium glutamicum* to the total volume of the seed medium and the medium containing *Corynebacterium glutamicum* after inoculation. In fermentation culture, the term "inoculum volume" refers to the percentage of the volume of the seed culture to the total volume of the seed culture and the fermentation medium after inoculation with the seed culture.
[0032] The term "OD" 600 "Refers to the optical density detected at a wavelength of 600nm"
[0033] To enable those skilled in the art to better understand the technical solutions described herein, some non-limiting embodiments are further disclosed below for further detailed explanation. All reagents used herein are commercially available or can be prepared by the methods described in this application. The terms "rpm" indicate rotational speed (revolutions per minute); "°C" indicates temperature (degrees Celsius); "L" indicates volume (liters); "mL" indicates volume (milliliters); "pH" indicates acidity or alkalinity; "g" indicates mass (grams); and "MPa" indicates pressure (megapascals).
[0034] Unless otherwise stated, the culture medium formulations used in the following examples are as follows: Attached Figure Description
[0035] Figure 1 Results of biotransformation of Corynebacterium glutamicum
[0036] Figure 2 Results of biotransformation of Corynebacterium glutamicum OMK-88 Detailed Implementation
[0037] Table 1. Corynebacterium glutamicum OMK-88 solid seed culture medium (g / L)
[0038]
[0039] Table 2. Corynebacterium glutamicum OMK-88 seed culture medium (g / L)
[0040]
[0041] Table 3. Corynebacterium glutamicum OMK-88 fermentation medium (g / L)
[0042]
[0043]
[0044] The inventors successfully screened a strain of *Corynebacterium glutamicum* capable of biotransforming L-homoserine and methylselenool (sodium) to produce L-selenomethionine. The laboratory conducted multiple rounds of conventional chemical mutagenesis on this strain using single and mixed chemical mutagenizers, while employing a rational and efficient strain screening method. Ultimately, a mutant strain, *Corynebacterium glutamicum* OMK-88, was screened that efficiently transforms L-homoserine and methylselenool or sodium methylselenool to produce L-selenomethionine. Based on this mutant, a corresponding fermentation synthesis method for L-selenomethionine was developed.
[0045] Fermentation culture process
[0046] (1) Plate activation
[0047] Take a tube of glycerol culture refrigerated at -80℃, streak the culture onto a beef extract peptone agar plate, and incubate the plate at 37℃ until the clones are full.
[0048] (2) Seed culture
[0049] Single clones were picked from the activated plate and inoculated into seed shake flasks. Corynebacterium glutamicum OMK-88 was cultured in shakers at 37°C and 200 rpm for 12 h. After microscopic examination showed no contamination, the clones were ready to be transferred to seed shake flasks.
[0050] (3) Fermentation culture and biotransformation
[0051] The seeds from the shake flasks were inoculated into the fermenter (shake flask) at an inoculum rate of 10% and cultured at 37°C. The pH was automatically controlled at 6.8-7.0 using 30% sodium hydroxide solution. Corynebacterium glutamicum OMK-88 was cultured for 12-16 hours, and the OD was measured using a spectrophotometer. 600 When OD 600 No further additions are made; proceed to the biotransformation stage. Add 70 g / L of substrate L-homoserine and 70 g / L of sodium methylselenool, or add sodium methylselenool in a fractional flow. Measure the sample every 4 hours. End the transformation when the substrate reaction is complete or ceases.
[0052] Example 1
[0053] Unless otherwise specified, in this embodiment, the seed culture medium and fermentation culture medium are the same as those in Tables 1, 2 and 3, containing the same components and concentrations as the Corynebacterium glutamicum OMK-88 culture medium.
[0054] Preparation of plate culture medium:
[0055] Wash and dry the petri dishes, then sterilize them for later use. Place the freshly sterilized but not yet solidified culture medium in a sterile box or laminar flow hood. Hold the Erlenmeyer flask in your left hand and flame the mouth of the flask. Pour in about 15 mL of sterilized or melted and cooled culture medium to 55°C. Quickly cover the dish with the lid, place it on the table, and gently rotate the petri dish to distribute the culture medium evenly on the bottom of the entire dish. After solidification, it becomes a plate culture medium.
[0056] The culture medium consists of the following components: 3 g / L beef extract, 10 g / L peptone, 15-25 g / L agar (15 g / L in this example), 5 g / L sodium chloride, and pH adjusted to 7.4-7.6 (7.4 in this example).
[0057] Seed preparation:
[0058] Under aseptic conditions, transfer -80℃ glycerol tubes of Corynebacterium glutamicum OMK-88 onto fresh, sterile beef extract peptone agar plates and incubate at 37℃ for 24 hours (mycelium).
[0059] Expanded cultivation:
[0060] Mycelia were picked and inoculated into a 3L bioreactor containing 1.8L of seed culture medium. The mixture was aerated and stirred at 300 rpm and cultured at 37℃ for 14-16 hours to obtain seed liquid.
[0061] The seed culture medium consisted of 20 g / L glucose, 15 g / L yeast extract, 10 g / L peptone, 2.0 g / L potassium dihydrogen phosphate, 2.5 g / L sodium chloride, and pH 7.0.
[0062] Fermentation and biotransformation:
[0063] Prepare the fermentation medium, which includes the following components: glucose 30 g / L, yeast extract 10.0 g / L, peptone 1.0 g / L, magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.2 g / L, sodium chloride 0.8 g / L, sulfuric acid 5.0 g / L, dipotassium hydrogen phosphate 2.4 g / L, and manganese sulfate 0.0001 g / L.
[0064] 10.2 L of the prepared fermentation medium was loaded into a 20 L bioreactor and sterilized at 121 °C for 30 min. Then, 1.8 L of the cultured seed culture was inoculated into the 20 L bioreactor for fermentation at 37 °C, with a stirring speed of 400 rpm and an aeration ratio of 1:0.4. After 20 h of fermentation, the microbial OD... 600 No further additions were made. Instead, 50 g / L of L-homoserine and 50 g / L of sodium methylselenool were added, initiating the biotransformation phase, which lasted for 16 hours. At the end of biotransformation, the concentration of L-selenomethionine in the fermentation broth was determined to be 70 g / L using HPLC.
[0065] Example 2
[0066] Unless otherwise specified, in this embodiment, the seed culture medium and fermentation culture medium are the same as those in Tables 1, 2 and 3, containing the same components and concentrations as the Corynebacterium glutamicum OMK-88 culture medium.
[0067] Preparation of plate culture medium:
[0068] Wash and dry the petri dishes, then sterilize them for later use. Place the freshly sterilized but not yet solidified culture medium in a sterile box or laminar flow hood. Hold the Erlenmeyer flask in your left hand and flame the mouth of the flask. Pour in about 15 mL of sterilized or melted and cooled culture medium to 60°C. Quickly cover the dish with the lid, place it on the table, and gently rotate the petri dish to distribute the culture medium evenly on the bottom of the entire dish. After solidification, it becomes a plate culture medium.
[0069] The culture medium consists of the following components: 3 g / L beef extract, 10 g / L peptone, 15-25 g / L agar (25 g / L in this example), 5 g / L sodium chloride, and pH adjusted to 7.4-7.6 (7.6 in this example).
[0070] Seed preparation:
[0071] Under aseptic conditions, transfer -80°C glycerol tubes containing Corynebacterium glutamicum OMK-88 onto fresh, sterile beef extract peptone agar plates and incubate at 37°C for 24 hours.
[0072] Expanded cultivation:
[0073] Mycelia were picked and inoculated into a 3L bioreactor containing 1.8L of seed culture medium. The mixture was aerated and stirred at 400 rpm and cultured at 37°C for 13 hours to obtain the seed liquid.
[0074] Fermentation and biotransformation:
[0075] 10.2 L of the prepared fermentation medium was transferred to a 20 L bioreactor and sterilized at 121 °C for 30 min. Then, 1.8 L of the cultured seed culture was inoculated into the 20 L bioreactor for fermentation at 30 °C, with a stirring speed of 500 rpm and an aeration ratio of 1:0.4. After 12 h of fermentation, the microbial OD... 600 No further additions were made. Then, 70 g / L of substrate L-homoserine and 70 g / L of sodium methylselenool were added, initiating the transformation phase. The transformation lasted for 16 hours until the concentration of the product L-selenomethionine ceased to increase, at which point the transformation ended. The concentration of L-selenomethionine in the fermentation broth was determined to be 91 g / L by HPLC at the end of the biotransformation.
[0076] The methods described in this application have been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this application to implement and apply the technology of this application. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within this application.
Claims
1. A type of Corynebacterium glutamicum ( Corynebacterium glutamicum ) OMK-88, Collection No.: CCTCC NO:M 20222075.
2. The application of Corynebacterium glutamicum OMK-88 as described in claim 1 in the preparation of L-selenomethionine, comprising fermenting the Corynebacterium glutamicum as described in claim 1 in a fermentation medium containing the reaction substrate to obtain L-selenomethionine.
3. The application according to claim 2, characterized in that, The fermentation substrate is L-homoserine and sodium methylselenool or methylselenool.
4. The application according to claim 3, characterized in that, The L-homoserine concentration is 50-70 g / L, and the sodium methylselenool or methylselenool concentration is 50-70 g / L.
5. The application according to claim 2, characterized in that, The fermentation medium includes a carbon source, a nitrogen source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, and ammonium sulfate. The carbon source includes glucose, and the nitrogen source includes one or more of yeast extract and peptone.
6. The application according to any one of claims 2-5, characterized in that, The preparation of L-selenomethionine includes the following steps: S1: Corynebacterium glutamicum OMK-88 was cultured in agar plates; S2: The plate mycelium obtained in step S1 is cultured in one or more stages; S3: The expanded culture seed liquid obtained in step S2 is placed in a fermentation medium containing the reaction substrate for fermentation to obtain L-selenomethionine.
7. The application according to claim 6, characterized in that, The pH of the plate culture medium in step S1 is 7.4-7.
6. The stirring speed of the scale-up culture in step S2 is 300-500 rpm, the culture temperature is 36.5-37.5℃, and the culture time is 13-16 h. The fermentation culture in step S3 is carried out at a temperature of 37℃, a stirring speed of 400-500 rpm, an aeration ratio of 1:0.1-1:0.4, and a fermentation time of 12-20 h. The reaction substrate L-homoserine and sodium methylselenool or methylselenool are added at once or in batches.