Yarrowia lipolytica, preparation, application and preparation method of erythritol

Abstract

The invention discloses Yarrowia lipolytica, a preparation, application and a preparation method of erythritol, and belongs to the technical field of microorganisms. The invention provides Yarrowia lipolytica which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on December 26, 2022, and the preservation number of the Yarrowia lipolytica is CGMCC (China General Microbiological Culture Collection Center) No.26283. The Yarrowia lipolytica is simple in culture condition and resistant to high temperature (35 DEG C), the conversion rate of erythritol synthesized from glucose reaches 71%, the yield of erythritol is high, byproducts are few, the yield is high, the product is easy to separate and purify, and the production cost of erythritol can be effectively reduced when the Yarrowia lipolytica is applied to industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to Yarrowia lipolytica, preparations, applications and a method for preparing erythritol. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Erythritol is a low-calorie sweetener that is widely used in food, medicine, cosmetics, and chemical industries. Erythritol is mainly obtained through fermentation of some yeasts, and Yarrowia lipolytica is currently the main erythritol production strain. However, in the prior art, when Yarrowia lipolytica produces erythritol, the conversion rate of glucose to erythritol is low (47%) and has no practical use value. At the same time, Yarrowia lipolytica belongs to aerobic fermentation, produces a lot of heat, and the fermentation temperature generally does not exceed 30°C. Therefore, in summer, there are problems such as cooling problems and contamination and tank inversion, which are important factors affecting the production cost of erythritol.

[0004] Some researchers used Yarrowia lipolytica as a synthetic chassis and edited the yeast genes through metabolic engineering to enhance its ability to synthesize erythritol and block the pathway for synthesizing byproducts. Although the strain can efficiently synthesize erythritol using carbon sources such as glucose as raw materials, does not synthesize byproducts (such as mannitol), and does not consume erythritol, its conversion rate from glucose to erythritol is only 62% at most, and the conversion rate needs to be further improved. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide Yarrowia lipolytica, preparations, applications and a method for preparing erythritol. The Yarrowia lipolytica provided by the present invention has a conversion rate of 71% in synthesizing erythritol from glucose, and has a high yield of erythritol.

[0006] In order to achieve the above object, the technical solution of the present invention is: The first aspect of the present invention provides a Yarrowia lipolytica ( 解脂耶氏酵母 ), the Yarrowia lipolytica was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 26, 2022, with the deposit number: CGMCC No.26283.

[0007] The second aspect of the present invention provides a preparation comprising the above-mentioned Yarrowia lipolytica ( 耶氏酵母属 解脂 ) or its fermentation broth.

[0008] The third aspect of the present invention provides a kind of above-mentioned Yarrowia lipolytica ( 耶氏酵母属 解脂 ) or the use of the above-mentioned preparation in the production of erythritol.

[0009] In some embodiments of the present invention, the use comprises the step of: 解脂耶氏酵母 ) to ferment to obtain a fermentation broth; and separate and purify the fermentation broth to obtain erythritol.

[0010] A fourth aspect of the present invention provides a method for preparing erythritol, comprising the following steps: The above-mentioned Yarrowia lipolytica ( 解脂耶氏酵母 ) to ferment to obtain a fermentation liquid; The fermentation broth is separated and purified to obtain erythritol.

[0011] In some embodiments of the present invention, the preparation method comprises: The above-mentioned Yarrowia lipolytica ( 解脂耶氏酵母 ) is inoculated into a culture medium and cultured to obtain seeds; The seeds were inoculated into the fermentation medium for fermentation. The initial fermentation temperature was controlled at 25-35°C and the initial pH value was 5.0-7.0. The fermentation was continued until the cell density OD 600 When the value is ≥20, adjust the pH to 3.1-4.0, continue fermentation and culture for 3-5 days to complete the fermentation; After fermentation, Yarrowia lipolytica ( 解脂耶氏酵母 ) is separated from the fermentation liquid, and the fermentation liquid is filtered, concentrated, and crystallized to obtain erythritol crystals.

[0012] Wherein, the initial fermentation temperature may be 25°C, 27°C, 29°C, 30°C, 32°C, 34°C or 35°C, etc.

[0013] Wherein, the initial pH may be 5.0, 5.5, 6.0 or 7.0, etc.

[0014] Wherein, the adjusted pH is 3.1, 3.5, 3.8 or 4.0, etc.

[0015] In some embodiments of the present invention, the culturing comprises the following steps: The above-mentioned Yarrowia lipolytica ( 解脂耶氏酵母 ) was inoculated into YPD medium, pH 6.9-7.1, 28-32°C, and cultured in a shaking incubator at 180-220 r / min for 18-19 h to obtain primary seeds; The first-level seeds are transferred to a seed tank containing YPD medium, and cultured at 28-32° C. and 480-520 rpm for 8-12 hours to obtain second-level seeds; The fermentation medium is treated at a temperature of 80-90°C for 20-40 min, and after cooling, the secondary seeds are inoculated for fermentation culture; The YPD culture medium comprises: 18-22 g / L of glucose, 18-22 g / L of peptone, and 8-12 g / L of yeast extract.

[0016] In some embodiments of the present invention, the fermentation medium comprises: 100-400 g / L of carbon source, 2-35 g / L of nitrogen source, 0-2.5 g / L of inorganic salt, 400-500 g / L of glucose and water; The carbon source is starch hydrolyzate; The nitrogen source is a mixture of diammonium phosphate, yeast powder and corn steep liquor powder; The inorganic salt is a mixture of magnesium sulfate, manganese sulfate, manganese chloride, copper sulfate, copper chloride, zinc sulfate and zinc chloride.

[0017] In some embodiments of the present invention, the concentration of the carbon source in the fermentation medium may be 100 g / L, 150 g / L, 200 g / L, 250 g / L, 300 g / L, 350 g / L or 400 g / L, etc.

[0018] In some embodiments of the present invention, the concentration of the nitrogen source in the fermentation medium may be 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L or 35 g / L, etc.

[0019] In some embodiments of the present invention, the concentration of the inorganic salt in the fermentation medium may be 0 g / L, 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L or 2.5 g / L, etc.

[0020] In some embodiments of the present invention, the fermentation medium comprises: 400-500 g / L glucose, 100-400 g / L starch hydrolyzate, 5 g / L diammonium phosphate, 15 g / L yeast powder, 15 g / L corn steep liquor powder, 0.25 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 0.25 g / L manganese chloride, 0.25 g / L copper sulfate, 0.25 g / L copper chloride, 0.25 g / L zinc sulfate, and 0.25 g / L zinc chloride.

[0021] In some embodiments of the present invention, the filtration comprises: separating and removing substances with a molecular weight greater than 1000 Da in the fermentation broth by a nanofiltration membrane to obtain a clear and transparent fermentation broth; The concentration comprises: evaporating and concentrating the fermentation liquid to a solid content of 50-80% by mass volume percentage to obtain a syrup rich in erythritol; The crystallization comprises: cooling the concentrated erythritol-rich syrup to below 10° C. at a rate of 1-5° C. / h, crystallizing erythritol, and centrifuging to obtain erythritol crystals.

[0022] The beneficial effects of the present invention are: The present invention provides a Yarrowia lipolytica ( 解脂耶氏酵母 ), the strain was screened from nature and is a non-GMO strain that meets the standards of food safety strains and avoids the risk of GMOs. At the same time, the culture conditions of this strain are simple, high temperature resistant (35°C), the conversion rate of glucose to erythritol reaches 71%, the erythritol yield is high, the by-products are few, the yield is high, and the product is easy to separate and purify. It can be used in industrial production to effectively reduce the production cost of erythritol. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 The colony morphology of Yarrowia lipolytica SYJZ-12 obtained in Example 1 of the present invention on a solid culture medium. DETAILED DESCRIPTION

[0025] Description of biological deposit: Culture Name: Yarrowia lipolytica 解脂耶氏酵母 ), deposited on December 26, 2022 in the General Microbiology Center of China Microorganism Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is: CGMCC No.26283.

[0026] The Yarrowia lipolytica mentioned in the present invention ( 解脂耶氏酵母 )SYJZ-12 is the strain with the deposit number of CGMCC No.26283.

[0027] The present invention discloses a method for preparing Yarrowia lipolytica, preparations, applications and erythritol. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired effect. 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 in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0028] Example 1 Screening of strains The samples collected from the natural environment were placed in YPD medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L), pH 7.0, temperature 30°C, and cultured in a shaking incubator at 200 r / min for 3 days. An appropriate amount of the sample was diluted and the dilution was spread on YPD solid medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, agar 15%) and cultured at 30°C for 3 days. The strains with larger colonies and morphology similar to yeast were selected, picked out with a sterile toothpick, and inoculated into a fermentation medium containing glucose (glucose 100 g / L, starch hydrolyzate 300 g / L, diammonium phosphate 5 g / L, yeast powder 15 g / L, corn steep liquor dry powder 15 g / L, magnesium sulfate 0.25 g / L, manganese sulfate 0.25 g / L, manganese chloride 0.25 g / L, copper sulfate 0.25 g / L, copper chloride 0.25 g / L, zinc sulfate 0.25 g / L, zinc chloride 0.25 g / L) in a 24-well deep-well plate, and the erythritol content in the fermentation broth was detected after culturing at 35°C and 200 r / min for 4 days.

[0029] Erythritol detection method: High performance liquid chromatography was used for detection, and the detector was a differential detector. The chromatographic column was an amino column, the mobile phase was acetonitrile: water (80:20), the flow rate was 1.0 mL / min, the column temperature was 40°C, and the injection volume was 10 μL.

[0030] By screening more than 2,000 strains from more than 200 samples, a high-activity yeast strain was finally screened from the corn farmland in Zhanhua, Shandong. The strain was identified as Yarrowia lipolytica ( 解脂耶氏酵母 ), the relevant 18SrDNA sequence is shown in SEQ ID NO. 1. The strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No. 26283.

[0031] Example 2: Test and verification of the high temperature resistance of Yarrowia lipolytica Because it is difficult to accurately control the temperature during industrial production, and there are problems such as cooling in summer, in order to further verify the Yarrowia lipolytica obtained in Example 1 ( 解脂耶氏酵母 )SYJZ-12 has high temperature resistance, the fermentation temperature was controlled at 35-45℃, and the yield of erythritol was measured.

[0032] 100 μL Yarrowia lipolytica ( 解脂耶氏酵母 SYJZ-12 was inoculated in 10 mL YPD medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L), pH 7.0, temperature 30°C, and cultured in a shaking incubator at 200 r / min for 18-19 h as seed solution.

[0033] 2 mL of seed liquid was inoculated into 100 mL of fermentation medium containing glucose (300 g / L glucose, 300 g / L starch hydrolyzate, 5 g / L diammonium phosphate, 15 g / L yeast powder, 15 g / L corn steep liquor powder, 0.25 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 0.25 g / L manganese chloride, 0.25 g / L copper sulfate, 0.25 g / L copper chloride, 0.25 g / L zinc sulfate, 0.25 g / L zinc chloride), and the fermentation temperatures were controlled at 30℃, 35℃, 40℃, and 45℃, respectively. The erythritol content in the fermentation broth was detected after culturing at 200 r / min for 4 days.

[0034] Table 1 Results of the high temperature resistance test of Yarrowia lipolytica SYJZ-12

[0035] As shown in Table 1, when the fermentation temperature is raised to 45°C, the conversion rate can also be maintained at 60%, indicating that the Yarrowia lipolytica obtained in Example 1 ( 解脂耶氏酵母 )SYJZ-12 has good temperature stability.

[0036] Example 3 Application of Yarrowia lipolytica in the synthesis of erythritol (1) Seed primary culture: 10 mL Yarrowia lipolytica ( 解脂耶氏酵母 )SYJZ-12 strain was inoculated into a 5 L shake flask containing 1 L YPD medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L), pH 7.0, temperature 30°C, and cultured in a shaking incubator at 200 r / min for 18-19 h to obtain primary seeds.

[0037] (2) Secondary seed culture: Transfer 2 L of the primary seeds into a 250 L seed tank containing 150 L of YPD medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) and culture at 30°C, 500 rpm for 10 h to obtain secondary seeds.

[0038] (3) The fermentation medium was treated at 80°C for 40 min. After cooling, all the secondary seeds were inoculated into the fermentation medium (2000 L fermenter) for culture. The formula of the fermentation medium was as follows: 500 g / L glucose, 300 g / L starch hydrolyzate, 5 g / L diammonium phosphate, 15 g / L yeast powder, 15 g / L corn syrup powder, 0.25 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 0.25 g / L manganese chloride, 0.25 g / L copper sulfate, 0.25 g / L copper chloride, 0.25 g / L zinc sulfate, and 0.25 g / L zinc chloride. The initial fermentation temperature was controlled at 35°C and the initial pH was 6.0. The fermentation was continued until the cell density OD 600 When the value was 20, the pH value was adjusted to 3.7; then the fermentation culture was continued for 4 days. After the fermentation was completed, the erythritol content reached 355 g / L and the conversion rate of erythritol was 71%. Other impurity polyols (ribitol, arabitol, mannitol, etc.) reached 28 g / L, with low impurity content, and the impurities could be fully removed through post-treatment.

[0039] (4) After the fermentation is completed, the yeast cells are separated from the fermentation liquid by ceramic membrane filtration, and then the macromolecular viscous substances with a molecular weight greater than 1000 Daltons in the fermentation liquid are removed by nanofiltration membrane separation to obtain a clear and transparent fermentation liquid.

[0040] The clear and transparent fermentation liquid is concentrated in an evaporator to a solid content of 70-80% by weight volume to obtain a syrup rich in erythritol; the syrup rich in erythritol is cooled to below 10°C at a rate of 3-5°C / h, and after crystallization is completed, centrifugation is performed to obtain erythritol crystals.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Yarrowia lipolytica Yarrowia lipolytica ), characterized in that The Yarrowia lipolytica was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on December 26, 2022, with the deposit number: CGMCC No.26283.

2. A preparation, characterized in that The Yarrowia lipolytica according to claim 1 ( Yarrowia lipolytica ) or its fermentation broth.

3. The Yarrowia lipolytica according to claim 1 Yarrowia lipolytica ) or the use of the preparation described in claim 2 in the production of erythritol.

4. The use according to claim 3, characterized in that The application comprises the step of culturing the Yarrowia lipolytica ( Yarrowia lipolytica ) to ferment to obtain a fermentation broth; and separate and purify the fermentation broth to obtain erythritol.

5. A method for preparing erythritol, characterized in that: The steps include: The Yarrowia lipolytica according to claim 1 ( Yarrowia lipolytica ) to ferment to obtain a fermentation liquid; The fermentation liquid is separated and purified to obtain erythritol.

6. The preparation method according to claim 5, characterized in that: The preparation method comprises: The Yarrowia lipolytica of claim 1 ( Yarrowia lipolytica ) is inoculated into a culture medium and cultured to obtain seeds; The seeds were inoculated into the fermentation medium for fermentation. The initial fermentation temperature was controlled at 25-35°C and the initial pH value was 5.0-7.

0. The fermentation was continued until the cell density OD 600 When the value is ≥20, adjust the pH to 3.1-4.0, continue fermentation and culture for 3-5 days to complete the fermentation; After fermentation, Yarrowia lipolytica ( Yarrowia lipolytica ) is separated from the fermentation liquid, and the fermentation liquid is filtered, concentrated, and crystallized to obtain erythritol crystals.

7. The preparation method according to claim 6, characterized in that: The culturing comprises the following steps: The Yarrowia lipolytica of claim 1 ( Yarrowia lipolytica ) was inoculated into YPD medium, pH 6.9-7.1, 28-32°C, and cultured in a shaking incubator at 180-220 r / min for 18-19 h to obtain primary seeds; The first-level seeds are transferred to a seed tank containing YPD medium, and cultured at 28-32° C. and 480-520 rpm for 8-12 h to obtain second-level seeds; The fermentation medium is treated at a temperature of 80-90°C for 20-40 min, and after cooling, the secondary seeds are inoculated for fermentation culture; The YPD culture medium comprises: 18-22 g / L of glucose, 18-22 g / L of peptone, and 8-12 g / L of yeast extract.

8. The preparation method according to claim 6, characterized in that: The fermentation medium comprises: 100-400 g / L of carbon source, 2-35 g / L of nitrogen source, 0-2.5 g / L of inorganic salt, 400-500 g / L of glucose and water; The carbon source is starch hydrolyzate; The nitrogen source is a mixture of diammonium phosphate, yeast powder and corn steep liquor powder; The inorganic salt is a mixture of magnesium sulfate, manganese sulfate, manganese chloride, copper sulfate, copper chloride, zinc sulfate and zinc chloride.

9. The preparation method according to claim 8, characterized in that: The fermentation medium comprises: 400-500 g / L of glucose, 100-400 g / L of starch hydrolyzate, 5 g / L of diammonium phosphate, 15 g / L of yeast powder, 15 g / L of corn steep liquor dry powder, 0.25 g / L of magnesium sulfate, 0.25 g / L of manganese sulfate, 0.25 g / L of manganese chloride, 0.25 g / L of copper sulfate, 0.25 g / L of copper chloride, 0.25 g / L of zinc sulfate and 0.25 g / L of zinc chloride.

10. The preparation method according to claim 6, characterized in that: The filtration comprises: separating and removing substances with a molecular weight greater than 1000 Da in the fermentation broth through a nanofiltration membrane to obtain a clear and transparent fermentation broth; The concentration comprises: evaporating and concentrating the fermentation liquid to a solid content of 50-80%, by mass volume percentage, to obtain a syrup rich in erythritol; The crystallization comprises: cooling the concentrated erythritol-rich syrup to below 10° C. at a rate of 1-5° C. / h, crystallizing erythritol, and centrifuging to obtain erythritol crystals.

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