Yarrowia lipolytica, formulations, uses and methods for preparing erythritol

Abstract

The application discloses a Yarrowia lipolytica, a preparation, application and a preparation method of erythritol, and belongs to the technical field of microorganisms. Yarrowia lipolytica The Yarrowia lipolytica is preserved in the China General Microbiological Culture Collection Center on December 26, 2022, and the preservation number is CGMCC No. 26283. The Yarrowia lipolytica Yarrowia lipolytica The Yarrowia lipolytica has simple culture conditions, high temperature resistance (35 DEG C), a glucose-to-erythritol conversion rate of 71%, high erythritol yield, few by-products, high yield, easy separation and purification of products, and can effectively reduce the production cost of erythritol in industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to Yarrowia lipolytica, a preparation, application and preparation method of erythritol. BACKGROUND

[0002] The information disclosed in this background section is only intended to increase an understanding of the general context of the present application and does not necessarily constitute admission to prior art of any form of suggestion that this information forms part of the prior art.

[0003] Erythritol is a low-calorie sweetener and is widely used in food, medicine, cosmetics, chemical industry and other fields. Erythritol is mainly obtained by fermentation of some yeast. Yarrowia lipolytica is the main erythritol production strain at present. However, the conversion rate of erythritol synthesized from glucose is low (47%) in the prior art, which has no practical value. At the same time, Yarrowia lipolytica belongs to aerobic fermentation, and the heat production is relatively high. The fermentation temperature is generally not more than 30℃, so there are problems such as cooling difficulty, contamination and tank dumping in summer, which is an important factor affecting the production cost of erythritol.

[0004] Some researchers use Yarrowia lipolytica as a synthetic chassis, and through metabolic engineering improvement means, the yeast is genetically edited to enhance its ability to synthesize erythritol and block the synthesis of by-products. Although this strain can efficiently synthesize erythritol from glucose and other carbon sources, it does not synthesize by-products (such as mannitol) and does not consume erythritol. However, the conversion rate of erythritol synthesized from glucose is only 62%, and the conversion rate needs to be further improved. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides Yarrowia lipolytica, a preparation, application and preparation method of erythritol. The conversion rate of erythritol synthesized from glucose by the Yarrowia lipolytica provided by the present application reaches 71%, and the erythritol yield is high.

[0006] In order to achieve the above-mentioned purposes, the technical scheme of the present application is as follows:

[0007] In a first aspect of the present application, a Yarrowia lipolytica is provided. Yarrowia lipolytica The Yarrowia lipolytica was preserved in the China General Microbiological Culture Collection Center on December 26, 2022, and the preservation number is CGMCC No. 26283.

[0008] In a second aspect of the present application, a preparation is provided, which comprises the above-mentioned Yarrowia lipolytica or fermentation liquor thereof. Yarrowia lipolytica The present application provides a Yarrowia lipolytica, a preparation, application and preparation method of erythritol. The conversion rate of erythritol synthesized from glucose by the Yarrowia lipolytica provided by the present application reaches 71%, and the erythritol yield is high.

[0009] In a third aspect of the present application, there is provided the Yarrowia lipolytica (Yarrowia lipolytica) as described above, Yarrowia lipolytica or the use of the preparation as described above in the production of erythritol.

[0010] In some embodiments of the present application, the use comprises fermenting the Yarrowia lipolytica (Yarrowia lipolytica) as described above in a culture medium containing glucose to obtain a fermentation liquor; and separating and purifying the fermentation liquor to obtain erythritol. Yarrowia lipolytica

[0011] In a fourth aspect of the present application, there is provided a preparation method of erythritol, comprising the following steps:

[0012] fermenting the Yarrowia lipolytica (Yarrowia lipolytica) as described above in a culture medium containing glucose to obtain a fermentation liquor; Yarrowia lipolytica

[0013] separating and purifying the fermentation liquor to obtain erythritol.

[0014] In some embodiments of the present application, the preparation method comprises:

[0015] inoculating the Yarrowia lipolytica (Yarrowia lipolytica) as described above into a culture medium to culture to obtain a seed; Yarrowia lipolytica

[0016] inoculating the seed into a fermentation culture medium to ferment, controlling the initial temperature of fermentation to be 25-35℃ and the initial pH value to be 5.0-7.0, and when the cell density OD 600 value is ≥20, adjusting the pH to be 3.1-4.0, continuing to ferment and culture for 3-5 days to complete the fermentation;

[0017] After the fermentation is completed, the Yarrowia lipolytica (Yarrowia lipolytica) is separated from the fermentation liquor, and the fermentation liquor is filtered, concentrated and crystallized to obtain erythritol crystals. Yarrowia lipolytica

[0018] The initial temperature of fermentation can be 25℃, 27℃, 29℃, 30℃, 32℃, 34℃ or 35℃, etc.

[0019] The initial pH value can be 5.0, 5.5, 6.0 or 7.0, etc.

[0020] The adjusted pH value can be 3.1, 3.5, 3.8 or 4.0, etc.

[0021] In some embodiments of the present application, the culture comprises the following steps:

[0022] inoculating the Yarrowia lipolytica (Yarrowia lipolytica) as described above into a culture medium to culture to obtain a seed; Yarrowia lipolytica ​​​​) inoculated into YPD medium, pH 6.9-7.1, 28-32℃, 180-220 r / min shaker culture for 18-19 h, to obtain a first seed;

[0023] The first seed is transferred to a seed tank containing YPD medium, 28-32℃, 480-520 rpm for 8-12 h to obtain a second seed;

[0024] The fermentation medium is treated at a temperature of 80-90℃ for 20-40 min, and after cooling, the second seed is inoculated for fermentation culture;

[0025] The YPD medium comprises: glucose 18-22 g / L, peptone 18-22 g / L, and yeast extract 8-12 g / L.

[0026] In some embodiments of the present application, the fermentation medium comprises: a carbon source 100-400 g / L, a nitrogen source 2-35 g / L, inorganic salt 0-2.5 g / L, glucose 400-500 g / L, and water;

[0027] The carbon source is starch hydrolysate;

[0028] The nitrogen source is a mixture of diammonium phosphate, yeast powder, and corn syrup dry powder;

[0029] The inorganic salt is a mixture of magnesium sulfate, manganese sulfate, manganese chloride, copper sulfate, copper chloride, zinc sulfate, and zinc chloride.

[0030] In some embodiments of the present application, the concentration of the carbon source in the fermentation medium can be 100 g / L, 150 g / L, 200 g / L, 250 g / L, 300 g / L, 350 g / L, or 400 g / L, etc.

[0031] In some embodiments of the present application, the concentration of the nitrogen source in the fermentation medium can 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.

[0032] In some embodiments of the present application, the concentration of the inorganic salt in the fermentation medium can 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.

[0033] In some embodiments of the present invention, the fermentation culture medium comprises: 400-500 g / L glucose, 100-400 g / L starch hydrolysate, 5 g / L diammonium hydrogen phosphate, 15 g / L yeast extract, 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.

[0034] In some embodiments of the present invention, the filtration includes: separating and removing substances with a molecular weight greater than 1000 Da from the fermentation broth through a nanofiltration membrane to obtain a clear and transparent fermentation broth;

[0035] The concentration includes: evaporating and concentrating the fermentation broth to a solid content of 50-80% by mass-volume percentage, to obtain a syrup rich in erythritol;

[0036] The crystallization process includes: cooling the concentrated erythritol-rich syrup to below 10°C at a rate of 1-5°C / h, crystallizing the erythritol, centrifuging to obtain erythritol crystals.

[0037] The beneficial effects of this invention are as follows:

[0038] This invention provides a lipolytic yeast ( Yarrowia lipolytica This strain, screened from nature, is a non-GMO strain that meets the standards for food safety strains, avoiding the risks associated with GMOs. Furthermore, this strain has simple cultivation conditions, is heat-resistant (35℃), achieves a glucose-to-erythritol conversion rate of 71%, and boasts high erythritol yield with few byproducts. Its high yield and easy product separation and purification make it suitable for industrial production, effectively reducing erythritol production costs. Attached Figure Description

[0039] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0040] Yarrowia lipolytica The colony morphology of *Yerovia lipophila* SYJZ-12 obtained in Example 1 of this invention on a solid culture medium. Detailed Implementation

[0041] Biological Preservation Instructions:

[0042] Culture name: Lipid-releasing Yeast ( Figure 1) was preserved in China General Microbiological Culture Collection Center on December 26, 2022, and the address of the preservation center is: No. 1, Beichen West Road, Yuan 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing; the preservation number is: CGMCC No. 26283.

[0043] The Yarrowia lipolytica mentioned in the present application refers to the strain with the preservation number of CGMCC No. 26283. Yarrowia lipolytica ) SYJZ-12 is the strain with the preservation number of CGMCC No. 26283.

[0044] The present application discloses a Yarrowia lipolytica, a preparation, an application and a preparation method of erythritol, and those skilled in the art can refer to the content of the present application to improve the process parameters as appropriate. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by 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 application, to realize and apply the present application technology.

[0045] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples.

[0046] Example 1: Screening of strains

[0047] The samples collected from the natural environment were placed in YPD culture medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L), pH 7.0, temperature 30℃, 200 r / min shaker culture for 3 days, and an appropriate amount of sample was diluted and spread on YPD solid culture medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, agar 15%) at 30℃ for 3 days; the strains with larger colonies and similar morphology to yeast were selected and inoculated into the fermentation medium (glucose 100 g / L, starch hydrolysate 300 g / L, diammonium phosphate 5 g / L, yeast powder 15 g / L, corn syrup 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 4 days of culture at 35℃ and 200 r / min.

[0048] Erythritol detection method: High performance liquid chromatography (HPLC) was used with 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℃, and the injection volume was 10 μL.

[0049] Through screening over 2000 strains from more than 200 samples, a highly active yeast strain was finally identified from cornfields in Zhanhua, Shandong Province. After identification, it was determined to be *Yerovia lipophila*. Yarrowia lipolytica The relevant 18S rDNA sequence is shown in SEQ ID NO.1. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26283.

[0050]

[0051] Example 2: High-Temperature Tolerance Test Verification of *Yerovia lipophila*

[0052] Because temperature is difficult to control precisely during industrial production, and there are challenges such as cooling down in summer, this study aimed to further verify the efficacy of the lipolytic yeast obtained in Example 1. Yarrowia lipolytica The high-temperature resistance of SYJZ-12 was tested, and the yield of erythritol was determined by controlling the fermentation temperature at 35-45℃.

[0053] 100 μL of lipolysin (Yerovia yeast) Yarrowia lipolytica SYJZ-12 was inoculated onto 10 mL of YPD medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract), and cultured at pH 7.0, 30℃, and 200 r / min in a shaker for 18-19 h to obtain the seed culture.

[0054] Two mL of seed culture was inoculated into 100 mL of fermentation medium containing glucose (300 g / L glucose, 300 g / L starch hydrolysate, 5 g / L diammonium hydrogen phosphate, 15 g / L yeast extract, 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). The fermentation temperatures were controlled at 30℃, 35℃, 40℃, and 45℃, and the culture was carried out at 200 r / min for 4 days. The erythritol content in the fermentation broth was then measured.

[0055] Table 1. Results of the high-temperature resistance test of Yeast Extract SYJZ-12

[0056]

[0057] As shown in Table 1, even when the fermentation temperature is increased to 45℃, the conversion rate can still be maintained at 60%, indicating that the lipolytic yeast obtained in Example 1 ( Yarrowia lipolytica SYJZ-12 has good temperature stability.

[0058] Example 3: Application of *Yerovia lipophila* in the synthesis of erythritol

[0059] (1) Primary culture of seeds: 10 mL of lipophilic yarrow yeast ( Yarrowia lipolytica Yarrowia lipolytica SYJZ-12 strain was inoculated into a 5 L shake flask containing 1 L of YPD medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract), and cultured at pH 7.0, temperature 30℃, and 200 r / min for 18-19 h to obtain primary seed culture.

[0060] (2) Seed secondary culture: All the first seed 2 L was transferred to a 250 L seed tank containing 150 L YPD medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L) and cultured at 30°C and 500 rpm for 10 h to obtain the secondary seed.

[0061] (3) The fermentation medium was treated at a temperature of 80°C for 40 min, and after cooling, the secondary seed was inoculated into the fermentation medium (2000 L fermenter) for culture. The formula of the fermentation medium was: glucose 500 g / L, starch hydrolysate 300 g / L, diammonium phosphate 5 g / L, yeast powder 15 g / L, corn syrup 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, and zinc chloride 0.25 g / L. The initial temperature of the fermentation was controlled at 35°C, and the initial pH value was 6.0. When the cell density OD 600 was 20, the pH value was adjusted to 3.7, and then the fermentation was cultured for 4 days. After the fermentation was completed, it was detected that the erythritol reached 355 g / L, and the conversion rate of erythritol was 71%. Other impurities such as polyol (ribitol, arabinitol, mannitol, etc.) reached 28 g / L, the impurity content was low, and the impurities could be fully removed through post-treatment.

[0062] (4) After the fermentation was completed, the yeast cells were separated from the fermentation broth by ceramic membrane filtration, and then the macromolecular viscous substances with a molecular weight greater than 1000 daltons in the fermentation broth were removed by nanofiltration membrane separation to obtain a clear and transparent fermentation broth.

[0063] The clear and transparent fermentation broth was concentrated in an evaporator to a solid content of 70-80% (w / v) to obtain a erythritol-rich syrup. The erythritol-rich syrup was cooled to below 10°C at a rate of 3-5°C / h, and after crystallization was completed, centrifugal separation was performed to obtain erythritol crystals.

[0064] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing erythritol, characterized by, The method comprises the following steps: The lipolytic yeast (Yerovia spp.) Yarrowia lipolytica SYJZ-12 was inoculated into the culture medium and cultured to obtain seeds; The seeds are inoculated into the fermentation medium for fermentation, the initial temperature of the fermentation is controlled at 25-35℃, the initial pH value is controlled at 5.0-7.0, and when the cell density OD 600 When the value is greater than or equal to 20, the pH value is adjusted to 3.1-4.0, the fermentation is continuously cultured for 3-5 days, and the fermentation is completed. After fermentation, the lipolytic yeast (Yerovia spp.) Yarrowia lipolytica SYJZ-12 was separated from the fermentation broth, and the fermentation broth was filtered, concentrated, and crystallized to obtain erythritol crystals; Yarrowia lipolytica (ATCC 20362) Yarrowia lipolytica SYJZ-12 was deposited with China General Microbiological Culture Collection Center on December 26, 2022, and the deposit number is CGMCC No. 26283. 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.

2. The production method according to claim 1, wherein The culturing comprises the following steps: The lipolytic yeast (Yerovia spp.) Yarrowia lipolytica SYJZ-12 was inoculated into YPD medium and cultured in a shaker at pH 6.9-7.1, 28-32℃, and 180-220 r / min for 18-19 h to obtain primary seeds; The primary seed is transferred into a seed tank containing YPD medium, and is cultured at 28-32 ℃ and 480-520 rpm for 8-12 h to obtain a secondary seed; The fermentation medium is treated at a temperature of 80-90 ℃ for 20-40 min, and then the secondary seed is inoculated into the fermentation medium after cooling; The YPD medium comprises: 18-22 g / L of glucose, 18-22 g / L of peptone and 8-12 g / L of yeast extract.

3. The production method according to claim 1, wherein The carbon source is starch hydrolysate; The nitrogen source is a mixture of diammonium phosphate, yeast powder and corn syrup dry powder; The inorganic salt is a mixture of magnesium sulfate, manganese sulfate, manganese chloride, copper sulfate, copper chloride, zinc sulfate and zinc chloride.

4. The production method according to claim 1, wherein The fermentation medium comprises: 400-500 g / L of glucose, 100-400 g / L of starch hydrolysate, 5 g / L of diammonium phosphate, 15 g / L of yeast powder, 15 g / L of corn syrup 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.

5. The production method according to claim 1, wherein The filtering comprises: removing substances with a molecular weight greater than 1000 Da in the fermentation liquid by separation through a nanofiltration membrane, to obtain clear and transparent fermentation liquid; The concentrating comprises: evaporating and concentrating the fermentation liquid to a solid content of 50-80%, mass percentage, to obtain a sugar syrup rich in erythritol; The crystallizing comprises: cooling the sugar syrup rich in erythritol obtained by concentration to below 10 ℃ at a rate of 1-5 ℃ / h, so that erythritol is crystallized, and then centrifugal separation is performed to obtain erythritol crystals.

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