Yarrowia lipolytica and uses thereof

Abstract

The application relates to the fermentation field and discloses a Yarrowia lipolytica and application thereof. The preservation number of the Yarrowia lipolytica is CGMCC No. 23718. The Yarrowia lipolytica has high fermentation stability, high tolerance to high temperature and high sugar concentration, and still has good erythritol production capacity under the conditions of high temperature and high initial sugar concentration; the Yarrowia lipolytica is used for producing erythritol, batch fermentation can be carried out with high initial sugar concentration, high content of erythritol is obtained, by-products in the fermentation are few, and the amount of cooling water or refrigerated water used for temperature control in the fermentation process can be reduced.

Description

Technical Field

[0001] This invention relates to the field of fermentation, specifically to a lipothermic yeast and its applications. Background Technology

[0002] Erythritol, also known as 1,2,3,4-butanetetrol, is a natural sugar alcohol found in small amounts in foods such as seaweed, mushrooms, pears, grapes, and watermelons, as well as in common fermented foods like soy sauce and wine. Erythritol is a white crystalline powder with a sweetness 65% that of sucrose. It has a pure sweetness and a refreshing sensation, and is highly stable to acid and heat. Compared to other functional sugar alcohols, it has a lower molecular weight, higher solution osmotic pressure, and hygroscopic properties. Erythritol has only one-tenth the calories of sucrose, is highly tolerable in the human body, and does not cause changes in blood sugar levels. It can be added as a sweetener to food and consumer products.

[0003] Currently, most industrial methods for producing erythritol use glucose as a raw material through microbial fermentation. In my country, the main microorganism used for erythritol production is *Yarrowia lipolytica*, whose optimal growth and fermentation temperature is 30-32℃. The commonly used fermentation method for erythritol production is fed-batch fermentation. In this process, erythritol-producing strains are cultured in a two-stage seed tank and then transferred to a fermenter. After fermentation reaches a certain stage, carbon sources and other components are added to the fermenter. Once the desired yield is achieved, fermentation ends and the extraction process begins. The disadvantages of fed-batch fermentation include the need for specialized feeding tanks and continuous sterilization systems, a relatively long cycle for obtaining high concentrations of erythritol, and susceptibility to microbial contamination. Currently, the initial sugar concentration for fed-batch or batch fermentation of erythritol is generally no higher than 35% (w / v). Higher initial sugar concentrations can cause excessive stress on the strains, leading to slow growth, low yield, and other problems. Furthermore, in industrial production, the metabolic heat, mechanical stirring heat, and system heat generated during strain production often lead to elevated fermentation system temperatures (often reaching 35-37℃), causing a significant decline in the strain's production performance due to high-temperature stress. Currently, industrial production mainly relies on cooling water circulation for fermenter temperature control. However, this is affected by seasonal environmental temperature variations, especially in summer, making it difficult to achieve stable temperature control around 32℃. Therefore, the economic losses caused by unstable production due to high temperatures are substantial. Thus, developing osmotically tolerant strains capable of withstanding high temperatures and high glucose concentrations is of great significance. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide a *Yarrowia lipolytica* yeast and its applications. This *Yarrowia lipolytica* yeast exhibits high fermentation stability and high tolerance to high temperatures and high sugar concentrations. Even under high temperature and high initial sugar concentration conditions, it still possesses good erythritol production capacity. Using this *Yarrowia lipolytica* yeast to produce erythritol allows for batch fermentation with higher initial sugar concentrations, yielding higher erythritol content, and reduces the amount of cooling water or chilled water used for temperature control during fermentation.

[0005] To achieve the above objectives, the first aspect of the present invention provides a Yarrowialipolytica, characterized in that the Yarrowialipolytica has the preservation number CGMCC No. 23718.

[0006] The second aspect of the present invention provides the application of the above-described Yarrowia lipolytica in the fermentation of erythritol.

[0007] A third aspect of the present invention provides a method for fermenting to produce erythritol, characterized in that the method includes: inoculating the above-mentioned Yeast lipolyticis into a fermentation medium for fermentation to obtain a fermentation broth containing erythritol.

[0008] The fourth aspect of the present invention provides the use of the above-described Yarrowia lipolytica and / or its fermentation products in the preparation of sweeteners.

[0009] The *Yarrowia lipolytica* yeast provided by this invention exhibits high fermentation stability, tolerating glucose concentrations above 400 g / L, making it suitable for high-sugar batch fermentation production of erythritol. Furthermore, *Yarrowia lipolytica* can withstand temperatures of 35-37°C while still producing erythritol effectively, reducing the amount of cooling or chilled water used for temperature control during fermentation. In addition, using *Yarrowia lipolytica* yeast of this invention for erythritol production results in fewer fermentation byproducts such as mannitol, arabinitol, and glycerol, leading to higher purity erythritol in the fermentation broth, which is more conducive to crystallization and separation. The fermentation broth contains fewer macromolecular impurities, and no white viscous impurities are generated during separation, facilitating separation and extraction operations, reducing waste liquid volume, and increasing erythritol yield.

[0010] Biological Preservation

[0011] The strain of this invention is classified as Yarrowia lipolytica and was deposited on November 4, 2021, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCC No. 23718.

[0012] The starting strain used to obtain the strain of this invention is classified as *Yarrowialipolytica*, and was deposited on November 4, 2021, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCC No. 23717. Detailed Implementation

[0013] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0014] The first aspect of this invention provides a Yarrowia lipolytica, which has the accession number CGMCC No. 23718.

[0015] A second aspect of the present invention provides the application of the above-described Yeast Extract in the fermentation of erythritol.

[0016] A third aspect of the present invention provides a method for fermenting to produce erythritol, the method comprising: inoculating the above-mentioned Yeast lipolytica into a fermentation medium for fermentation to obtain a fermentation broth containing erythritol.

[0017] In some embodiments of the present invention, the fermentation conditions include: a fermentation temperature of 30-37°C and a fermentation time of 60-130 hours.

[0018] In some embodiments of the present invention, in order to further increase the yield of erythritol, the fermentation can be carried out at 30-32°C for 12-36 hours, and then the temperature can be raised to 35-37°C for 48-100 hours.

[0019] In some embodiments of the present invention, the method further includes: controlling the dissolved oxygen content in the fermentation broth to 10-35 vol% during the fermentation process. In this invention, the method for controlling the dissolved oxygen content in the fermentation broth can be a method commonly used in the art, such as controlling the aeration rate of the fermenter or the rotation speed of the fermenter, which can be adjusted according to actual conditions by those skilled in the art.

[0020] In this invention, the fermentation medium can be a commonly used culture medium in the art for fermenting erythritol.

[0021] In some embodiments of the present invention, the fermentation medium contains glucose. Preferably, the glucose content is 150-450 g relative to 1 L of the fermentation medium. More preferably, in order to produce a higher content of erythritol, the glucose content is 350-415 g relative to 1 L of the fermentation medium.

[0022] In some preferred embodiments of the present invention, the fermentation medium contains glucose, a nitrogen source, optional inorganic salts, and optional phytic acid; wherein, relative to 1L of the fermentation medium, the glucose content is 350-450g, the nitrogen source content is 5-30g, the inorganic salt content is 0-8g, and the phytic acid content is 0-0.5g.

[0023] In some embodiments of the present invention, the nitrogen source is at least one selected from yeast extract powder, yeast paste, corn steep liquor powder, ammonium citrate, ammonium sulfate, and diammonium hydrogen sulfate.

[0024] In some embodiments of the present invention, the inorganic salt is at least one of potassium dihydrogen phosphate, magnesium sulfate, and copper chloride.

[0025] In some embodiments of the present invention, the initial pH value of the fermentation medium is 5.5-6.5.

[0026] Unless otherwise specified, the solvent for the culture medium in this invention is deionized water, and the preparation method is a method commonly used in the art.

[0027] In this invention, unless otherwise specified, the preparation method of the culture medium can be a conventional choice in the art.

[0028] In some embodiments of the present invention, in order to save the raw material cost of the fermentation medium, the fermentation medium is prepared by adding a nitrogen source, optional inorganic salts, and optional phytic acid to the glucose solution.

[0029] The inventors of this invention have discovered that when using the *Yarrowia lipolytica* yeast described in this invention to ferment erythritol, the carbon source in the fermentation medium can be directly glucose solution, which can reduce raw material costs.

[0030] In this invention, the method for preparing glucose solution by saccharification of high-concentration starch liquid is as follows: Starch is mixed with water to prepare a starch slurry with a concentration of 40-45% (w / w). The pH is adjusted to 5.5-6.0. 0.1-0.25 g / kg (based on dry starch) of thermostable α-amylase is added, followed by spray liquefaction at a temperature of 108-115℃. The spray-liquefied solution is then laminar-flow liquefaction at 95-98℃ for 1.5-3 hours, followed by pH adjustment to 4.0-4.5 and temperature reduction to 58-62℃ before entering a saccharification tank. Saccharifying enzyme is added to the saccharification tank at a dosage of 0.25-0.45 g / kg (based on dry starch), and saccharification is carried out for 32-40 hours to obtain the glucose solution.

[0031] In this invention, those skilled in the art can select the mass concentration of the glucose solution according to actual needs, which will not be elaborated here.

[0032] In some embodiments of the present invention, the lipophilic yeast is inoculated in the form of a seed liquid.

[0033] In this invention, the method for preparing the seed liquid can be a conventional preparation method in the art, such as primary seed culture or secondary seed culture. Those skilled in the art can choose the culture method according to the actual situation.

[0034] In this invention, the primary seed culture can be a conventional method in the art, for example, it may include: inoculating glycerol bacteria into an activation medium and culturing at 28-32°C and 180-240 rpm for 16-30 hours. In this invention, the "glycerol bacteria" refers to *Yarrowia lipolyticis* preserved in glycerol, with a viable count generally of 10-1. 7 CFU / mL or higher. In this invention, the inoculum amount of the "glycerol bacteria" can be a conventional inoculum amount in the art, for example, 3‰ (v / v).

[0035] In this invention, the secondary seed culture can be a conventional method in the art. In some embodiments of this invention, the secondary seed culture is as follows: first, the glycerol bacteria or slant-preserved bacterial strain is inoculated into an activation medium and cultured at 28-32℃ and 180-240 rpm for 12-24 h; then, it is inoculated into a seed culture medium at an inoculation rate of 5-15% (v / v) and cultured at 28-32℃ and 180-350 rpm for 16-24 h to obtain the seed solution.

[0036] In some embodiments of the present invention, the activation culture medium can be a commonly used culture medium for activating Yeast Extract. Preferably, the activation culture medium contains 50-150 g / L glucose, 10-30 g / L yeast powder or yeast extract, and 0.2-1 g / L potassium dihydrogen phosphate.

[0037] In this invention, the seed culture medium can be any conventionally selected in the art, such as the fermentation culture medium as described above. Preferably, in order to improve the efficiency of seed liquid preparation, the seed culture medium contains 50-300 g / L glucose, 10-30 g / L yeast extract, 0.2-1 g / L potassium dihydrogen phosphate, and pH 5.5-6.5.

[0038] In some embodiments of the present invention, the seed culture medium is prepared by adding yeast extract and potassium dihydrogen phosphate to a glucose solution and adjusting the pH to 5.5-6.5. The glucose solution is prepared by the high-concentration starch liquid saccharification method described above, and will not be repeated here.

[0039] In this invention, the OD of the seed liquid 600 The value can be selected within a wide range; preferably, the OD value of the seed solution is... 600 The value is 8-25.

[0040] In this invention, the inoculation amount of the seed solution can be a commonly used inoculation amount in the art. In some embodiments of this invention, the inoculation amount of the seed solution is 5-15% (v / v).

[0041] A fourth aspect of the present invention provides the use of the above-described Yeast Extract and / or its fermentation products in the preparation of sweeteners.

[0042] The present invention will be described in detail below through embodiments.

[0043] In the examples below, unless otherwise specified, all reagents and raw materials used are commercially available.

[0044] Acclimation culture medium: glucose 400 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, ammonium citrate 5 g / L, pH 6;

[0045] Screening medium: glucose 450 g / L, yeast extract 15 g / L, agar 20 g / L;

[0046] Activation medium: glucose 150 g / L, yeast extract 15 g / L, potassium dihydrogen phosphate 0.5 g / L;

[0047] In the following examples, 42wt% glucose solution was prepared by saccharification of high-concentration starch liquid. The specific method was as follows: starch and water were mixed to prepare a starch slurry with a starch concentration of 42% (w / w). The pH was adjusted to 5.7-5.8, and 0.2 g / kg (based on dry starch) of thermostable α-amylase (purchased from Genentech (China) Biotechnology Co., Ltd.) was added before spray liquefaction at 115°C. The spray-liquefied solution was then laminarized at 100°C for 2 hours, and the pH was adjusted to 4.2-4.3. The temperature was then lowered to 60°C before entering the saccharification tank. Saccharifying enzyme (specifically Highdex Ultra1.0 complex saccharifying enzyme, purchased from Nanjing Bestgene Biotechnology Co., Ltd.) was added to the saccharification tank at a dosage of 0.4 g / kg (based on dry starch). After saccharification for 40 hours, glucose solution was obtained.

[0048] The concentration of glucose in the fermentation broth (supernatant) was detected by a biosensor: SBA-40E biosensor, D-glucase membrane, injection volume 25 μL;

[0049] The contents of erythritol, mannitol, arabinitol, and glycerol (all referring to the contents in the fermentation broth (supernatant)) were determined by liquid chromatography: an Agilent Technologies 1260 Infinity II chromatograph and RID detector were used, an Aminex HPX-87H Column 300×7.8mm separation column was used, and the mobile phase was 0.005M sulfuric acid.

[0050] Glycerin bacteria refers to *Yarrowia lipolyticis* preserved in glycerol, with a viable count of 10-1. 7 cfu / mL.

[0051] Glucose consumption refers to the glucose concentration in the fermentation medium minus the glucose concentration in the fermentation broth after fermentation.

[0052] The sugar alcohol conversion rate is the ratio of the mass of erythritol produced during fermentation to the mass of glucose consumed.

[0053] Example 1

[0054] This embodiment illustrates the mutagenesis, domestication, and fermentation test of Yarrowia lipolytica CGMCC No. 23718.

[0055] (1) Strains mutagenesis screening and domestication

[0056] The CoY1-MT (CGMCC No. 23717) strain was cultured to the logarithmic growth phase using activation medium, washed once with physiological saline, and then resuspended in physiological saline to obtain the OD. 600A bacterial suspension of 0.5 μL was subjected to alternating ARTP (Ambient Tolerancing Plasma) mutagenesis and acclimatization. The resulting strain was serially diluted and plated onto screening plates until single colonies emerged. Single colonies were picked and incubated overnight (20 h) on activation medium, then transferred to acclimatization medium at a 10% (v / v) inoculation rate and incubated at 37°C and 220 rpm. OD200 was measured after 24 h. 600 Values ​​were selected based on the fastest growth rate (OD) over 24 hours. 600 The 10 strains with the highest values ​​were numbered 1#-10#.

[0057] (2) Fermentation test

[0058] Fermentation tests were conducted on strains 1#-10# together with the starting strain as follows: The preserved glycerol bacteria were inoculated into activation medium at 3‰ v / v and activated at 30℃ and 200rpm for 20h. Then, 10% (v / v) was inoculated into 50ml of fermentation medium (the composition of the fermentation medium is: glucose 400g / L, yeast extract 5g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.2g / L, ammonium citrate 5g / L, pH 6) and cultured at 37℃ and 240rpm. Samples were taken every 24h to detect residual sugar (i.e., the concentration of glucose in the fermentation broth) and to replenish the evaporated water, and the amount of glucose consumed was calculated. Near the end of fermentation (24 hours before the fermentation endpoint; specifically, the method for determining this is as follows: after sampling and testing the glucose concentration of the fermentation broth every 24 hours, estimate the fermentation rate, and determine the fermentation endpoint (depletion of glucose) based on the glucose concentration and fermentation rate; 24 hours before the fermentation endpoint is the end of fermentation), sample and test the glucose concentration every 4 hours. When the glucose concentration is less than 1 g / L, stop fermentation and sample to test the erythritol content (i.e., erythritol yield).

[0059] The starting strain consumed only about 210 g / L of glucose and produced 122 g / L of erythritol after 132 h of fermentation, with about 5 g / L of mannitol as a byproduct. The glucose content showed no significant change in the last two samples. In contrast, strains 1-10 consumed more than 350 g / L of glucose in the culture medium after 132 h, yielding more than 195 g / L of erythritol. The best strain, strain 3, exhausted all glucose in 132 h, yielding 219.5 g / L of erythritol, with only 1.6 g / L of mannitol as a byproduct. Arabitol and glycerol were not detected. This strain was named CoY1-OT, with the preservation number CGMCC No. 23718.

[0060] Example 2

[0061] This embodiment is used to verify the fermentation performance of CoY1-OT in producing erythritol under the conditions of high initial sugar concentration, maintaining the optimal growth temperature of the strain in the early stage of fermentation, and maintaining high temperature only in the fermentation stage of erythritol production.

[0062] In this embodiment, the seed culture medium is: glucose 265g / L, yeast extract 20g / L, potassium dihydrogen phosphate 0.5g / L, pH 6;

[0063] The fermentation medium consisted of: 415 g / L glucose, 6 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate, 5 g / L ammonium sulfate, and pH 6.

[0064] Take the preserved CoY1-OT glycerol bacteria and inoculate it into 50 mL of activation medium at 3‰ v / v. Incubate overnight at 30℃ and 200 rpm (16 h). Then, transfer it to 300 mL of seed culture medium at 10% (v / v) inoculation rate and incubate at 30℃ and 240 rpm for 16 h (OD). 600 =20), to obtain seed liquid.

[0065] All seed culture was inoculated into a 5L fermenter containing fermentation medium. After inoculation, the fermenter was filled with 60% liquid volume, the rotation speed was 200-1000 rpm, and the aeration rate was 0.5-1 vvm (L / min·L). Dissolved oxygen was controlled at 20-25 vol%. The fermentation temperature was controlled at 32℃ for the first 24 hours, and then increased to 37℃. The glucose concentration in the fermentation broth was sampled every 12 hours. The fermentation rate was estimated based on the glucose concentration. The fermentation endpoint (glucose depletion) was determined based on the glucose concentration and fermentation rate. The glucose concentration was sampled every 0.5 hours for 12 hours before the fermentation endpoint.

[0066] Fermentation ended when the glucose concentration in the fermentation broth was less than 1 g / L. After fermentation, the content of erythritol and mannitol (a byproduct) in the fermentation broth was measured, and the sugar alcohol conversion rate was calculated.

[0067] Fermentation ended after 121 hours. The erythritol content in the fermentation broth was 235.6 g / L, the mannitol content of the byproduct was 1.2 g / L, and the sugar alcohol conversion rate of erythritol production was 58.9%.

[0068] The fermentation broth was treated using a centrifugation-microfiltration-nanofiltration process. First, the fermentation broth was centrifuged, and the supernatant was then subjected to microfiltration and nanofiltration. The processing conditions were: centrifugation at 4000 rpm for 15 min at 25°C; microfiltration using a ceramic membrane at an operating pressure of 0.15 MPa and a temperature of 30°C; and nanofiltration with a molecular weight cutoff of 250-300 Da at an operating pressure of 1 MPa and a temperature of 30°C. The microfiltration permeation-to-cutoff volume ratio of the CoY1-OT fermentation broth was 76% per unit time, and the nanofiltration permeation-to-cutoff volume ratio was 70% per unit time. The concentrated side of the fermentation broth after nanofiltration showed less liquid and no white viscous impurities, which is more conducive to subsequent crystallization and separation.

[0069] Example 3

[0070] This embodiment illustrates the performance of CoY1-OT in producing erythritol at high temperature in a fermentation medium prepared with glucose solution obtained by saccharification with high-concentration starch solution.

[0071] The preserved CoY1-OT glycerol bacteria were inoculated into 150 mL of activation medium at a 3‰ v / v inoculation rate for activation culture and cultured at 30℃ and 200 rpm for 20 h (OD = 10) to obtain seed culture. This seed culture was then inoculated into a 2 L fermenter containing fermentation medium at a 10% (v / v) inoculation rate. The fermenter's volume after inoculation was 60% by volume (the fermentation medium composition was: 42 wt% glucose solution 950 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L). The fermentation broth was prepared with the following concentrations: ammonium citrate 5 g / L, phytic acid 0.2 g / L, pH 5.7. The fermentation temperature was controlled at 37℃, the rotation speed was 200-1000 rpm, the aeration rate was 0.5 vvm (L / min·L), and the dissolved oxygen content was controlled at 20-25 vol%. The glucose concentration in the fermentation broth was sampled every 12 hours. The fermentation rate was estimated based on the glucose concentration. The fermentation endpoint (glucose depletion) was determined based on the glucose concentration and the fermentation rate. The glucose concentration was sampled every 1 hour for 12 hours before the fermentation endpoint.

[0072] Fermentation ended when the glucose concentration in the fermentation broth was less than 1 g / L. After fermentation, the content of erythritol and mannitol (a byproduct) in the fermentation broth was measured, and the sugar alcohol conversion rate was calculated.

[0073] Fermentation was completed after 126 hours. The erythritol content in the fermentation broth was 216.2 g / L, the mannitol content of the byproduct was 1.9 g / L, and the sugar alcohol conversion rate of erythritol production was 57.8%.

[0074] Example 4

[0075] This embodiment is used to illustrate and verify the performance of CoY1-OT in producing erythritol using high-concentration sugar solution as the main raw material under the condition of poor temperature control during the simulated fermentation process, which leads to an increase in temperature.

[0076] In this embodiment, the seed culture medium is prepared by adding yeast extract, potassium dihydrogen phosphate, and water to a 42wt% glucose solution, and adjusting the pH to 5.7. The composition ratio of the seed culture medium is: 630 g / L of 42wt% glucose solution, 15 g / L of yeast extract, 0.5 g / L of potassium dihydrogen phosphate, and pH 5.7.

[0077] CoY1-OT cultured on a slant was inoculated into 350 mL of activation medium and cultured overnight (16 h). It was then transferred to a 5 L fermenter containing seed culture medium. After inoculation, the volume of the fermenter was 70% by volume, the fermentation temperature was controlled at 30 °C, the rotation speed was constant at 350 rpm, the aeration rate was 0.5 vvm, and the culture was carried out for 12 h (OD = 18) to obtain the seed culture.

[0078] All seed culture was inoculated into a 70L fermenter containing fermentation medium. After inoculation, the volume of fermentation medium was 55% by volume (the composition of the fermentation medium was: 1000g / L 42wt% glucose solution, 5g / L yeast extract, 1g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate, 0.1mg / L copper chloride, 5g / L ammonium citrate, pH 5.7). The rotation speed was 200-800rpm, and the aeration rate was 0.3-0.8vvm (L / min·L) to maintain dissolved oxygen at 20-25vol%. The fermentation temperature was controlled at 32℃ for the first 30 hours, and then increased to 35℃. The glucose concentration in the fermentation broth was sampled every 12 hours to estimate the fermentation rate. The fermentation endpoint (glucose depletion) was determined based on the glucose concentration and fermentation rate. The glucose concentration was sampled every 0.5 hours for 12 hours before the fermentation endpoint.

[0079] Fermentation ended when the glucose concentration in the fermentation broth was less than 1 g / L. After fermentation, the content of erythritol and mannitol (a byproduct) in the fermentation broth was measured, and the sugar alcohol conversion rate was calculated.

[0080] Fermentation ended after 112.5 hours. The erythritol content in the fermentation broth reached 233.9 g / L, the mannitol content of the byproduct was 0.7 g / L, and the sugar alcohol conversion rate of erythritol production was 57.6%.

[0081] Example 5

[0082] This example illustrates the erythritol production performance of CoY1-OT under normal fermentation temperature of Yarrowia lipolytica and high initial sugar concentration.

[0083] Fermentation was carried out according to the method of Example 4, except that the fermentation temperature was controlled at 32°C.

[0084] Fermentation ended after 114 hours. The content of erythritol in the fermentation broth was 235.3 g / L, and the byproduct mannitol was basically undetectable. The sugar alcohol conversion rate of erythritol production was 58.0%.

[0085] Example 6

[0086] This example illustrates the erythritol production performance of CoY1-OT at normal initial sugar concentrations.

[0087] Fermentation was carried out according to the method of Example 4, except that the seed culture medium had the following composition: 240 g / L 42 wt% glucose solution, 15 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, pH 5.7; and the fermentation medium had the following composition: 790 g / L 42 wt% glucose solution, 5 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.1 mg / L copper chloride, 5 g / L ammonium citrate, pH 5.7.

[0088] After 90 hours of fermentation, the erythritol content in the fermentation broth was 182.9 g / L, the mannitol content of the byproduct was 0.8 g / L, and the sugar alcohol conversion rate of erythritol production was 58.8%.

[0089] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A type of lipophilic yeast ( Yarrowia lipolytica ), characterized in that, The preservation number of this *Yarlium lipophilum* is CGMCC No. 23718.

2. The application of the *Yarrowia lipolytica* as described in claim 1 in the fermentation production of erythritol.

3. A method for fermenting erythritol, characterized in that, The method includes: inoculating the *Yarrowia lipolytica* as described in claim 1 into a fermentation medium for fermentation to obtain a fermentation broth containing erythritol.

4. The method according to claim 3, wherein, The fermentation conditions include: a fermentation temperature of 30-37℃ and a fermentation time of 60-130 h.

5. The method according to claim 4, wherein, The method further includes controlling the dissolved oxygen content in the fermentation broth to 10-35 vol during the fermentation process.

6. The method according to any one of claims 3-5, wherein, The fermentation medium contains glucose; The glucose content is 150-450 g relative to 1 L of the fermentation medium.

7. The method according to claim 6, wherein, The glucose content is 350-415 g relative to 1 L of the fermentation medium.

8. The method according to claim 3, wherein, The initial pH of the fermentation medium is 5.5-6.

5.

9. The method according to claim 3, wherein, The lipophilic yeast was inoculated using a seed culture.

10. The use of the Yeast Lysimachia christinae according to claim 1 and / or its fermentation products in the preparation of erythritol.