Yarrowia lipolytica and application thereof

Abstract

The invention relates to a saccharomyces lipolytica strain, a breeding method and application. The Yarrowia lipolytica strain is preserved in the China Center for Type Culture Collection (CCTCC) on July 22, 2024, and the preservation number of the Yarrowia lipolytica strain is CCTCC NO: M 20241641. When the arrowia lipolytica strain is used for preparing the single-cell protein, the conversion rate of the single-cell protein reaches 40% or above, a continuous fermentation process is adopted, the production efficiency is improved by 50% or above, and the production efficiency reaches 2.5 g / L / h or above.

Description

Technical Field

[0001] The present application relates to the field of biotechnology microorganisms, and in particular to a strain of Yarrowia lipolytica and its application. Background Art

[0002] Single-cell protein, also known as microbial protein, mainly refers to single-cell organisms such as bacteria, yeast, and algae. The current demand for protein is very huge. At the same time, the production capacity of acetic acid is growing.

[0003] In order to cope with the problems of overcapacity and insufficient market demand, some companies have begun to explore new downstream products, such as acetic acid hydrogenation to produce ethanol, in order to expand market demand and alleviate the pressure of overcapacity.

[0004] Enterprises are responding to this challenge through technological innovation and market expansion. The development of a single-cell protein technology using acetate as a carbon source has important practical significance. Summary of the invention

[0005] Based on this, it is necessary to provide at least one Yarrowia lipolytica strain and its application.

[0006] In a first aspect of the present application, a strain of Yarrowia lipolytica is provided. The strain is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20241641 and a deposit date of July 22, 2024.

[0007] In a second aspect of the present application, a culture is provided, comprising the Yarrowia lipolytica as described in the first aspect.

[0008] In a third aspect of the present application, there is provided a use of the Yarrowia lipolytica as described in the first aspect or the culture medium as described in the second aspect in preparing single-cell protein.

[0009] In a fourth aspect of the present application, a method for producing single-cell protein is provided, comprising: fermenting and culturing the Yarrowia lipolytica described in the first aspect to prepare the single-cell protein.

[0010] The fifth aspect of the present application provides a method for screening strains that efficiently utilize acetic acid to produce single-cell protein.

[0011] Compared with the traditional technology, the beneficial effects of the technical solution in this application include at least:

[0012] 1. Provide a new strain screening method, which uses microorganisms in distiller's grains in Meishan, Sichuan to screen out strains that can efficiently utilize acetic acid to produce single-cell protein. This method not only simplifies the strain screening process and improves the screening efficiency, but also the screened strains have higher acetic acid utilization and single-cell protein production efficiency, thereby improving production efficiency and economic benefits.

[0013] 2. The method for producing single-cell protein using acetic acid by the strain provided in this application can be combined with optimized fermentation conditions and process control to achieve high-density culture and high-efficiency single-cell protein production. Compared with traditional technologies, the control and optimization of the fermentation process in this method is more precise and efficient, thereby improving production efficiency and economic benefits.

[0014] 3. The strain and production method of the present application do not require genetic engineering modification, thus avoiding the risks and complexity that may be brought about by genetic engineering technology, and also reducing production costs. At the same time, the strain and production method of the present application have good stability and reproducibility, and are suitable for large-scale production.

[0015] In summary, compared with traditional technologies, this application provides a more efficient, stable and economical single-cell protein production method with significant advantages and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0017] Figure 1 The strain screening process in one embodiment of the present application is shown.

[0018] A Yarrowia lipolytica strain was deposited in China Center for Type Culture Collection (CCTCC), deposit address: Wuhan University, Wuhan, China, with deposit number: CCTCC NO:M 20241641, and deposit date: July 22, 2024. DETAILED DESCRIPTION

[0019] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0021] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0022] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0023] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0024] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0025] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0026] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0027] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0028] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0029] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0030] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0031] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0032] One aspect of the present application provides a strain of Yarrowia lipolytica, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20241641 and a deposit date of July 22, 2024.

[0033] Another aspect of the present application provides a composition comprising Yarrowia lipolytica as described above.

[0034] The composition may be, for example, a culture obtained by inoculating the above-mentioned Yarrowia lipolytica into a liquid culture medium and performing fermentation culture.

[0035] Another aspect of the present application provides use of the Yarrowia lipolytica or culture as described above in preparing single-cell protein.

[0036] Another aspect of the present application provides a method for producing single-cell protein, which comprises: fermenting and culturing the Yarrowia lipolytica as described above to prepare the single-cell protein.

[0037] In some embodiments, the method comprises isolating the single cell protein from a culture as described above.

[0038] In some embodiments, the fermentation medium for fermentation culture comprises: 0.1 g / L~2 g / L potassium chloride, 0.2 g / L~2 g / L magnesium sulfate heptahydrate, 0.2 g / L~2 g / L calcium chloride, 1 g / L~5 g / L potassium dihydrogen phosphate, 1 g / L~5 g / L sodium hydrogen phosphate, and 1 ml / L~5 ml / L trace element stock solution, and the balance is water; wherein the trace element stock solution comprises: 5 g / L~10 g / L citric acid, 20 g / L~25 g / L ferrous sulfate heptahydrate, 18 g / L~21 g / L zinc sulfate heptahydrate, 5 g / L~6 g / L manganese sulfate monohydrate, 4 g / L~8 g / L copper sulfate pentahydrate, 0.2 g / L~0.4 g / L ammonium molybdate tetrahydrate, 0.6 g / L~0.8 g / L cobalt chloride hexahydrate and 0.2 g / L~0.5 g / L boric acid; the pH of the fermentation medium is 7.2~7.5.

[0039] In some embodiments, the concentration of potassium chloride in the fermentation medium is 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2.0 g / L, or a range or value between any two values.

[0040] In some embodiments, the concentration of magnesium sulfate heptahydrate in the fermentation medium is 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2.0 g / L, or a range or value between any two values.

[0041] In some embodiments, the concentration of calcium chloride in the fermentation medium is 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2.0 g / L, or a range or value between any two values.

[0042] In some embodiments, the concentration of potassium dihydrogen phosphate in the fermentation medium is 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, or a range or value between any two values.

[0043] In some embodiments, the concentration of disodium hydrogen phosphate in the fermentation medium is 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, or a range or value between any two values.

[0044] In some embodiments, the concentration of the trace element stock solution in the fermentation medium is 1 mL / L, 1.5 mL / L, 2 mL / L, 2.5 mL / L, 3 mL / L, 3.5 mL / L, 4 mL / L, 4.5 mL / L, 5 mL / L, or a range or value between any two values.

[0045] In some embodiments, the concentration of citric acid in the trace element stock solution is 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, or a range or value between any two values.

[0046] In some embodiments, the concentration of ferrous sulfate heptahydrate in the trace element storage solution is 20 g / L, 20.5 g / L, 21 g / L, 21.5 g / L, 22 g / L, 22.5 g / L, 23 g / L, 23.5 g / L, 24 g / L, 24.5 g / L, 25 g / L, or a range or value between any two values.

[0047] In some embodiments, the concentration of zinc sulfate heptahydrate in the trace element stock solution is 18 g / L, 18.5 g / L, 19 g / L, 19.5 g / L, 20 g / L, 20.5 g / L, 21 g / L, or a range or value between any two values.

[0048] In some embodiments, the concentration of manganese sulfate monohydrate in the trace element stock solution is 5 g / L, 5.1 g / L, 5.2 g / L, 5.3 g / L, 5.4 g / L, 5.5 g / L, 5.6 g / L, 5.7 g / L, 5.8 g / L, 5.9 g / L, 6 g / L, or a range or value between any two values.

[0049] In some embodiments, the concentration of copper sulfate pentahydrate in the trace element storage solution is 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, or a range or value between any two values.

[0050] In some embodiments, the concentration of ammonium molybdate tetrahydrate in the trace element stock solution is 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, or a range or value between any two values.

[0051] In some embodiments, the concentration of cobalt chloride hexahydrate in the trace element stock solution is 0.6 g / L, 0.65 g / L, 0.7 g / L, 0.75 g / L, 0.8 g / L, or a range or value between any two values.

[0052] In some embodiments, the concentration of boric acid in the trace element stock solution is 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, 0.5 g / L, or a range or value between any two values.

[0053] In some embodiments, the fermentation medium comprises 0.2 g / L potassium chloride, 0.5 g / L magnesium sulfate heptahydrate, 1 g / L calcium chloride, 4 g / L potassium dihydrogen phosphate, 5 g / L sodium dihydrogen phosphate, 1 ml / L trace element stock solution, and water; wherein the trace element stock solution comprises: 6 g / L citric acid, 25 g / L ferrous sulfate heptahydrate, 20 g / L zinc sulfate heptahydrate, 5 g / L manganese sulfate monohydrate, 5 g / L copper sulfate pentahydrate, 0.3 g / L ammonium molybdate tetrahydrate, 0.7 g / L cobalt chloride hexahydrate and 0.5 g / L boric acid, and the balance is water; the pH of the fermentation medium is 7.4.

[0054] In some embodiments, the method comprises:

[0055] (1) Inoculate the seed liquid of Yarrowia lipolytica into the fermentation medium at a volume ratio of 0.1% to 10%;

[0056] (2) Cultivate with stirring at 29℃~31℃, with a stirring speed of 100rpm~1000rpm; ventilation volume of 0.1 vvm~3vvm, and control the dissolved oxygen at 15%~20%.

[0057] The volume of the seed liquid to the fermentation medium may be a conventional proportion in the art. In some embodiments, the seed liquid accounts for 1% to 9% of the volume of the fermentation medium. In some embodiments, the seed liquid accounts for 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% of the volume of the fermentation medium, or a range or value between any two values. In some embodiments, the seed liquid accounts for 2% to 6% of the volume of the fermentation medium.

[0058] In some embodiments, the OD of the seed solution is 600 3 or more.

[0059] In some embodiments, during the culture process, the pH is controlled at 6.5-7.5, the acetic acid concentration is 1 g / L-20 g / L, and the ammonia nitrogen concentration is 1 g / L-5 g / L.

[0060] In some embodiments, the pH during the culture process is controlled to be 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or a range or value between any two values.

[0061] In some embodiments, the concentration of acetate during the culture process is controlled to be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, or a range or value between any two values. In some embodiments, the concentration of acetate during the culture process is controlled to be 2 g / L to 15 g / L. In some embodiments, the concentration of acetate during the culture process is controlled to be 3 g / L to 5 g / L.

[0062] In some embodiments, the concentration of ammonia nitrogen during the culture process is controlled to be 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, or a range or value between any two values. In some embodiments, the concentration of ammonia nitrogen during the culture process is controlled to be 3 g / L to 4 g / L.

[0063] In some embodiments, the pH, acetic acid concentration and ammonia nitrogen concentration are controlled by adding 10% to 100% (v / v) acetic acid solution and 10% to 100% (v / v) ammonia water during the culture process.

[0064] In another aspect of the present application, a method for screening strains that efficiently utilize acetic acid to produce single-cell protein is provided, and the method involves strain screening using distiller's grains from Meishan area of ​​Sichuan.

[0065] In some embodiments, the method comprises:

[0066] The lees collected in Meishan, Sichuan, were added with 0.1% to 10% (m / m) acetic acid aqueous solution at a ratio of 1 g / L to 10 g / L, and the culture medium was prepared by shaking at 28°C to 32°C and 150 rpm to 250 rpm for 10 h to 20 h.

[0067] The culture solution is applied to a plate prepared with a screening medium, cultured at 29° C. to 31° C. for 15 h to 48 h, and red or yellow-orange colonies are picked; streaking is performed on the plate prepared with the screening medium, and larger colonies are picked for shake flask fermentation and continuous subculture to prepare a strain that efficiently utilizes acetic acid to produce single-cell protein;

[0068] Wherein, the screening medium comprises: 10 g / L~20 g / L peptone, 1 g / L~20 g / L sodium acetate, 0.5 g / L~2 g / L phenol red or 0.01 g / L~0.05 g / L neutral red, and 15 g / L~20 g / L agar;

[0069] The culture medium for shake flask fermentation contains: 10 g / L~20 g / L peptone and 10 g / L~50 g / L sodium acetate.

[0070] In some embodiments, the concentration of the acetic acid aqueous solution is 0.1 (m / m), 0.2 (m / m), 0.3 (m / m), 0.4 (m / m), 0.5 (m / m), 0.6 (m / m), 0.7 (m / m), 0.8 (m / m), 0.9 (m / m), 1% (m / m), 2% (m / m), 3% (m / m), 4% (m / m), 5% (m / m), 6% (m / m), 7% (m / m), 8% (m / m), 9% (m / m), 10% (m / m), or a value or range between any two values. In some embodiments, the concentration of the acetic acid aqueous solution is 1% to 8% (m / m). Exemplarily, in some embodiments, the concentration of the acetic acid aqueous solution is 3% to 6% (m / m).

[0071] In some embodiments, the mass volume ratio of vinasse to acetic acid aqueous solution is 2 g / L to 8 g / L. Exemplarily, the mass volume ratio is 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, or a value or range between any two values. In some embodiments, the mass volume ratio is 4 g / L to 6 g / L.

[0072] In some embodiments, the concentration of peptone in the screening medium is 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, or a range or value between any two values.

[0073] In some embodiments, the concentration of sodium acetate in the screening medium is 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, or a range or value between any two values. In some embodiments, the concentration of sodium acetate in the screening medium is 5 g / L to 15 g / L. In some embodiments, the concentration of sodium acetate is 10 g / L to 14 g / L.

[0074] In some embodiments, the concentration of phenol red in the screening medium is 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2 g / L, or a range or value between any two values. In some embodiments, the concentration of phenol red in the screening medium is 1.01 g / L.

[0075] In some embodiments, the concentration of neutral red in the screening medium is 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, or a range or value between any two values.

[0076] In some embodiments, the concentration of agar in the screening medium is 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, or a range or value between any two values.

[0077] In some embodiments, the concentration of peptone in the culture medium for shake flask fermentation is 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, or a range or value between any two values.

[0078] In some embodiments, the concentration of sodium acetate in the culture medium for shake flask fermentation is 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L g / L, 48 g / L, 49 g / L, 50 g / L, or a range or value between any two values. In some embodiments, the concentration of sodium acetate in the culture medium for shake flask fermentation is 15 g / L to 40 g / L. In some embodiments, the concentration of sodium acetate in the culture medium for shake flask fermentation is 15 g / L to 20 g / L.

[0079] In some embodiments, the screening method comprises the following steps:

[0080] 1) Prepare 0.1%~10% (m / m) acetic acid aqueous solution, without adjusting pH, sterilize at 121℃ for 20min, and cool to room temperature for later use;

[0081] 2) Add the vinasse collected from Meishan, Sichuan to the above-mentioned acetic acid aqueous solution at a ratio of 1 g / L to 10 g / L, and shake at 30°C and 200 rpm for 10 h to 20 h;

[0082] 3) Take 0.2 mL of the aqueous solution obtained in step 2) and apply it on the screening medium: 20 g of peptone, 1 g ~ 20 g of sodium acetate, 1.01 g of phenol red (or 0.03 g of neutral red), 20 g of agar, dilute to 1000 mL with water, adjust the pH to 7.4, and inactivate at 121°C for 20 min.

[0083] 4) Spread the plate and incubate in a 30℃ incubator for 30h, pick the red colonies (or yellow-orange) on the plate, streak on the above screening plate, pick the larger colonies and inoculate them in the evolution shake flask: 20g of peptone, 10 g~50 g of sodium acetate, dilute to 1000mL with water, adjust pH to 7.4, inactivate at 121℃, 20min. Incubate at 30℃, 200rpm for 15h, take 0.2mL of the culture medium and inoculate it in a 100mL evolution shake flask, continue to passage, obtain strains with higher acetic acid utilization rate, mix the culture with 50% glycerol in a 1:1 ratio, and dispense into cryopreservation tubes at -80℃ or liquid nitrogen for storage.

[0084] In some embodiments, the treatment time on the shaking table in step 2) is 10 h to 18 h. Exemplarily, the treatment time is 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, or a value or range between any two values. In some embodiments, the treatment time on the shaking table in step 2) is 14 h to 16 h.

[0085] Some examples are provided below.

[0086] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0087] Main raw material information of the embodiment of the present invention:

[0088] Yeast powder and peptone were purchased from OXOID, and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0089] Cell dry weight detection method:

[0090] Weigh the dried centrifuge tube and calculate it as ml;

[0091] Place one milliliter of the culture solution to be tested into the above centrifuge tube, centrifuge at 13,000 rpm, and remove the supernatant; control the oven temperature at 105°C, and dry the above centrifuge tube containing bacteria until the mass no longer changes, which is counted as m2.

[0092] It can be seen that the dry weight of cells contained in 1 mL of culture medium is m=m2-m1.

[0093] Example 1 Strain Screening

[0094] The lees come from the winery in Meishan, Sichuan. The screening method is as follows:

[0095] 1) Prepare 5% (m / m) acetic acid aqueous solution, without adjusting pH, sterilize at 121°C for 20 min, and cool to room temperature for later use;

[0096] 2) Add 5 g / L of the vinasse collected from Meishan, Sichuan to the acetic acid aqueous solution and shake at 30°C and 200 rpm for 15 h;

[0097] 3) Take 0.2 mL of the above aqueous solution and spread it on the screening medium: 20 g of peptone, 10 g of sodium acetate, 1.01 g of phenol red, 20 g of agar, dilute to 1000 mL with water, adjust the pH to 7.4, and inactivate at 121°C for 20 min.

[0098] 4) Spread the plate and incubate in a 30℃ incubator for 30h. Pick the red colonies on the plate, streak them on the screening plate, pick the larger colonies and inoculate them into evolution shake flasks (see Figure 1 ): 20g peptone, 15g sodium acetate, dilute to 1000mL with water, adjust pH to 7.4, inactivate at 121℃, 20min. Cultivate at 30℃, 200rpm for 15h, take 0.2mL of culture medium and inoculate into 100mL evolution shake flask, continue to passage, and obtain strains with high acetic acid utilization. ITS sequencing of the strain confirmed that it is Yarrowia lipolytica.

[0099] 5) Mix the culture medium with 50% (v / v) glycerol in a 1:1 ratio and dispense into cryovials for storage at -80°C or in liquid nitrogen.

[0100] After screening, 10 strains, namely 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9# and 10#, were obtained, and their efficiency in producing single-cell protein using acetic acid was further determined.

[0101] Example 2

[0102] Shake bottle seeds:

[0103] 1) Take the bacteria from the glycerol tube and inoculate 0.2 mL into a 100 mL YPD shake flask: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L. Cultivate at 30°C, 200 rpm for 15 h, or until OD 600 When the concentration reaches 3 or above, it is used as seed liquid to inoculate the fermentation tank.

[0104] Fermentation culture:

[0105] 1) Fermentation medium: 0.2 g / L potassium chloride, 0.5 g / L magnesium sulfate heptahydrate, 1 g / L calcium chloride, 4 g / L potassium dihydrogen phosphate, 5 g / L sodium hydrogen phosphate, 1 ml / L trace element stock solution, the balance is water; ammonia water to adjust pH to 7.4, trace element composition: 6 g / L citric acid, 25 g / L ferrous sulfate heptahydrate, 20 g / L zinc sulfate heptahydrate, 5 g / L manganese sulfate monohydrate, 5 g / L copper sulfate pentahydrate, 0.3 g / L ammonium molybdate tetrahydrate, 0.7 g / L cobalt chloride hexahydrate and 0.5 g / L boric acid, the balance is water.

[0106] 2) Inoculate the seed liquid into the fermentation medium at a ratio of 5% (v / v), culture at 30°C, stir at 100-1000 rpm, aerate at 0.1-3 vvm, control the dissolved oxygen at 20%, add 50% (v / v) acetic acid solution and concentrated ammonia water during the culture process, control the pH to 7.4, and keep the acetic acid residue at 2 g / L and the ammonia nitrogen at 1 g / L. Cultivate for 40 h, and stop the culture when the cell dry weight reaches 100 g / L.

[0107] It was determined that the production rates and conversion rates of single-cell protein produced by strains 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9# and 10# using acetic acid fermentation are shown in Table 1.

[0108] Table 1

[0109]

[0110] The data in Table 1 show that the production rate of strain 6# can reach 2.5 g / L / h and the conversion rate can reach 40%. The strain was deposited and obtained the deposit number: CCTCC NO:M 20241641.

[0111] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0112] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A strain of Yarrowia lipolytica, which was deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20241641 and a deposit date of July 22, 2024.

2. A culture, characterized in that It contains the Yarrowia lipolytica according to claim 1.

3. Use of the Yarrowia lipolytica according to claim 1 or the culture according to claim 2 in the preparation of single-cell protein.

4. A method for producing single cell protein, characterized in that: It includes: The Yarrowia lipolytica described in claim 1 is fermented and cultured to prepare single-cell protein.

5. The method according to claim 4, characterized in that The fermentation medium used for fermentation culture contains: 0.1 g / L~2 g / L potassium chloride, 0.2 g / L~2 g / L magnesium sulfate heptahydrate, 0.2 g / L~2 g / L calcium chloride, 1 g / L~5 g / L potassium dihydrogen phosphate, 1 g / L~5 g / L disodium hydrogen phosphate, and 1 ml / L~5 ml / L trace element stock solution, and the balance is water; Wherein, the trace element stock solution comprises: 5 g / L~10 g / L citric acid, 20 g / L~25 g / L ferrous sulfate heptahydrate, 18 g / L~21 g / L zinc sulfate heptahydrate, 5 g / L~6 g / L manganese sulfate monohydrate, 4 g / L~8 g / L copper sulfate pentahydrate, 0.2 g / L~0.4 g / L ammonium molybdate tetrahydrate, 0.6 g / L~0.8 g / L cobalt chloride hexahydrate and 0.2 g / L~0.5 g / L boric acid; The pH of the fermentation medium is 7.2-7.5; Optionally: The fermentation medium comprises 0.2 g / L potassium chloride, 0.5 g / L magnesium sulfate heptahydrate, 1 g / L calcium chloride, 4 g / L potassium dihydrogen phosphate, 5 g / L sodium dihydrogen phosphate, 1 ml / L trace element stock solution, and water; wherein the trace element stock solution comprises: 6 g / L citric acid, 25 g / L ferrous sulfate heptahydrate, 20 g / L zinc sulfate heptahydrate, 5 g / L manganese sulfate monohydrate, 5 g / L copper sulfate pentahydrate, 0.3 g / L ammonium molybdate tetrahydrate, 0.7 g / L cobalt chloride hexahydrate and 0.5 g / L boric acid, and the balance is water; the pH of the fermentation medium is 7.

4.

6. The method according to claim 4 or 5, characterized in that The method comprises: The seed liquid of Yarrowia lipolytica is inoculated into the fermentation medium at a volume ratio of 0.1% to 10%; Cultivate with stirring at 29℃~31℃, stirring speed 100rpm~1000rpm; ventilation volume 0.1 vvm~3vvm, control dissolved oxygen 15%~20%; Optionally, the seed solution satisfies one or more of the following conditions 1) to 2): 1) the seed liquid accounts for 1% to 9% of the volume of the fermentation medium; optionally 2% to 6%; and, 2) OD of the seed solution 600 3 or more.

7. The method according to claim 6, characterized in that During the culture process, the pH was controlled at 6.5-7.5, the acetic acid concentration was 1 g / L-20 g / L, and the ammonia nitrogen concentration was 1 g / L-5 g / L; in: The pH during the culture process is optionally controlled to 7.4; During the culture process, the concentration of acetic acid may be optionally controlled to be 2 g / L to 15 g / L, and may further be optionally controlled to be 3 g / L to 5 g / L; The ammonia nitrogen concentration during the culture process can be optionally controlled to 3 g / L~4 g / L.

8. The method according to claim 7, characterized in that The pH, acetic acid concentration and ammonia nitrogen concentration were controlled by adding 10%~100% (v / v) acetic acid solution and 10%~100% (v / v) ammonia water during the culture process.

9. A method for screening a strain that efficiently utilizes acetic acid to produce single-cell protein, characterized in that: It includes: The lees collected in Meishan, Sichuan, were added with 0.1% to 10% (m / m) acetic acid aqueous solution at a ratio of 1 g / L to 10 g / L, and the culture medium was prepared by shaking at 28°C to 32°C and 150 rpm to 250 rpm for 10 h to 20 h. The culture solution is applied to a plate prepared with a screening medium, cultured at 29° C. to 31° C. for 15 h to 48 h, and red or yellow-orange colonies are picked; streaking is performed on the plate prepared with the screening medium, and larger colonies are picked for shake flask fermentation and continuous subculture to prepare a strain that efficiently utilizes acetic acid to produce single-cell protein; Wherein, the screening medium comprises: 10 g / L~20 g / L peptone, 1 g / L~20 g / L sodium acetate, 0.5 g / L~2g / L phenol red or 0.01 g / L~0.05 g / L neutral red, and 15 g / L~20 g / L agar; The culture medium for shake flask fermentation contains: 10 g / L~20 g / L peptone and 10 g / L~50 g / L sodium acetate.

10. The method according to claim 9, characterized in that It satisfies one or more of the following conditions i) to iii): i) the concentration of the acetic acid aqueous solution is 1% to 8% (v / v); ii) the concentration of sodium acetate in the screening medium is 5 g / L to 15 g / L; and, iii) The concentration of sodium acetate in the culture medium used for shake flask fermentation is 15 g / L~40 g / L.

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