Yarrowia lipolytica strains and methods of fermentative production of gamma-decalactone or delta-decalactone

Abstract

The application provides a Yarrowia lipolytica strain and a method for preparing gamma-decalactone and delta-decalactone. The Yarrowia lipolytica strain is named Y.lipolytica OMK-95. The Y.lipolytica OMK-95 is used for fermenting to prepare gamma-decalactone and delta-decalactone, and the efficiency is high, and the method meets the requirements of green industrial manufacturing.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a *Yarrowia lipolytica* strain and a method for fermenting and preparing γ-decyl lactone or δ-decyl lactone. Background Technology

[0002] R-propyldecanolide is a compound, a colorless to pale yellow clear liquid, with the molecular formula C. 10 H 18 O2 has a strong coconut and peach-like fruity aroma and is mainly used in fragrances and flavorings.

[0003] Eudelactone is an edible flavoring with creamy, coconut, and peach-like fruity aromas. It is used as a food flavoring, daily-use flavoring, and other functional additive. Besides being synthesized artificially, it is also found in coconut and raspberry.

[0004] Early lactone fragrances were all synthesized chemically. A typical example is the discovery by Kula et al. in 1996, who obtained (R)-(-)-1,3-nonanediol by ozone decomposition of ricinoleic acid, and then cyclized it to form γ-decyl lactone while maintaining the configuration of the chiral hydroxyl group. Chemical synthesis methods require a large number of chemical raw materials, and the synthesized γ-decyl lactone is not a natural fragrance. With the US, Europe, and my country defining fragrances obtained through enzymatic catalysis or microbial fermentation as natural fragrances, the biosynthesis of lactone fragrances has become an important research direction in the fragrance industry, and industrial production has been gradually achieved both domestically and internationally. After a long period of research and development, representative achievements emerged in the late 20th century. In 1991, Cardillo et al. synthesized gamma-dodecyl lactone and butyl lactone by microbial metabolism using hydroxy long-chain fatty acids or long-chain fatty acid peroxides as raw materials and Pichia pastoris. In 1996, Nicant et al. produced 9.5 g / L gamma-decyl lactone by fermenting methyl ricinoleate with Y. lipolytica PO1d for 75 h. In 2000, Rabenhorst and Gatfield synthesized a mixture of gamma-decyl lactone and hydroxyl lactone at 11 g / L by fermenting castor oil with Y. lipolytica HR145 for 70 h. Jung-Ung's research group used 10-hydroxystearic acid or 12-hydroxystearic acid as raw materials to obtain 51 g / L (R)-propyldodecyl lactone, 28 g / L (R)-propyldecyl lactone, and 12 g / L (R)-propylbutyrolactone through β-oxidation in Waltomyceslipofer cells. These products have certain industrial application value, but the separation and purification of the three lactones produced are quite difficult, especially the separation of (R)-propyldodecyl lactone and (R)-propyldecyl lactone.

[0005] The main methods for preparing gamma-decyl lactone by bioenzymatic methods are as follows: (1) Baeyer-Villiger oxidation of cyclic ketone compounds catalyzed by BVMO monooxygenase; (2) kinetic resolution of racemic alkyl lactone compounds catalyzed by hydrolytic enzymes; (3) selective oxidation of the terminal hydroxyl group of diol compounds by alcohol dehydrogenases, the resulting hydroxyaldehyde cyclization to form a hemiacetal, which is then further oxidized to generate lactone. Using these biosynthetic methods, the process conditions are mild, and chiral products can be obtained. A typical example is the mechanism of gamma-decyl lactone synthesis from castor oil hydroxy fatty acids. The main component of castor oil is 12-hydroxy-9-octadecenoic acid. Microorganisms shorten the carbon chain through four β-oxidations, and then lactonize it to generate gamma-decyl lactone. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for synthesizing two natural substances, R-gamma-decanolide and doxy-decanolide, from castor oil via the metabolism of castor oil by the microorganism Y. lipolytica OMK-95. This provides a low-cost, efficient, and environmentally friendly industrial manufacturing method for the synthesis of natural gamma-decanolide and doxy-decanolide, offering a novel approach, particularly for the synthesis of doxy-decanolide. Doxy-decanolide, an important flavoring agent, possesses creamy, coconut, and peach-like fruity aromas and is widely used in the food and other fields. Non-chemical, green synthesis methods are an important source of these substances.

[0007] On the one hand, the present invention provides a strain of Y. lipolytica OMK-95, which has been deposited at the Microbial Culture Collection Center of Wuhan University with accession number CCTCC M 20231427.

[0008] The Yarrowia lipolytica strain described in this invention, named Yarrowia lipolytica OMK-95, was deposited at the China Center for Type Culture Collection, Wuhan University on August 10, 2023, with accession number CCTCC M 20231427.

[0009] On the other hand, the present invention also provides a method for preparing gamma-decyl lactone or doxy-decyl lactone, characterized in that gamma-decyl lactone or doxy-decyl lactone is obtained by biological fermentation culture using Y. lipolytica OMK-95, accession number CCTCC M 20231427.

[0010] In some embodiments, the fermentation medium comprises carbon compounds, nitrogen compounds, and inorganic salts, wherein the carbon compounds include at least castor oil, and the concentration of the castor oil is 10-60 g / L; preferably, the concentration of the castor oil is 30-40 g / L.

[0011] In some embodiments, the carbon compound further includes other carbon sources, including one or more combinations of sugars, sugar alcohols, alcohols, organic acids, complex mixtures, and vegetable oils; preferably, the carbon compound further includes one or more combinations of glucose, glycerol, mannitol, yeast extract, yeast powder, and malt extract; preferably, the carbon compound includes glucose and yeast powder; preferably, the concentration of the other carbon sources is 5-30 g / L.

[0012] In some embodiments, the nitrogen compound includes one or more combinations of nitrates, ammonium salts, yeast powder, yeast extract, soybean flour, and corn steep liquor; preferably, the nitrogen compound includes one or more combinations of ammonium sulfate, urea, and peptone; preferably, the concentration of the nitrogen compound is 15-30 g / L.

[0013] In some embodiments, the inorganic salt includes one or more of borates, carbonates, chlorides, molybdates, nitrates, phosphates, and sulfates; the metallic portion of the salt includes one or more of calcium, iron, potassium, cobalt, copper, magnesium, cobalt, sodium, or zinc; preferably, the inorganic salt includes magnesium sulfate and potassium dihydrogen phosphate; preferably, the concentration of the inorganic salt is 5-20 g / L.

[0014] In some embodiments, the fermentation medium further includes trace elements.

[0015] In some embodiments, the method involves inoculating seeds into a fermentation medium for fermentation, controlling the fermentation temperature at 26-30℃, the pH at 6-7, the stirring speed at 300-600 rpm, and the aeration ratio at 1:0.3-0.8.

[0016] In some embodiments, castor oil is added to the fermentation system once or multiple times; preferably, castor oil is added during fermentation cycles of 50-60h and 80-90h; preferably, castor oil is added until the concentration of castor oil in the fermentation system is 10-60g / L.

[0017] Beneficial effects

[0018] 1. The strain Y. lipolytica OMK-95 provided by this invention has a high yield of gamma-decyl lactone in fermentation, up to 35.2 g / L; it can also produce d-decyl lactone, an important flavoring agent.

[0019] 2. The method for producing propyl-decyl lactone or d-decyl lactone by fermentation using Y. lipolytica OMK-95 provided by this invention is simple and suitable for industrial production.

[0020] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0021] In the following content, all figures disclosed herein, whether or not the words "approximately" or "about" are used, are approximate values. The value of each figure may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%, etc. Whenever a figure with a value of N is disclosed, any figure with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction.

[0022] The carbon compounds, i.e. carbon sources, described in this invention are nutrients that provide microorganisms with the carbon elements required for their growth and reproduction. Examples of such carbon compounds include, but are not limited to, sugars, sugar alcohols, alcohols, organic acids, complex mixtures, and vegetable oils; specific examples may include, but are not limited to, glucose, dextrin, sucrose, monosodium glutamate, molasses, glycerol, lactic acid, maltodextrin, maltose, and castor oil.

[0023] The nitrogen compounds, or nitrogen sources, described in this invention refer to nutrients that provide the nitrogen element necessary for the growth and reproduction of microorganisms. Examples of nitrogen sources include, but are not limited to, yeast extract, yeast powder, yeast paste, tryptone, monosodium glutamate, ammonium chloride, ammonium sulfate, soybean peptone, soybean flour, corn flour, peanut meal, and cottonseed meal. In some embodiments, the "nitrogen source" is preferably yeast extract, yeast powder, yeast paste, tryptone, ammonium chloride, or ammonium sulfate, or any combination thereof.

[0024] The inorganic salts described in this invention refer to mineral salts among inorganic compounds, such as borates, carbonates, chlorides, molybdates, nitrates, phosphates, and sulfates. The metallic components include calcium, iron, potassium, cobalt, copper, magnesium, cobalt, sodium, or zinc. Examples of the inorganic salts include, but are not limited to: sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, ferric sulfate heptahydrate, calcium chloride dihydrate, potassium chloride, sodium sulfate, and disodium hydrogen phosphate.

[0025] The vegetable oils described in this invention are selected from the group consisting of: avocado oil, European fir seed oil, Victory acacia seed oil, kiwifruit seed oil, amaranth seed oil, peanut oil, *Palmus tinctoria* seed oil, *Palmus tinctoria* seed oil, *Palmus tinctoria* seed oil, *Palmus tinctoria* fruit oil, *Palmus tinctoria* kernel oil, cultivated oat kernel oil, white mustard seed oil, rapeseed seed oil, shea butter, shea butter oil, calendula seed oil, red scabra seed oil, *Tetrapanax papyrifera* seed oil, camellia seed oil, *Camellia yunnanensis* seed oil, and camellia seed oil. Hemp seed oil, hemp seed / stem oil, rapeseed oil, safflower seed oil, chlorella oil, watermelon seed oil, lime seed oil, sweet orange seed oil, grapefruit seed oil, cocoa coconut oil, cocoa coconut seed butter, Arabica coffee seed oil, chlorella oil (Biotech), American hazelnut seed oil, European hazelnut seed oil, cantaloupe seed oil, cucumber seed oil, zucchini seed oil, African palm oil, palm oil Fruit oil, wild soybean (soybean) oil, herbaceous cottonseed oil, upland cottonseed oil, sunflower seed oil, macadamia seed oil, macadamia nut seed oil, mango seed oil, mango seed oil, lemon balm seed oil, microalgae oil, moringa seed oil, arabic seed oil, evening primrose (night-blooming jasmine) oil, olive (olive) fruit oil, Olus oil, babassu seed oil, beautiful Albania palm kernel oil, cultivated rice (rice) bran / germ oil, cultivated rice (rice) bran oil, cultivated rice germ oil, cultivated rice lipids, cultivated rice seed oil Poppy seed oil, passion fruit seed oil, avocado butter, avocado oil, sweet almond oil, apricot kernel oil, peach kernel oil, pomegranate seed oil, apple seed oil, ricinoleic acid / caproic acid / capric acid / capric acid triglycerides, castor seed oil, rosehip oil, musk rose seed oil, raspberry seed oil, sesame seed butter, soybean glycerides, cocoa bean seed butter, large-flowered cocoa seed butter, wheat bran lipids, wheat germ oil, European grape seed oil, corn germ oil, and corn oil. In some embodiments, the vegetable oil is selected from the group consisting of: peanut oil, linseed oil, cottonseed oil, rice bran oil, coconut oil, camellia seed oil, rapeseed oil, palm fruit oil, corn oil, sunflower seed oil, soybean oil, castor oil, or sesame oil. Attached Figure Description

[0026] Figure 1 The display shows the concentration of fermentation products from Y. lipolytica OMK-95 in Example 1.

[0027] Figure 2 The display shows the concentration of fermentation products from Y. lipolytica OMK-95 in Example 2.

[0028] Figure 3The figures show the concentrations of fermentation products from wild-type Y. lipolytica in Comparative Example 1; γ-Decalactone is γ-decanolide; δ-Decalactone is δ-decanolide; Time / h is time / hour. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to provide a more detailed description of the present invention.

[0030] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0031] In some embodiments, the Y. lipolytica strain of the present invention is named Y. lipolytica OMK-95 and has the accession number CCTCC M 20231427.

[0032] In some embodiments, the present invention utilizes Y. lipolytica OMK-95 for bio-fermentation culture to obtain gamma-decanolide or doxy-decanolide.

[0033] In some embodiments, the fermentation medium comprises carbon compounds, nitrogen compounds, and inorganic salts, wherein the carbon compounds include at least castor oil at a concentration of 10-60 g / L.

[0034] In some embodiments, the concentration of castor oil in the fermentation medium is 20-50 g / L.

[0035] In some embodiments, the concentration of castor oil in the fermentation medium is 30-40 g / L.

[0036] In some embodiments, the concentration of castor oil in the fermentation medium is 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L.

[0037] In some embodiments, the carbon compound further includes other carbon sources, including one or more combinations of sugars, sugar alcohols, alcohols, organic acids, complex mixtures, and vegetable oils;

[0038] In some embodiments, the carbon compound further includes one or more combinations of glucose, glycerol, mannitol, yeast extract, yeast powder, and wort.

[0039] In some embodiments, the carbon compound includes glucose and yeast extract.

[0040] In some embodiments, the concentration of the other carbon source is 5-30 g / L.

[0041] In some embodiments, the concentration of the other carbon source is 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, or 30 g / L.

[0042] In some embodiments, the concentration of glucose is 15-25 g / L.

[0043] In some embodiments, the concentration of the yeast powder is 3-10 g / L.

[0044] In some embodiments, the other carbon source includes glucose and yeast extract, wherein the concentration of glucose is 20 g / L and the concentration of yeast extract is 5 g / L.

[0045] In some embodiments, the nitrogen compound includes one or more combinations of nitrates, ammonium salts, yeast powder, yeast extract, soybean flour, and corn steep liquor.

[0046] In some embodiments, the nitrogen compound includes one or more combinations of ammonium sulfate, urea, and peptone.

[0047] In some embodiments, the nitrogen compound includes ammonium sulfate and urea.

[0048] In some embodiments, the concentration of the nitrogen compound is 15-30 g / L.

[0049] In some embodiments, the concentration of the nitrogen compound is 15 g / L, 20 g / L, 25 g / L, or 30 g / L.

[0050] In some embodiments, the concentration of ammonium sulfate is 5-15 g / L.

[0051] In some embodiments, the concentration of urea is 5-10 g / L.

[0052] In some embodiments, the concentration of peptone is 10-20 g / L.

[0053] In some embodiments, the inorganic salt includes one or more of borates, carbonates, chlorides, molybdates, nitrates, phosphates, and sulfates; the metallic portion of the salt includes one or more of calcium, iron, potassium, cobalt, copper, magnesium, cobalt, sodium, or zinc.

[0054] In some embodiments, the inorganic salt includes magnesium sulfate and potassium dihydrogen phosphate.

[0055] In some embodiments, the concentration of the inorganic salt is 5-20 g / L.

[0056] In some embodiments, the concentration of the inorganic salt is 10-20 g / L.

[0057] In some embodiments, the concentration of the inorganic salt is 5 g / L, 10 g / L, 15 g / L, or 20 g / L.

[0058] In some embodiments, the concentration of magnesium sulfate is 2-5 g / L.

[0059] In some embodiments, the concentration of potassium dihydrogen phosphate is 10-15 g / L.

[0060] In some embodiments, the fermentation medium further includes trace elements.

[0061] In some embodiments, the method involves inoculating seeds into a fermentation medium for fermentation, controlling the fermentation temperature at 26-30℃, the pH at 6-7, the stirring speed at 300-600 rpm, and the aeration ratio at 1:0.3-0.8.

[0062] In some embodiments, the ventilation ratio is 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7 or 1:0.8.

[0063] Adequate aeration is essential during the production process. This allows for the design of reactors suitable for use with the present invention. Generally, according to the present invention, all bioreactors suitable for aerobic methods and known to those skilled in the art can be used. Preferably, any apparatus suitable for aerobic immersion methods can be used. That is, according to the present invention, containers with or without mechanical mixing devices can be used.

[0064] In some embodiments, castor oil is added to the fermentation system once or multiple times.

[0065] In some embodiments, castor oil is added during fermentation cycles of 50-60 hours and 80-90 hours.

[0066] In some embodiments, castor oil is added to bring the concentration of castor oil in the fermentation system to 10-60 g / L.

[0067] In some embodiments, castor oil is added to the fermentation system to a concentration of 20-50 g / L.

[0068] In some embodiments, castor oil is added to bring the concentration of castor oil in the fermentation system to 30-40 g / L.

[0069] In some embodiments, castor oil is added to the fermentation system at a concentration of 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L.

[0070] Supplementing the culture medium at the beginning of inoculation, during growth, or after growth can be done either by adding it all at once or by adding it sequentially throughout the process.

[0071] Screening and isolation of Y. lipolytica OMK-95 strain of the present invention:

[0072] Y. lipolytica strains preserved in the laboratory were cultured to the logarithmic growth phase. 10 ml of the culture was centrifuged, and the culture was resuspended three times with pH 7.0 phosphate buffer. The washed strain was then resuspended in 9 ml of pH 7.0 phosphate buffer, and 1 ml of nitrosoguanidine solution was added to achieve a nitrosoguanidine concentration of 1 mg / ml. The bacterial suspension was placed in a shaker and incubated at 30°C for 20 min to induce mutagenesis. After centrifugation, the strain was washed three times with physiological saline. The strain was then spread onto an isolation medium with castor oil as the sole carbon source and incubated at 30°C for 48 h. Filter paper was punched into 5 mm pieces and soaked in a solution of equal volumes of 0.7% nitroaniline hydrochloric acid solution, 1% sodium nitrite solution, and 5% sodium carbonate solution. Finally, the paper pieces were tightly covered onto single colonies in the isolation dish, and strains exhibiting yellow spots were selected as the initial screening strains.

[0073] Shake-flask fermentation experiments were conducted on the initially screened strains. Gas chromatography was used to identify strains producing high levels of gamma- and doxy-decanoic acid lactones. Multiple rounds of conventional chemical mutagenesis were performed on the strain using single and mixed chemical mutagen reagents. Simultaneously, a reasonable and efficient strain screening method was employed, ultimately identifying a strain capable of efficiently metabolizing castor oil to produce gamma- and doxy-decanoic acid lactones. This strain was named *Y. lipolytica* OMK-95 and is deposited at the Microbial Culture Collection Center of Wuhan University, accession number CCTCC M 20231427.

[0074] Culture medium formulation:

[0075] The solid seed culture medium for Y. lipolytica OMK-95 is shown in Table 1, the liquid seed culture medium is shown in Table 2, and the fermentation culture medium is shown in Table 3.

[0076] Table 1. Y. lipolytica OMK-95 solid seed culture medium

[0077]

[0078] Table 2. Y. lipolytica OMK-95 liquid seed culture medium

[0079]

[0080]

[0081] Table 3. Y. lipolytica OMK-95 fermentation medium

[0082]

[0083] Cultivation process:

[0084] (1) Plate activation

[0085] Take a tube of glycerol culture stored at -80℃ and streak it onto a prepared and sterilized agar plate (solid culture medium). Place the plate in a 30℃ incubator and incubate until the clones are full.

[0086] (2) Seed culture

[0087] Single clones were picked from the activated plate and inoculated into seed shake flasks (liquid culture medium). Y. lipolytica OMK-95 was cultured in a shaker at 28-30℃ and 200rpm for 12-24h. After microscopic examination showed no contamination, the clones were ready to be transferred to fermentation shake flasks.

[0088] (3) Fermentation culture

[0089] Inoculate the shake flask seeds at a 10% inoculum into the fermenter or shake flask (fermentation medium), and incubate at 28°C. The pH of the fermentation broth is automatically controlled at 6.0-7.0 using ammonia solution. Culture in Y. lipolytica OMK-95 for 24-36 hours, and measure the OD using a spectrophotometer. 600 When OD 600 The growth gradually increases, entering the late logarithmic phase and the fermentation stage. Samples are taken every 12 hours, and fermentation is stopped when the free fatty acids in the fermentation broth are completely metabolized or no longer metabolized.

[0090] Example 1

[0091] Seed culture medium and fermentation culture medium are shown in Tables 1-3.

[0092] Seed culture: Under aseptic conditions, transfer -80℃ glycerol tubes of Y. lipolytica OMK-95 onto fresh, sterile beef extract peptone agar plates (solid medium, Table 1) and incubate at 28℃ for 24-48 h. Pick the bacterial colony and inoculate it into a 3L bioreactor containing 1.8L of seed culture medium (liquid medium, Table 2), aerate and stir at 300 rpm, and incubate at 28℃ for 12-24 h to obtain the seed culture.

[0093] Fermentation Cultivation: 10.2 L of the prepared fermentation medium (fermentation medium, Table 3) was added to a 20 L bioreactor and sterilized at 121 °C for 30 min. Then, 1.8 L of the cultured seed culture was inoculated into the 20 L bioreactor for fermentation. The fermentation temperature was 28 °C, the stirring speed was 400 rpm, and the aeration ratio was 1:0.5. After 20-30 h of fermentation, the microbial OD... 600The growth was slow, indicating the start of the bio-fermentation stage, which lasted for 106 hours. The pH of the fermentation broth was adjusted to 1-2 with an inorganic acid, such as 30% sulfuric acid, and vortexed for 3-10 minutes. Extraction was performed with an equal volume of ethyl acetate. The organic phase was analyzed by GC, revealing a concentration of 13.5 g / L for propyldecanolide and 0.5 g / L for dextrose lactone. The results are shown below. Figure 1 .

[0094] Example 2

[0095] Seed preparation: Y. lipolytica OMK-95 was transferred from -80℃ glycerol tubes to fresh, sterile beef extract peptone agar plates (solid medium, Table 1) under sterile conditions and incubated at 30℃ for 48 h. The bacterial growth was then inoculated into a 3L bioreactor containing 1.8L of seed culture medium (liquid medium, Table 2), aerated and stirred at 400 rpm, and incubated at 28℃ for 22 h to obtain the seed culture.

[0096] Fermentation of castor oil: 10.2 L of the prepared fermentation medium (fermentation medium, Table 3) was loaded into a 20 L bioreactor and sterilized at 121 °C for 30 min. Then, 1.8 L of the cultured seed liquid was inoculated into the 20 L bioreactor for fermentation. The fermentation temperature was 28 °C, the stirring speed was 500 rpm, and the aeration ratio was 1:0.5. After 26 h of fermentation, the microbial OD... 600 The growth was slow, indicating the start of the fermentation stage. Castor oil was added at 56 hours of fermentation until the concentration reached 30 g / L, and again at 86 hours, for a total fermentation time of 112 hours. The pH of the fermentation broth was adjusted to 1-2 using an inorganic acid, such as 20% hydrochloric acid, and vortexed for 3-10 minutes. Extraction was performed with an equal volume of ethyl acetate. The organic phase was analyzed by GC, revealing a concentration of 35.2 g / L for decanolide at the pro-gamma position and 1.5 g / L for decanolide at the delta position. The results are shown in [Figure number missing]. Figure 2 .

[0097] Comparative Example 1

[0098] The culture method and conditions were the same as in Example 1, except that strain Y. lipolytica OMK-95 was replaced with wild-type strain Y. lipolytica. Ultimately, 500 mg / L of R-propyldecanolide was produced, but no d-decanolide was formed, as shown in the results. Figure 3 .

[0099] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A strain of Yersinia lipophila ( Yarrowia Lipolytica OMK-95, characterized in that, The preservation number of the Yersinia lipophila OMK-95 is CCTCC NO: M 20231427.

2. A method for preparing γ-decanolide or δ-decanolide, characterized in that, γ-decanolide or δ-decanolide was obtained by bio-fermentation using the Yersinia lipolytica OMK-95 as described in claim 1.

3. The method according to claim 2, characterized in that, The culture medium for the bio-fermentation includes carbon compounds, nitrogen compounds, and inorganic salts, wherein the carbon compounds include at least castor oil, and the concentration of the castor oil is 10-60 g / L.

4. The method according to claim 3, characterized in that, The concentration of the castor oil is 30-40 g / L.

5. The method according to claim 3, characterized in that, The carbon compounds also include one or more combinations of glucose, glycerol, mannitol, and wort.

6. The method according to claim 3, characterized in that, The concentration of the carbon compound is 5-30 g / L.

7. The method according to claim 3, characterized in that, The nitrogen compound includes one or more combinations of ammonium sulfate, urea, and peptone.

8. The method according to claim 3, characterized in that, The concentration of the nitrogen compound is 15-30 g / L.

9. The method according to claim 3, characterized in that, The inorganic salts include magnesium sulfate and potassium dihydrogen phosphate.

10. The method according to claim 3, characterized in that, The concentration of the inorganic salt is 5-20 g / L.

11. The method according to claim 3, characterized in that, The culture medium for bio-fermentation also includes trace elements.

12. The method according to any one of claims 2-11, characterized in that, The method involves inoculating the Yeast OMK-95 seed into a fermentation medium for fermentation, controlling the fermentation temperature at 26-30℃, the pH at 6-7, the stirring speed at 300-600 rpm, and the aeration ratio at 1:0.3-0.

8.

13. The method according to claim 12, characterized in that, During the fermentation process, castor oil is added to the fermentation system once or multiple times.

14. The method according to claim 13, characterized in that, Castor oil was added during the 50-60h and 80-90h fermentation cycles.

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