WO2021254076A1 - 一种产油酵母及其应用 - Google Patents

一种产油酵母及其应用 Download PDF

Info

Publication number
WO2021254076A1
WO2021254076A1 PCT/CN2021/094555 CN2021094555W WO2021254076A1 WO 2021254076 A1 WO2021254076 A1 WO 2021254076A1 CN 2021094555 W CN2021094555 W CN 2021094555W WO 2021254076 A1 WO2021254076 A1 WO 2021254076A1
Authority
WO
WIPO (PCT)
Prior art keywords
fermentation
glucose
xylose
fermentation system
oleaginous yeast
Prior art date
Application number
PCT/CN2021/094555
Other languages
English (en)
French (fr)
Inventor
孙付保
周秋利
胡芸
任洪艳
孙驰贺
Original Assignee
江南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2021254076A1 publication Critical patent/WO2021254076A1/zh
Priority to US17/674,913 priority Critical patent/US20220170058A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention relates to an oleaginous yeast and its application, and belongs to the technical field of microorganisms.
  • microorganisms such as yeasts, bacteria, molds and microalgae can use carbohydrates, hydrocarbons and oils as carbon sources to accumulate oils. Any microorganisms that accumulate oils more than 20% of the dry cell weight can be called oil-producing microorganisms.
  • Microbial oils especially those produced by yeast, have a fatty acid composition similar to vegetable oils, mainly C16 and C18 fatty acids, such as palmitic acid, stearic acid, and oleic acid. Microbial oils can replace expensive animal and vegetable oils as raw materials for the production of biodiesel, and have a positive effect on promoting large-scale industrial production of biodiesel.
  • oil-producing microorganisms also have the advantages of short fermentation cycle, unaffected by seasonal climate, and wide utilization of carbon sources. They have good development prospects and will play an important role in the development of the future biodiesel industry.
  • the high cost of cultivating microbial carbon sources and the low yield of microbial oils limit the development of microbial oils.
  • Biomass is rich in carbohydrates (cellulose, hemicellulose), and glucose can be obtained after full hydrolysis, which can meet the carbon source requirements for the growth and metabolism of oleaginous yeast.
  • the use of these cellulosic sugar-containing hydrolysates to cultivate oleaginous yeast may greatly reduce the production cost of microbial oils and provide the possibility to realize large-scale production of microbial oils.
  • the hydrolysate of agricultural and forestry biomass is not only rich in glucose, but also monosaccharides such as xylose.
  • xylose monosaccharides
  • This mechanism will lead to technical problems such as a long fermentation cycle using hydrolysate, low utilization of cellulose resources, and high production costs. Therefore, obtaining high-yield oleaginous yeast that can simultaneously utilize glucose and xylose mixed sugar has become an important direction in the field of microbial lipid technology.
  • the invention aims to provide a method for cultivating high-oil-yielding yeast using cellulosic hydrolysate to ferment to produce microbial oil.
  • wild oily yeast ZZ-46 was used as the starting strain, a certain concentration of cerulenin and TTC were used as screening factors to screen the mutants, and Nile red fluorescence detection was used to detect The mutant strains were initially screened for high-throughput screening, and finally a high-yield oleaginous yeast with stable genetic traits was obtained.
  • the cellulosic hydrolysate can be directly used to ferment to produce oil, so that the cultivation of oil-producing microorganisms can more efficiently use the raw materials from lignocellulose, improve the technical and economic efficiency of microbial oil production, and have broad application prospects.
  • An oleaginous yeast (Trichosporon dermatis) provided by the present invention has been deposited in the China Center for Type Culture Collection on May 21, 2020, and the deposit number is CCTCC NO: M 2020139.
  • the present invention provides a method for simultaneously utilizing xylose and glucose.
  • the oleaginous yeast is added to a system containing xylose and glucose.
  • the oleaginous yeast is fermented at 22-28°C for 7-9 days.
  • the oleaginous yeast is added to the fermentation system to ferment to produce oil.
  • the carbon source in the fermentation system comes from the hydrolyzed mixed sugar solution of lignocellulosic biomass raw materials.
  • the preparation method of the hydrolyzed mixed sugar liquid is:
  • the lignocellulosic biomass includes agricultural straws and forestry processing residue waste materials.
  • the oleaginous yeast with an OD 600 of 6-8 is added to the fermentation system at an addition amount of 5%-10%.
  • the loading volume of the fermentation broth in the fermentation system is 12%-20%.
  • feeding is carried out during the fermentation process, and the feeding is supplemented with a mixture of glucose and xylose at a final concentration of 10-20 g/L, and the ratio of glucose and xylose in the mixture is (1-2 ):1.
  • the oleaginous yeast is fermented at 22-28°C and pH 6-8 for 7-9 days.
  • the present invention provides a method for improving oil yield, which comprises adding the oleaginous yeast to a fermentation system to ferment to produce oil.
  • the fermentation system contains a carbon source, and the carbon source is xylose and glucose.
  • the fermentation system contains a carbon source, and the carbon source comes from the hydrolyzed mixed sugar solution of lignocellulosic biomass raw materials.
  • the oleaginous yeast seed liquid with an OD 600 of 6-8 is added to the fermentation system at an addition amount of 5%-10%.
  • the loading volume of the fermentation broth in the fermentation system is 12%-20%, and the fermentation is carried out at 22-28°C and pH 6-8 for 7-10 days.
  • feeding is carried out during the fermentation process; the feeding is supplemented with a mixture of glucose and xylose at a final concentration of 10-20 g/L, and the ratio of glucose and xylose in the mixture is (1-2 ):1.
  • the fermentation system contains a carbon source, the carbon sources are xylose and glucose; the carbon source comes from the hydrolyzed mixed sugar solution of lignocellulosic biomass raw materials; the OD 600 is 6-8.
  • the filling amount of the fermentation broth in the fermentation system is 12% ⁇ 20%, and the fermentation is carried out at 22 ⁇ 28°C and pH6 ⁇ 8 for 7 ⁇ 10 days; supplementation is carried out during the fermentation process.
  • the supplement is to supplement a mixture of glucose and xylose at a final concentration of 10-20 g/L, and the ratio of glucose to xylose in the mixture is (1-2):1.
  • the present invention also protects the application of the oleaginous yeast, or a method for simultaneously utilizing xylose and glucose, or a method for increasing the oil yield in the production of oil in the fields of food, medicine, agriculture, and chemical industry.
  • Agricultural and forestry lignocellulosic biomass waste is a renewable resource with a high content of cellulose and hemicellulose. It has a wide range of sources and low cost. However, its hydrolysate contains mixed sugars of glucose and xylose. Sugar-cultured microorganisms usually have a glucose effect, resulting in a long fermentation cycle and low efficiency.
  • the high-yield oily yeast obtained by the present invention can simultaneously use glucose and xylose to accumulate a large amount of oil, and the sugar utilization efficiency and production intensity are improved. After 8 days of fed-batch fermentation, the oil output can reach 31.33g/L and the oil content is 60.83%. .
  • the waste resources can be reused, and the production cost is reduced, which has broad application prospects.
  • Trichosporon dematis provided by the present invention, classified and named as Trichosporon dermatis L7, has been deposited in the China Center for Type Culture Collection on May 21, 2020, and the deposit number is CCTCC NO: M 2020139, the deposit address is Wuhan University, Wuhan, China.
  • Figure 1 shows the simultaneous sugar consumption curve of the starting bacteria ZZ-46.
  • Figure 2 shows the simultaneous sugar consumption curve of strain L7.
  • YPD solid medium yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar 15g/L.
  • YPD seed medium yeast extract 10g/L, peptone 20g/L, glucose 20g/L.
  • Re-screening fermentation medium carbon source: glucose 46.67g/L, xylose 23.33g/L; nitrogen source: yeast extract 0.75g/L, NH 4 Cl 0.1g/L; inorganic salt: MgCl 2 ⁇ 6H 2 O 1g/L, Na 2 SO 4 0.1g/L; Phosphate buffer: KH 2 PO 4 11.8g/L, K 2 HPO 4 ⁇ 3H 2 O 3.7g/L; Trace element: CaCl 2 ⁇ 2H 2 O 40mg /L, FeSO 4 ⁇ 7H 2 O 5.5mg/L, citric acid monohydrate 5.2mg/L, ZnSO 4 ⁇ 7H 2 O 1mg/L, MnSO4 ⁇ H 2 O 0.76mg/L, 18mol H 2 SO 4 1.84 ⁇ 10 -3 mg/L, ready for use after sterilization.
  • the wild oil-producing yeast strain used in the examples is Trichosporon.dermatis ZZ-46, which is preserved in the Nanyang City Industrial Microorganism Strain Collection, and the strain number is NICC 30027.
  • Enzymatic hydrolysis was carried out with mechanical stirring at a speed of 150r ⁇ min -1 , a temperature of 50°C, and enzymatic hydrolysis for 72 hours. After the enzymolysis is completed, centrifuge at 10000r ⁇ min- 1 for 5min, and take the supernatant to determine the concentration of glucose and xylose.
  • the content of the mixed sugar (208g/L) of the bagasse hydrolysate obtained according to the above method is: glucose 140g/L and xylose 68g/L. According to fermentation needs, the hydrolysate is diluted 3 times to 70g/L with deionized water, or 2.6 times to 80g/L, and the pH is adjusted to 6 with NaOH.
  • ARTP mutagenesis Take 10ul of wild fat yeast ZZ-46 bacterial suspension in the ultra-clean workbench and spread it on a sterile metal slide, and place the metal slide in the groove of the stage of the ARTP mutagenesis instrument operating room. Inside, nitrogen is used as the working gas, the set power is 100W, the processing distance is 2mm, the gas flow rate is 10L/min, and the processing time is 140s;
  • Shake flask re-screening pick a single colony of the high-yielding strains obtained from the initial screening and inoculate it in YPD seed medium, culture at 140r/min at 25°C for 36h, and inoculate it in 50mL basic fermentation medium at 10% of the inoculum , 25°C, 140r/min fermentation for 7 days.
  • the bacteria were collected by centrifugation of the fermentation broth, and the biomass of the bacteria, the fat output of the fermentation broth and the fat content of the bacteria were determined. The results are shown in Table 1.
  • the strain L7 with better comprehensive effect was screened out and sent to the China Type Culture Collection for preservation.
  • the strain L7 was subcultured (the cultivation method was the same as the re-screening), and the biomass, oil production and oil content of the first to seventh generations were tested. It can be seen from Table 2 that the biomass, oil production and oil content of the mutant strains are relatively stable. The results show that the strain has good genetic stability.
  • the bagasse hydrolysate is diluted to 70g/L total sugar, the pH is adjusted to 6, and no other nitrogen sources, inorganic salts and other substances are added.
  • Fermentation of bagasse hydrolysate Inoculate a single colony of strain L7 in YPD seed medium, culture for 36h at 25°C and 140r/min, to an OD 600 of 7.6, and then inoculate 50/250mL (ie In a 250mL Erlenmeyer flask, 50mL fermentation medium) bagasse hydrolysate medium was fermented at 140r/min at 25°C for 7 days.
  • strain fermentation to produce oil The strain L7 was fermented in the bagasse hydrolysate fermentation medium, and its biomass, oil production and oil content were 18.87g/L and the biomass, oil production and oil content of the starting strain ZZ-46, respectively. 9.26g/L and 49.07%, the starting strain ZZ-46 was 15.21g/L, 6.76g/L and 44.46%. The biomass and oil yield were increased by 24.06% and 36.98% respectively compared with the starting strain.
  • Example 3 Fermentation medium using bagasse hydrolysate as carbon source
  • the bagasse hydrolysate is used as a carbon source fermentation medium component: the bagasse hydrolysate is diluted to 70g/L total sugar, pH is adjusted to 6, and nitrogen source is added: yeast extract 0.75g/L, NH 4 Cl 0.1g/L ; Inorganic salt: MgCl 2 ⁇ 6H 2 O 1g/L, Na 2 SO 4 0.1g/L; Phosphate buffer: KH 2 PO 4 11.8g/L, K 2 HPO 4 ⁇ 3H 2 O 3.7g/L; trace Quantity elements: CaCl 2 ⁇ 2H 2 O 40mg/L, FeSO 4 ⁇ 7H 2 O 5.5mg/L, citric acid monohydrate 5.2mg/L, ZnSO 4 ⁇ 7H 2 O 1mg/L, MnSO4 ⁇ H 2 O 0.76mg /L, 18mol H 2 SO 4 1.84 ⁇ 10 -3 mg/L.
  • Strain fermentation to produce oil Inoculate a single colony of strain L7 in YPD seed medium, culture it at 25°C and 140r/min for 36 hours until OD 600 is 7.6, and then inoculate 50/250mL bagasse at 10% of the inoculum Fermented in the hydrolysate medium at 140r/min and 25°C for 7 days.
  • Strain L7 after being fermented in a fermentation medium with bagasse hydrolysate as a carbon source, its biomass, oil yield and oil content reached 22.12 g/L, 11.18 g/L and 50.54%, respectively.
  • Example 4 Strain L7 fermented to produce oil under optimized conditions in shake flasks
  • inorganic salt MgCl 2 ⁇ 6H 2 O 1g/L , Na 2 SO 4 0.1g/L
  • Phosphate buffer KH 2
  • Strain fermentation to produce oil inoculate a single colony of strain L7 in YPD seed medium, culture at 22°C and 140r/min for 36h, until OD 600 is 7.6, and then inoculate 40/250mL bagasse at 10% of the inoculum In the hydrolysate medium, the pH was adjusted to 6,140r/min, 22°C, and the fermentation was carried out for 8 days.
  • the specific method is: inoculate a single colony of strain L7 in YPD seed medium, culture for 36h at 25°C and 140r/min, to an OD 600 of 7.6, and then inoculate 40/250mL bagasse hydrolyzed at 10% of the inoculum Fermented in liquid culture medium at 140r/min and 22°C for a total of 9 days.
  • Feeding 3 times Feeding on the 2nd, 3rd and 4th day respectively;
  • Feeding 4 times Feeding on the 2nd, 3rd, 4th and 5th day respectively;
  • the results of the fermentation are shown in Table 3.
  • the results show that the oil yield and oil content obtained from two feedings are the largest, which are 20.21g/L and 57.93%, respectively.
  • the biomass is 34.89g/L, which is higher than the batch shake flask culture. Increase of 44%, 11% and 31%.
  • Example 6 Fermentation of strain L7 in a 5L fermentor to produce oil
  • Strain fermentation to produce oil Inoculate a single colony of strain L7 in YPD seed medium, culture it at 25°C and 140r/min for 36 hours to an OD600 of 7.6, and then inoculate 2L of bagasse hydrolysate at a 10% inoculum amount.
  • In the base adjust the pH to 6, 22°C, and ferment for 5 days; during the fermentation process, use 2M NaOH to adjust the pH to keep the pH stable at 6, the speed is related to DO, and the DO is maintained above 30%.
  • silicone is added. The foaming agent controls the foam produced during the fermentation process.
  • Strain fermentation to produce oil Inoculate a single colony of strain L7 in YPD seed medium, culture it at 25°C and 140r/min for 36 hours to an OD600 of 7.6, and then inoculate 2L of bagasse hydrolysate at a 10% inoculum amount.
  • the pH is adjusted to 6, 22°C for fermentation.
  • use 2M NaOH to adjust the pH, keep the pH stable at 6, and the rotation speed is related to the DO, keeping the DO above 30%.
  • Silicone defoamer is added during the fermentation process to control the foam produced during the fermentation process.
  • Example 5 Specific implementation methods refer to Example 4, the difference is that the total sugar of 80g/L is replaced with 50g/L, 60g/L, 70g/L, 90g/L, 100g/L, and the biomass and oil yield obtained after fermentation are compared with The oil content is shown in Table 5.
  • Example 4 Specific implementation methods refer to Example 4, the difference is that the nitrogen source soy peptone is replaced with NH 4 Cl, (NH 4 ) 2 SO 4 , yeast extract, fish meal peptone, NH 4 Cl + yeast extract, and the biomass obtained after fermentation , Oil production and oil content are shown in Table 6.
  • the amount, oil production and oil content are shown in Table 7.
  • Example 4 Specific implementation methods refer to Example 4, the difference is that the inoculum amount 10% is replaced with 5%, 7.5%, 12.5%, 15%, and the biomass, oil yield and oil content obtained after fermentation are shown in Table 8.
  • Example 4 Specific implementation methods refer to Example 4, the difference is that the liquid volume 40/250mL is replaced with 30/250mL, 50/250mL, 60/250mL, 70/250mL, the biomass, oil yield and oil content obtained after fermentation are divided into Table 9.
  • Example 4 Specific implementation methods refer to Example 4, the difference is that pH 6 is replaced with pH 4, pH 5, pH 7, and pH 8.
  • the biomass, oil yield, and oil content obtained after fermentation are shown in Table 10.
  • Example 4 Specific implementation methods refer to Example 4, the difference is that the temperature 22°C is replaced with 25°C, 28°C, and 31°C, and the biomass, oil yield and oil content obtained after fermentation are shown in Table 11.
  • Example 4 Specific implementation methods refer to Example 4, the difference is that the fermentation time of 8 days is replaced with 7 days and 9 days, and the biomass, oil yield and oil content obtained after fermentation are shown in Table 12.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Cell Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种产油酵母及其应用，该产油酵母为德马特丝孢酵母(Trichosporon dermatis)，已于2020年5月21日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020139。

Description

一种产油酵母及其应用 技术领域
本发明涉及一种产油酵母及其应用，属于微生物技术领域。
背景技术
某些微生物如酵母、细菌、霉菌和微藻能够利用碳水化合物、碳氢化合物和油脂作为碳源积累油脂，凡是油脂积累量超过细胞干重的20％的微生物，即可称为产油微生物。微生物油脂，特别是酵母所产油脂，其脂肪酸组成与植物油脂类似，以C16和C18系脂肪酸为主，如棕榈酸、硬脂酸、油酸等。微生物油脂可以代替昂贵的动植物油脂作为生产生物柴油的原料，对促进生物柴油大规模工业化生产具有积极作用。此外，产油微生物还具有发酵周期短、不受季节气候影响、碳源利用广等优点，具有良好的发展前景，将在未来生物柴油产业的发展中发挥重要作用。但培养微生物碳源成本高和微生物油脂产量低等问题限制了微生物油脂的发展。
我国是农业大国，每年大约会产生7亿吨亿秸秆类生物质，加上林业加工剩余物，我国每年产生的农林纤维类生物质剩余物高达20亿吨，但是我国并没有对这些生物质进行有效利用。生物质中含有丰富的碳水化合物(纤维素、半纤维素)，充分水解后能得到葡萄糖，可以满足产油酵母生长代谢对碳源的要求。利用这些含纤维质糖的水解液培养产油酵母，有可能大幅度降低微生物油脂的生产成本，为实现规模化生产微生物油脂提供可能。
此外，农林生物质水解液中不仅含有丰富的葡萄糖，还会得到木糖等单糖。然而，大多数微生物代谢过程中存在葡萄糖效应，即当培养基中含有葡萄糖和木糖时，会优先利用葡萄糖，只有当葡萄糖浓度较低时才开始利用木糖。这种机制会导致利用水解液发酵周期长，纤维素资源的利用率低，生产成本高等技术问题。因此，获得能够同时利用葡萄糖、木糖混合糖的高产油脂酵母，已经成为微生物油脂技术领域的重要方向。
发明内容
本发明旨在提供一种利用纤维质水解液培养高产油脂酵母发酵生产微生物油脂的方法。为克服上述油脂酵母产油不高的不足，以野生油脂酵母ZZ-46为出发菌，以一定浓度的浅蓝菌素和TTC作为筛选因子对突变体进行初筛，利用尼罗红荧光检测对初筛突变菌株进行高通量筛选，最终获得遗传性状稳定的高产油脂酵母。并且可直接利用纤维质水解液发酵生产油脂，使产油微生物培养更高效地利用木质纤维素来源的原料，提高微生物油脂生产的技术经济性，具有广阔的应用前景。
本发明提供的一株产油酵母(Trichosporon dermatis)，已于2020年5月21日保藏于中 国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020139。
本发明提供了一种同时利用木糖和葡萄糖的方法，将所述产油酵母加入含有木糖和葡萄糖的体系中。
在一种实施方式中，将所述产油酵母在22～28℃发酵7～9天。
在一种实施方式中，将所述产油酵母加入发酵体系发酵产油。
在一种实施方式中，所述发酵体系中的碳源来自于木质纤维素类生物质原料的水解混合糖液。
在一种实施方式中，所述水解混合糖液的制备方法为：
(1)碱催化常压甘油有机溶剂预处理木质纤维素类生物质原料：称取90～150g干燥的木质纤维素类生物质原料装入1000～5000mL三口烧瓶中，再加入900～1500g甘油和0.2％(w/w)NaOH固体；将装有基质的三口烧瓶放入恒温加热套中，同时机械搅拌使基质混合均匀并200～250℃保持8～12min；反应结束后，将1000～1500mL自来水倒入烧瓶中使基质充分解离，然后用G1砂芯漏斗过滤，接着用1500～2000mL自来水对滤饼洗涤两次并抽滤，最终所得滤饼即是木质纤维素类生物质基质。将基质分为两部分，一部分晾晒至含水量45～55％，于2～4℃保存；另一部分于100～110℃烘箱烘至绝重；
(2)预处理后基质的酶解：称取含水量为45～55％的基质10～20g装于150～250mL圆底烧瓶中，加入2～5FPU·g -1干基的纤维素酶0.5～1.0mL(使用柠檬酸缓冲液将原酶液稀释至55～65FPU·g -1)以及添加剂PEG 4000 180～200mg、曲拉通X-100 350～400mg、茶皂素180～200mg、牛血清白蛋白180～200mg、木聚糖酶20～25mg，用柠檬酸缓冲液(50mM，pH 4.5～5.0)补至45～55mL；在酶解第10～12h、22～24h、34～36h时分别补充3.0～3.5g、2.5～3.0g、2.5～3.0g干基；在45～55℃下，酶解68～78h；酶解结束后8000～10000r·min -1离心4～6min。
在一种实施方式中，所述木质纤维素类生物质包括农业秸秆和林业加工剩余物类废弃物原料。
在一种实施方式中，将OD 600为6～8的所述产油酵母以5％～10％的添加量加入发酵体系。
在一种实施方式中，所述发酵体系中的总糖含量为70～90g/L；氮源为大豆蛋白胨、NH 4Cl或酵母提取物；碳/氮＝(273～373)∶1。
在一种实施方式中，所述发酵体系中发酵液的装液量为12％～20％。
在一种实施方式中，在发酵过程中进行补料，所述补料是补加终浓度10～20g/L为葡萄糖和木糖的混合物，混合物中葡萄糖和木糖的比例为(1～2)∶1。
在一种实施方式中，所述将所述产油酵母在22～28℃、pH6～8发酵7～9天。
本发明提供了一种提高油脂产量的方法，将所述产油酵母加入发酵体系发酵产油。
在一种实施方式中，所述发酵体系中含有碳源，碳源为木糖和葡萄糖。
在一种实施方式中，所述发酵体系中含有碳源，碳源来自于木质纤维素类生物质原料的水解混合糖液。
在一种实施方式中，将OD 600为6～8的所述产油酵母种子液以5％～10％的添加量加入发酵体系。
在一种实施方式中，所述发酵体系中的总糖含量为70～90g/L；氮源为大豆蛋白胨、NH 4Cl或酵母提取物；碳/氮＝(273～373)∶1。
在一种实施方式中，所述发酵体系中发酵液的装液量为12％～20％，在22～28℃、pH6～8发酵7～10天。
在一种实施方式中，在发酵过程中进行补料；所述补料是补加终浓度10～20g/L为葡萄糖和木糖的混合物，混合物中葡萄糖和木糖的比例为(1～2)∶1。
在一种实施方式中，所述发酵体系中含有碳源，碳源为木糖和葡萄糖；碳源来自于木质纤维素类生物质原料的水解混合糖液；将OD 600为6～8的所述产油酵母种子液以5％～10％的添加量加入发酵体系；所述发酵体系中的总糖含量为70～90g/L；氮源为大豆蛋白胨、NH 4Cl或酵母提取物；碳/氮＝(273～373)∶1；所述发酵体系中发酵液的装液量为12％～20％，在22～28℃、pH6～8发酵7～10天；在发酵过程中进行补料，所述补料是补加终浓度10～20g/L为葡萄糖和木糖的混合物，混合物中葡萄糖和木糖的比例为(1～2)∶1。
本发明还保护所述产油酵母，或一种同时利用木糖和葡萄糖的方法，或一种提高油脂产量的方法在食品、医药、农业、化工领域生产油脂中的应用。
本发明的有益效果：
农林木质纤维素类生物质废弃物，是一种纤维素和半纤维素含量较高的可再生资源，来源广泛，成本低廉，但因其水解液中含有葡萄糖和木糖的混合糖，而混合糖培养微生物通常会出现葡萄糖效应，造成发酵周期长，效率低。本发明所获的高产油脂酵母，可以同时利用葡萄糖和木糖积累大量油脂，糖利用效率和生产强度提高，在分批补料发酵8天后，油脂产量可达31.33g/L，油脂含量60.83％。使得废弃的资源可再利用，且降低生产成本，具有广阔的应用前景。
生物材料保藏
本发明所提供的德马特丝孢酵母，分类命名为德马特丝孢酵母L7 Trichosporon dermatis L7，已于2020年5月21日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020139，保藏地址为中国.武汉.武汉大学。
附图说明
图1为出发菌ZZ-46的同步耗糖曲线。
图2为菌株L7的同步耗糖曲线。
图3为菌株L7在5L发酵罐中分批补料发酵结果，补料碳源葡萄糖/木糖＝2∶1。
图4为菌株L7在5L发酵罐中分批补料发酵结果，补料碳源葡萄糖/木糖＝1∶1。
具体实施方式
YPD固体培养基：酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，琼脂15g/L。
YPD种子培养基：酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L。
复筛发酵培养基：碳源：葡萄糖46.67g/L、木糖23.33g/L；氮源：酵母提取物0.75g/L、NH 4Cl 0.1g/L；无机盐：MgCl 2·6H 2O 1g/L、Na 2SO 4 0.1g/L；磷酸缓冲液：KH 2PO 4 11.8g/L、K 2HPO 4·3H 2O 3.7g/L；痕量元素：CaCl 2·2H 2O 40mg/L、FeSO 4·7H 2O 5.5mg/L、一水合柠檬酸5.2mg/L、ZnSO 4·7H 2O 1mg/L、MnSO4·H 2O 0.76mg/L、18mol H 2SO 4 1.84×10 -3mg/L，灭菌后备用。
实施例中使用的野生产油酵母菌株为德马特丝孢酵母Trichosporon.dermatis ZZ-46，保藏于南阳市工业微生物菌种保藏中心，菌株编号NICC 30027。
甘蔗渣水解液的制备：
(1)碱催化常压甘油有机溶剂预处理甘蔗渣
称取100g干燥的甘蔗渣装入5000mL三口烧瓶中，再加入1000g甘油和0.2％(w/w)NaOH固体。将装有基质的三口烧瓶放入恒温加热套中，同时机械搅拌使基质混合均匀并240℃保持10min。反应结束后，将1500mL自来水倒入烧瓶中使基质充分解离，然后用G1砂芯漏斗过滤，接着用2000mL自来水对滤饼洗涤两次并抽滤，最终所得滤饼即是甘蔗渣基质。将基质分为两部分，一部分晾晒至含水量50％，于4℃保存；另一部分于105℃烘箱烘至绝重。
(2)预处理后基质的酶解
称取含水量为50％的基质16g装于250mL圆底烧瓶中，加入3FPU·g -1干基的纤维素酶0.875mL(使用柠檬酸缓冲液将原酶液稀释至60FPU·g -1)以及添加剂PEG 4000 192mg、曲拉通X-100 356mg、茶皂素193mg、牛血清白蛋白190mg、木聚糖酶21mg，用柠檬酸缓冲液(50mM，pH 4.8)补至50mL。在酶解第12h、24h、36h时分别补充3.5g、3g、3g干基。采用机械搅拌进行酶解，转速150r·min -1，温度50℃，酶解72h。酶解结束后10000r·min - 1离心5min，取上清测定葡萄糖和木糖的浓度。
(3)甘蔗渣水解液的稀释
根据上述方法获得的甘蔗渣水解液混合糖(208g/L)的含量为：葡萄糖140g/L、木糖68g/L。依据发酵需要将该水解液用去离子水稀释3倍至70g/L，或稀释2.6倍至80g/L，并用NaOH将pH调至6。
实施例1：菌株的突变及筛选
(1)菌悬液的制备：保藏的野生产油酵母菌株经活化后，挑取单菌落于YPD种子培养基中，25℃，140r/min培养36h，取1mL菌液8000r离心5min收集菌体，以无菌生理盐水洗涤三次，在涡旋仪上混匀使菌体分散，调整菌悬液浓度为OD 600＝1的菌悬液，备用；
(2)ARTP诱变：在超净工作台中取10ul野生油脂酵母ZZ-46菌悬液涂布在无菌金属载片上，将金属载片置于ARTP诱变仪操作室载物台的凹槽内，以氮气为工作气体，设定功率为100W，处理距离2mm，气体流量10L/min，处理时间140s；
(3)平板预筛选：将经过处理的菌悬液用无菌生理盐水稀释10 -2稀释度，然后涂布于含有1.67×10 -3％(w/v)的TTC和4.107×10 -6mol/L浅蓝菌素的筛选平板上，置于25℃恒温培养箱中避光培养2天；
(4)高通量筛选：挑取筛选平板上大且红的单菌落于48孔板中培养(1mL培养基/孔)，在25℃、200r/min的摇床中培养2天，将100uL的菌液移到96孔板利用酶标仪检测OD 600nm的吸光值表征细胞浓度。每个孔板中再添加5uL的尼罗红溶液，混匀后避光染色5min，用荧光强度表征油脂产量。检测的发射波长为485nm，吸收波长为595nm。荧光强度为：测出的样品荧光强度减去没有加尼罗红的样品背景荧光强度；
(5)DES诱变：通过ARTP诱变获得的高产突变菌株经过活化和种子液培养制得菌悬液，取OD 600＝1的菌悬液2mL，加入2mL pH 7.0的磷酸缓冲液，再加入0.2mL 50％硫酸二乙酯-乙醇溶液，25℃条件下振荡70min。处理结束后，向反应混合物中加入25％的硫代硫酸钠1mL终止反应；
(6)DES诱变后的筛选：DES处理后的菌悬液按照步骤(3)和(4)进行平板预筛选和高通量筛选。
(7)摇瓶复筛：将初筛得到的高产菌株挑取单菌落接种于YPD种子培养基中，25℃，140r/min培养36h，按10％的接种量接种于50mL基础发酵培养基中，25℃，140r/min发酵7天。发酵液离心收集菌体，测定菌体生物量、发酵液油脂产量以及菌体油脂含量，结果如表1所示。
根据测定的相关指标，筛选出综合效果较好的菌株L7，并将其送至中国典型培养物保藏中心保藏。
表1 复筛菌株生产性能
Figure PCTCN2021094555-appb-000001
将菌株L7进行传代培养(培养方法同复筛)，检测了第一代到第七代的生物量、油脂产量和油脂含量。由表2可知，突变菌株的生物量、油脂产量和油脂含量都较稳定。结果表明，该菌株具有良好的遗传稳定性。
表2菌株L7传代稳定性
Figure PCTCN2021094555-appb-000002
实施例2：利用甘蔗渣水解液发酵
甘蔗渣水解液发酵培养基组分：甘蔗渣水解液稀释至总糖70g/L，pH调至6，未添加其他氮源、无机盐等物质。
甘蔗渣水解液发酵：将菌株L7的单菌落接种于YPD种子培养基中，25℃、140r/min条件下培养36h，至OD 600为7.6，然后按照10％的接种量接种50/250mL(即在250mL的锥形瓶装50mL发酵培养基)甘蔗渣水解液培养基中，140r/min，25℃发酵7天。
菌株发酵产油：将菌株L7，在甘蔗渣水解液发酵培养基中发酵，其生物量、油脂产量和油脂含量与出发菌株ZZ-46生物量、油脂产量和油脂含量分别为18.87g/L、9.26g/L和49.07％，出发菌株ZZ-46为15.21g/L、6.76g/L和44.46％，其中生物量、油脂产量相比出发菌株分别提高了24.06％、36.98％。
实施例3：利用甘蔗渣水解液作为碳源的发酵培养基
甘蔗渣水解液作为碳源的发酵培养基组分：甘蔗渣水解液稀释至总糖70g/L，pH调至6， 添加氮源：酵母提取物0.75g/L、NH 4Cl 0.1g/L；无机盐：MgCl 2·6H 2O 1g/L、Na 2SO 4 0.1g/L；磷酸缓冲液：KH 2PO 4 11.8g/L、K 2HPO 4·3H 2O 3.7g/L；痕量元素：CaCl 2·2H 2O 40mg/L、FeSO 4·7H 2O 5.5mg/L、一水合柠檬酸5.2mg/L、ZnSO 4·7H 2O 1mg/L、MnSO4·H 2O 0.76mg/L、18mol H 2SO 4 1.84×10 -3mg/L。
菌株发酵产油：将菌株L7的单菌落接种于YPD种子培养基中，在25℃、140r/min条件下培养36h，至OD 600为7.6，然后按照10％的接种量接种50/250mL甘蔗渣水解液培养基中，140r/min，25℃发酵7天。
菌株L7，在甘蔗渣水解液作为碳源的发酵培养基中发酵后，其生物量、油脂产量和油脂含量分别达到22.12g/L、11.18g/L和50.54％。
实施例4：菌株L7在摇瓶优化条件下发酵产油
发酵培养基组分：甘蔗渣水解液稀释至总糖80g/L，并且添加大豆蛋白胨作为氮源，保持C/N＝273∶1，此外还添加无机盐：MgCl 2·6H 2O 1g/L、Na 2SO 4 0.1g/L；磷酸缓冲液：KH 2PO 4 11.8g/L、K 2HPO 4·3H 2O 3.7g/L；痕量元素：CaCl 2·2H 2O 40mg/L、FeSO 4·7H 2O 5.5mg/L、一水合柠檬酸5.2mg/L、ZnSO 4·7H 2O 1mg/L、MnSO4·H 2O 0.76mg/L、18mol H 2SO 4 1.84×10 -3mg/L。
菌株发酵产油：将菌株L7的单菌落接种于YPD种子培养基中，在22℃、140r/min条件下培养36h，至OD 600为7.6，然后按照10％的接种量接种40/250mL甘蔗渣水解液培养基中，pH调至6，140r/min，22℃、发酵8天。
其生物量、油脂产量和油脂含量达到26.73g/L、14.00g/L和52.37％。
实施例5：摇瓶条件下补料分批发酵
具体实施条件参见实施例4，在此基础上，设计了四种补料方式：初始补料点为发酵的第二天，补料次数分别为2、3、4、5次，每隔一天补料一次，每次补料量为15g/L(补料为葡萄糖/木糖＝2∶1的混合糖)。
具体方式为：将菌株L7的单菌落接种于YPD种子培养基中，在25℃、140r/min条件下培养36h，至OD 600为7.6，然后按照10％的接种量接种40/250mL甘蔗渣水解液培养基中，140r/min，22℃发酵，一共发酵9天。
补料2次：分别在第2天和第3天补料；
补料3次：分别在第2天、第3天和第4天补料；
补料4次：分别在第2天、第3天、第4天和第5天补料；
补料5次：分别在第2天、第3天、第4天、第5天和第6天补料。
每次补料添加终浓度为15g/L的混合糖(葡萄糖/木糖＝2∶1)。
发酵结果如表3，结果显示，补料两次所获得的油脂产量和油脂含量最大，分别是20.21g/L和57.93％，此时生物量为34.89g/L，较分批摇瓶培养分别提高44％、11％和31％。
表3 不同补料次数下菌株L7的产油性能
Figure PCTCN2021094555-appb-000003
实施例6：L7菌株在5L发酵罐发酵产油
(1)发酵罐分批发酵
具体发酵培养基组分和实施方式参见实施例4。
菌株发酵产油：将菌株L7的单菌落接种于YPD种子培养基中，在25℃、140r/min条件下培养36h，至OD600为7.6，然后按照10％的接种量接种2L甘蔗渣水解液培养基中，pH调至6，22℃、发酵5天；发酵过程中用2M NaOH调节pH，保持pH稳定在6，转速和DO相关联，保持DO在30％以上，发酵过程中添加有机硅消泡剂控制发酵过程中产生的泡沫。
发酵结果显示：生物量、油脂产量和油脂含量分别为33.33g/L、18.11g/L和54.34％
(2)发酵罐分批补料培养
采用葡萄糖/木糖＝2∶1，或葡萄糖/木糖＝1∶1的补料液进行补料发酵。
菌株发酵产油：将菌株L7的单菌落接种于YPD种子培养基中，在25℃、140r/min条件下培养36h，至OD600为7.6，然后按照10％的接种量接种2L甘蔗渣水解液培养基中，pH调至6，22℃进行发酵。发酵过程中用2M NaOH调节pH，保持pH稳定在6，转速和DO相关联，保持DO在30％以上。发酵过程中添加有机硅消泡剂控制发酵过程中产生的泡沫。
在第2和3天进行补料，补料为终浓度20g/L的混糖(葡萄糖/木糖＝2∶1，或葡萄糖/木糖＝1:1)。
在发酵至第8天时，油脂含量达到最大，其中当葡萄糖/木糖＝＝2∶1时，油脂产量能达到31.33g/L。
表4 不同比例补料下菌株L7的产油性能
Figure PCTCN2021094555-appb-000004
对比例1
具体实施方式参见实施例4，区别在于，将80g/L的总糖替换为50g/L、60g/L、70g/L、90g/L、100g/L，发酵后得到的生物量、油脂产量与油脂含量分见表5。
表5 不同总糖含量下菌株L7的产油性能
Figure PCTCN2021094555-appb-000005
对比例2
具体实施方式参见实施例4，区别在于，将氮源大豆蛋白胨替换为NH 4Cl、(NH 4) 2SO 4、酵母提取物、鱼粉蛋白胨、NH 4Cl+酵母提取物，发酵后得到的生物量、油脂产量与油脂含量分见表6。
表6 不同氮源条件下菌株L7的产油性能
Figure PCTCN2021094555-appb-000006
对比例3
具体实施方式参见实施例4，区别在于，将C/N＝273∶1替换为123∶1、223∶1、323∶1、373∶1、423∶1、523∶1，发酵后得到的生物量、油脂产量与油脂含量分见表7。
表7 不同C/N条件下菌株L7的产油性能
Figure PCTCN2021094555-appb-000007
对比例4
具体实施方式参见实施例4，区别在于，将接种量10％替换为5％、7.5％、12.5％、15％，发酵后得到的生物量、油脂产量与油脂含量分见表8。
表8 不同接种量下菌株L7的产油性能
Figure PCTCN2021094555-appb-000008
对比例5
具体实施方式参见实施例4，区别在于，将装液量40/250mL替换为30/250mL、50/250mL、60/250mL、70/250mL，发酵后得到的生物量、油脂产量与油脂含量分见表9。
表9 不同装液量下菌株L7的产油性能
Figure PCTCN2021094555-appb-000009
对比例6
具体实施方式参见实施例4，区别在于，将pH 6替换为pH 4、pH 5、pH 7、pH 8，发酵后得到的生物量、油脂产量与油脂含量分见表10。
表10 不同pH下菌株L7的产油性能
Figure PCTCN2021094555-appb-000010
对比例7
具体实施方式参见实施例4，区别在于，将温度22℃替换为25℃、28℃、31℃，发酵后得到的生物量、油脂产量与油脂含量分见表11。
表11 不同发酵温度下菌株L7的产油性能
Figure PCTCN2021094555-appb-000011
Figure PCTCN2021094555-appb-000012
对比例8
具体实施方式参见实施例4，区别在于，将发酵时间8天替换为7天、9天，发酵后得到的生物量、油脂产量与油脂含量分见表12。
表12 不同发酵时间下菌株L7的产油性能
Figure PCTCN2021094555-appb-000013
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (12)

  1. 一株产油酵母，分类命名为德马特丝孢酵母(Trichosporon dermatis)，已于2020年5月21日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020139。
  2. 一种同时利用木糖和葡萄糖产油脂的方法，其特征在于，将权利要求1所述产油酵母加入含有木糖和葡萄糖的体系中。
  3. 根据权利要求2所述的方法，其特征在于，将所述产油酵母在22～28℃发酵7～10天。
  4. 一种提高油脂产量的方法，其特征在于，将权利要求1所述产油酵母加入发酵体系发酵产油。
  5. 根据权利要求4所述的方法，其特征在于，所述发酵体系中含有碳源，碳源为木糖和葡萄糖。
  6. 根据权利要求4所述的方法，其特征在于，所述发酵体系中含有碳源，碳源来自于木质纤维素类生物质原料的水解混合糖液。
  7. 根据权利要求4所述的方法，其特征在于，将OD 600为6～8的所述产油酵母种子液以5％～10％的添加量加入发酵体系。
  8. 根据权利要求4所述的方法，其特征在于，所述发酵体系中的总糖含量为70～90g/L；氮源为大豆蛋白胨、NH 4Cl或酵母提取物；碳/氮＝(273～373)∶1。
  9. 根据权利要求4所述的方法，其特征在于，所述发酵体系中发酵液的装液量为12％～20％，在22～28℃、pH6～8发酵7～10天。
  10. 根据权利要求4所述的方法，其特征在于，在发酵过程中进行补料；所述补料是补加终浓度10～20g/L为葡萄糖和木糖的混合物，混合物中葡萄糖和木糖的比例为(1～2)∶1。
  11. 根据权利要求4所述的方法，其特征在于，所述发酵体系中含有碳源，碳源为木糖和葡萄糖；碳源来自于木质纤维素类生物质原料的水解混合糖液；将OD 600为6～8的所述产油酵母种子液以5％～10％的添加量加入发酵体系；所述发酵体系中的总糖含量为70～90g/L；氮源为大豆蛋白胨、NH 4Cl或酵母提取物；碳/氮＝(273～373)∶1；所述发酵体系中发酵液的装液量为12％～20％，在22～28℃、pH6～8发酵7～10天；在发酵过程中进行补料，所述补料是补加终浓度10～20g/L为葡萄糖和木糖的混合物，混合物中葡萄糖和木糖的比例为(1～2)∶1。
  12. 权利要求1所述产油酵母在食品、医药、农业、化工领域生产油脂中的应用。
PCT/CN2021/094555 2020-06-15 2021-05-19 一种产油酵母及其应用 WO2021254076A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/674,913 US20220170058A1 (en) 2020-06-15 2022-02-18 Oleaginous Yeast and its application

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010546363.0A CN111635867B (zh) 2020-06-15 2020-06-15 一种产油酵母及其应用
CN202010546363.0 2020-06-15

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/674,913 Continuation US20220170058A1 (en) 2020-06-15 2022-02-18 Oleaginous Yeast and its application

Publications (1)

Publication Number Publication Date
WO2021254076A1 true WO2021254076A1 (zh) 2021-12-23

Family

ID=72327044

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/094555 WO2021254076A1 (zh) 2020-06-15 2021-05-19 一种产油酵母及其应用

Country Status (3)

Country Link
US (1) US20220170058A1 (zh)
CN (1) CN111635867B (zh)
WO (1) WO2021254076A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115521951A (zh) * 2022-10-12 2022-12-27 中国科学院青岛生物能源与过程研究所 一种对废酿酒酵母泥进行诱导培养以积累油脂的方法
CN115521951B (zh) * 2022-10-12 2024-05-31 中国科学院青岛生物能源与过程研究所 一种对废酿酒酵母泥进行诱导培养以积累油脂的方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111635867B (zh) * 2020-06-15 2022-03-15 江南大学 一种产油酵母及其应用
CN112694980A (zh) * 2020-11-16 2021-04-23 南京工业大学 一株产油酵母菌及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102634549A (zh) * 2012-04-28 2012-08-15 中国科学院广州能源研究所 一种微生物油脂的生产方法
CN103642858A (zh) * 2013-11-22 2014-03-19 中国科学院广州能源研究所 一种调控油脂酵母发酵产生的微生物油脂的脂肪酸组成的方法
CN111635867A (zh) * 2020-06-15 2020-09-08 江南大学 一种产油酵母及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102634549A (zh) * 2012-04-28 2012-08-15 中国科学院广州能源研究所 一种微生物油脂的生产方法
CN103642858A (zh) * 2013-11-22 2014-03-19 中国科学院广州能源研究所 一种调控油脂酵母发酵产生的微生物油脂的脂肪酸组成的方法
CN111635867A (zh) * 2020-06-15 2020-09-08 江南大学 一种产油酵母及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HOU-MIN LI ; HONG-TAO DU ; WEI LIU ; ZHE WAN ; RUO-YU LI: "Microbiological Characteristics of Medically Important Trichosporon Species", MYCOPATHOLOGIA, KLUWER ACADEMIC PUBLISHERS, DO, vol. 160, no. 3, 1 October 2005 (2005-10-01), Do , pages 217 - 225, XP019259944, ISSN: 1573-0832 *
HUANG CHAO, XUE-FANG CHEN, LIAN XIONG, XIN-DE CHEN, LONG-LONG MA: "Oil production by the yeast Trichosporon dermatis cultured in enzymatic", BIORESOURCE TECHNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 110, 24 January 2012 (2012-01-24), AMSTERDAM, NL , pages 711 - 714, XP055882137, ISSN: 0960-8524, DOI: 10.1016/j.biortech.2012.01.077 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115521951A (zh) * 2022-10-12 2022-12-27 中国科学院青岛生物能源与过程研究所 一种对废酿酒酵母泥进行诱导培养以积累油脂的方法
CN115521951B (zh) * 2022-10-12 2024-05-31 中国科学院青岛生物能源与过程研究所 一种对废酿酒酵母泥进行诱导培养以积累油脂的方法

Also Published As

Publication number Publication date
US20220170058A1 (en) 2022-06-02
CN111635867A (zh) 2020-09-08
CN111635867B (zh) 2022-03-15

Similar Documents

Publication Publication Date Title
Hickert et al. Fermentation kinetics of acid–enzymatic soybean hull hydrolysate in immobilized-cell bioreactors of Saccharomyces cerevisiae, Candida shehatae, Spathaspora arborariae, and their co-cultivations
Guerfali et al. Single cell oil production by Trichosporon cutaneum and lignocellulosic residues bioconversion for biodiesel synthesis
Wang et al. Effect of fermentation conditions on L-lactic acid production from soybean straw hydrolysate
CN102041235B (zh) 一株耐高温酿酒酵母菌及其应用
Shrestha et al. Ethanol production via in situ fungal saccharification and fermentation of mild alkali and steam pretreated corn fiber
WO2021254076A1 (zh) 一种产油酵母及其应用
WO2010072093A1 (zh) 一种纤维素乙醇的生产方法
Liu et al. Consolidated bioprocess for bioethanol production with alkali-pretreated sugarcane bagasse
CN102864188A (zh) 一种木质纤维素生产生物柴油的方法
CN109706089A (zh) 一种耐受抑制物的木糖发酵菌株及其构建方法和应用
CN102250974A (zh) 一种微生物油脂的制备方法
CN113308387B (zh) 联产不饱和脂肪酸和类胡萝卜素的菌株及其应用
Lin et al. Microbial lipid production by oleaginous yeast in D-xylose solution using a two-stage culture mode
CN102174433A (zh) 一株高抗逆性贝氏梭菌及其应用
WO2016173262A1 (zh) 一种农林生物质废弃物浓醪发酵产纤维素乙醇的方法
Xue et al. Microbial lipid production from AFEX™ pretreated corn stover
CN102766615B (zh) 一种利用芽孢杆菌制备纤维素酶的方法
Sun et al. Highly efficient microbial lipid synthesis from co-fermentation of enzymatic hydrolysate of sugarcane bagasse by a Trichosporon dermatis mutant
CN104789492A (zh) 巨大芽孢杆菌菌株及其应用
Shokrkar et al. Exploring strategies for the use of mixed microalgae in cellulase production and its application for bioethanol production
CN101591684A (zh) 一种固体发酵木质纤维素原料产微生物油脂的方法
CN104762229A (zh) 一种枯草芽孢杆菌菌株及其应用
Hideno et al. Utilization of spent sawdust matrix after cultivation of Grifola frondosa as substrate for ethanol production by simultaneous saccharification and fermentation
Sopandi et al. Sugar consumption in mono and co-culture Saccharomyces cerevisiae and others selected microorganism for bioethanol production from stream rice husk medium
CN111411141B (zh) 一种菌酶共酵玉米秸秆生产微生物油脂的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21825081

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21825081

Country of ref document: EP

Kind code of ref document: A1