CN114921504B - Method for producing microbial grease and carotenoid through variable-temperature fermentation - Google Patents
Method for producing microbial grease and carotenoid through variable-temperature fermentation Download PDFInfo
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- 230000004151 fermentation Effects 0.000 title claims abstract description 86
- 235000021466 carotenoid Nutrition 0.000 title claims abstract description 25
- 150000001747 carotenoids Chemical class 0.000 title claims abstract description 25
- 230000000813 microbial effect Effects 0.000 title claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
- 239000004519 grease Substances 0.000 title claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 25
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- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
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- 238000009776 industrial production Methods 0.000 abstract description 3
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- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
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- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
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- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
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- 230000007365 immunoregulation Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 208000018937 joint inflammation Diseases 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229940095102 methyl benzoate Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 239000001053 orange pigment Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000001054 red pigment Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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- 239000012488 sample solution Substances 0.000 description 1
- 235000009657 tetraterpenes Nutrition 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000001052 yellow pigment Substances 0.000 description 1
Classifications
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- 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
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for producing microbial oil and carotenoid by variable-temperature fermentation, which comprises the steps of preparing inclined plane single colony, activating seeds, fermenting and culturing, and carrying out temperature regulation and control in stages in the fermenting and culturing process, and specifically comprises the following steps: the first stage: the duration is 36-60h, and the fermentation temperature is 30 ℃; and a second stage: the fermentation time is 6h, and the fermentation temperature is 25 ℃; and a third stage: the duration is 54-78h, and the fermentation temperature is 22 ℃; the total duration of the fermentation was 120h. The method for producing microbial grease and carotenoid by fermenting rhodosporidium through variable temperature can simultaneously realize high strain growth, high microbial grease content and high carotenoid content, reduce fermentation cost, improve fermentation efficiency, reduce production cost and bring great economic benefit to industrial production.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation pharmacy, and relates to a method for producing microbial oil and carotenoid by variable-temperature fermentation.
Background
The microbial oil is also called single-cell oil, and is produced by using yeast, mould, bacterial and algae and other microbes under certain conditions by using carbohydrate, hydrocarbon and common oil as carbon source, nitrogen source and inorganic salt as auxiliary materials. Under suitable conditions, some microorganisms produce and store more than 20% of their total biomass, and strains with such phenotypes are called oleaginous microorganisms.
The microbial oil contains a large amount of unsaturated fatty acid, which is a fatty acid constituting fat in a living body and is essential for the body. The unsaturated fatty acid has different double bond numbers, and is divided into monounsaturated fatty acid and polyunsaturated fatty acid. In the food fat, the monounsaturated fatty acid comprises oleic acid, polyunsaturated fatty acid comprises linoleic acid, linolenic acid, arachidonic acid, etc., and the human body can not synthesize the linoleic acid and the linolenic acid, and must be supplemented from the food. As an important organic compound, the method is widely used for regulating blood fat, clearing thrombus, participating in immunoregulation, improving joint inflammation, relieving pain and the like, so that the production of unsaturated fatty acid has strong economic benefit.
Carotenoids are a general term for an important class of natural pigments, commonly found in yellow, orange or red pigments of animals, higher plants, fungi, algae. It is a 40 carbon isoprenoid polymer, namely a tetraterpene compound. Typical carotenoids are formed from 8 isoprene units joined end to end. The color of carotenoids varies with the number of conjugated double bonds. The greater the number of conjugated double bonds, the more the color shifts to red.
In the current fermentation process of rhodosporidium toruloides, the temperature of a fermentation tank in a culture stage is mostly about 30 ℃, and the optimal temperatures of two paths of the rhodosporidium toruloides co-production microbial oil and carotenoid are different, so that the yields of the two products are influenced by the influence of the temperature. However, at present, no temperature-variable fermentation process of rhodosporidium toruloides exists, the fermentation culture temperature and duration have influence on the growth amount of strains, the content of microbial oil and the content of carotenoid, and the thallus is produced quickly, so that the thallus is attenuated early and the accumulation of final tank release titer is not facilitated, so that the realization of high strain growth amount, high microbial oil content and high carotenoid content is not easy.
Disclosure of Invention
The invention aims to provide a method for producing microbial oil and carotenoid by variable-temperature fermentation.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for producing microbial oil and carotenoid by variable-temperature fermentation comprises the steps of preparing inclined plane single colony, activating seeds, fermenting and culturing, and carrying out temperature regulation and control in stages in the culturing process of a fermentation tank, wherein the method comprises the following steps:
the first stage: the duration is 36-60h, and the fermentation temperature is 30 ℃;
and a second stage: the fermentation time is 6h, and the fermentation temperature is 25 ℃;
and a third stage: the duration is 54-78h, and the fermentation temperature is 22 ℃;
the total duration of the fermentation was 120h.
As a preferred embodiment, microbial oils and carotenoids are produced by fermentation using Rhodosporidium toruloides as a fermentation strain.
As a preferred embodiment, rhodosporidium toruloides (Rhodosporidium toruloides) Z11 is used as a fermentation strain for the fermentative production of microbial oils and carotenoids.
As a preferred embodiment, the carbon source for the fermentation culture is glucose.
As a preferred embodiment, the initial concentration of the carbon source at the time of fermentation culture is 70g/L.
As a preferred embodiment, the carbon source concentration in the first stage of fermentation culture is 70g/L, and the carbon source concentration in the second and third stages is 40g/L.
As a preferred embodiment, the pH of the fermentation system is maintained at 6 during the fermentation culture by adjusting the pH with citric acid.
As a preferred embodiment, the aeration ratio during the fermentation culture is 1:1vvm, stirring speed is 600rpm, and dissolved oxygen is controlled to be not less than 70%.
As a preferred embodiment, the first stage time length of fermentation culture is 48 hours, and the fermentation temperature is 30 ℃; the second stage is 6 hours long, and the fermentation temperature is 25 ℃; the third stage is 66h, and the fermentation temperature is 22 ℃.
The method for producing microbial grease and carotenoid by fermenting rhodosporidium through variable temperature can simultaneously realize high strain growth, high microbial grease content and high carotenoid content, reduce fermentation cost, improve fermentation efficiency, reduce production cost and bring great economic benefit to industrial production.
Detailed Description
The strain used in the invention is as follows: rhodosporidium toruloides (Rhodosporidium toruloides) Z11 (published in applicant's prior patent application CN 113308387A, accession number is CCTCC NO: M2021226.
The unsaturated fatty acid analysis method adopted by the invention comprises the following steps:
gas Chromatography (GC) was used: after the fermentation-finished bacterial liquid (Z11) is completely collected and freeze-dried for 48 hours, 20mg of strain freeze-dried powder is weighed into a 15mL glass tube, 1.5mL of normal hexane, 0.5mL of methyl benzoate with concentration of 2mg/mL are added into the mixture, and 2mL of 15% H is added into the mixture 2 SO 4 (esterification) in methanol.
The pretreated sample was placed in an environment of 100deg.C and continuously shaken for 2 hours. Then cooled on ice for 10min, 1mL ddH was added 2 O, the rotation speed was centrifuged at 2500rpm for 5min. The supernatant (fatty acid methyl ester extract) was prepared and placed in a GC flask for gas phase detection.
The carotenoid extraction method adopted by the invention comprises the following steps:
using a spectrophotometer (OD) 455 ): the bacterial liquid after fermentation is duplicated at 10000rpm for 3min; one part of bacterial sludge is dried at 105 ℃ overnight to constant weight and is used for detecting the biomass M; 400uL of 3M HCl is added into one part of bacterial sludge, and the bacterial sludge is subjected to water bath for 3min and then ice water bath for 3min,13000rpm and 5min; removing supernatant, adding 300uL acetone into thallus, leaching for 1 hr, collecting supernatant with carotenoid, preserving, repeating this operation for 3-4 times until thallus is colorless, mixing acetone, adding 0.5mL deionized water and petroleum ether, centrifuging, removing colorless layer, and measuring OD with spectrophotometer 455 Finally, carotenoid C (ug/g) = (OD) 455 *V(mL)*10 4 ) 2680 x M (g); (V is the sample solution volume)
The following specific examples illustrate the invention in further detail.
The formulation of the slant medium (g/L) in the examples is: glucose 10g/L, yeast powder 20g/L, peptone 20g/L and agar powder 20g/L. Sterilizing at pH6 and 121deg.C for 15min;
the formula of the seed culture medium (g/L) is as follows: 10g/L glucose, 20g/L yeast powder, 20g/L, pH peptone and sterilizing at 121 ℃ for 15min;
the formula of the fermentation medium (g/L) is as follows: the initial sugar (glucose) concentration is 70g/L for the first 0-48 hr, the sugar supplementing concentration is 40g/L for the last 48 hr-120 hr, ammonium sulfate is 0.1g/L, and yeast extract is 0.75g/L, KH 2 PO 4 0.4g/L、MgSO 4 .7H 2 O3.07 g/L, trace element ZnSO 4 、CaCl 2 、MnCl 2 、CuSO 4 0.192X 10 respectively -7 g/L、0.167g/L、0.54×10 -4 g/L、0.16×10 -4 g/L. Sterilizing at 121deg.C for 15min at pH 6.0.
The table shows the effect of different temperatures on the fermentation product of rhodosporidium toruloides, the other operations being identical during the fermentation in examples 1-5, in particular:
example 1
(1) Activating strains: after the strain is subjected to plate streaking in a slant culture medium, performing activation culture in an aerobic incubator at 30 ℃ for 48 hours; until a single colony is grown;
(2) Seed culture: placing in a test tube filled with 5mLYPD culture solution, and culturing at 30deg.C in a shaker at 180rpm for 28 hr as seed solution;
(3) Culturing in a fermentation tank: the activated seed liquid is inoculated into a fermentation tank, the inoculation amount is 10% (v/v), the stirring speed is 600rpm, the fermentation culture is carried out at 18 ℃, and the full program control pH is 6.0 by using citric acid.
Example 2
Example 2 differs from example 1 only in that the temperature of the fermentation culture is 22 ℃.
Example 3
Example 3 differs from example 1 only in that the temperature of the fermentation culture is 25 ℃.
Example 4
Example 4 differs from example 1 only in that the temperature of the fermentation culture is 30 ℃.
Example 5
Example 5 differs from example 1 only in that the temperature of the fermentation culture is 37 ℃.
TABLE 1 fermentation results for examples 1-5
Examples 6 to 10
Examples 6 to 10 differ from examples 1 to 5 only in that the staged temperature swing fermentation is controlled at the time of fermentation culture:
example 6:
culturing in a fermentation tank: inoculating the activated seed liquid into a fermentation tank, fermenting and culturing for 0-24h at 30 ℃ with the inoculum size of 10% (v/v) and the stirring speed of 600rpm, changing the temperature to 25 ℃ and fermenting and culturing for 24-30h, changing the temperature to 22 ℃ and fermenting and culturing for 30-120h, and using citric acid to realize full program control pH value of 6.0.
Example 7:
culturing in a fermentation tank: inoculating the activated seed liquid into a fermentation tank, fermenting and culturing for 0-36h at 30 ℃ with the inoculum size of 10% (v/v) and the stirring speed of 600rpm, fermenting and culturing for 42-120h at 22 ℃ with the temperature of 25 ℃ after changing the temperature, and fully controlling the pH to 6.0 with citric acid.
Example 8:
culturing in a fermentation tank: inoculating the activated seed liquid into a fermentation tank, fermenting and culturing for 0-48h at 30 ℃ with the inoculum size of 10% (v/v) and the stirring speed of 600rpm, fermenting and culturing for 54-120h at 22 ℃ with the temperature of 25 ℃ after changing the temperature to be 48-54h, and fully controlling the pH to be 6.0 by using citric acid.
Example 9:
culturing in a fermentation tank: inoculating the activated seed liquid into a fermentation tank, fermenting and culturing for 0-60h at 30 ℃ with the inoculum size of 10% (v/v) and the stirring speed of 600rpm, fermenting and culturing for 66-120h at 22 ℃ with the temperature of 25 ℃ and then fermenting and culturing for 66-120h with citric acid full program control pH of 6.0.
Example 10:
culturing in a fermentation tank: inoculating the activated seed liquid into a fermentation tank, fermenting and culturing for 0-72h at 30 ℃ with the inoculum size of 10% (v/v) and the stirring speed of 600rpm, fermenting and culturing for 78-120h at 22 ℃ with the temperature of 25 ℃ after changing the temperature, and fully controlling the pH to 6.0 with citric acid.
TABLE 2 fermentation results for examples 5-10
In summary, in the temperature-variable fermentation process of rhodosporidium toruloides, compared with the fermentation result in the prior art, the yield of microbial oil and carotenoid of rhodosporidium toruloides is improved, and when the fermentation conditions are as follows: 0-48h, controlling the temperature at 30 ℃, the sugar supplementing concentration at 70g/L and 48-54h, controlling the temperature at 25 ℃, the sugar supplementing concentration at 40g/L and 54-120h, controlling the temperature at 22 ℃, controlling the pH at 6 by using citric acid in the whole fermentation tank, wherein the rhodosporidium toruloides OD can reach 150.45, the microbial oil can reach 54.65%, and the yield of the carrots can reach 30.8mg/g. The result is far higher than the fermentation result in the prior art, further improves the production efficiency, reduces the production cost, and can bring considerable economic benefit for industrial production of the rhodosporidium toruloides co-produced microbial oil and carotenoid.
Claims (7)
1. A method for producing microbial oil and carotenoid by variable-temperature fermentation comprises the steps of preparing inclined plane single colony, activating seeds, fermenting and culturing, and is characterized in that the temperature regulation and control are carried out in stages in the fermenting and culturing process, and specifically comprises the following steps:
the first stage: the duration is 36-60h, and the fermentation temperature is 30 ℃;
and a second stage: the fermentation time is 6h, and the fermentation temperature is 25 ℃;
and a third stage: the duration is 54-78h, and the fermentation temperature is 22 ℃;
the total duration of fermentation is 120h;
using rhodosporidium toruloidesRhodosporidium toruloides) Z11 is used as a fermentation strain for fermentation production of microbial grease and carotenoid.
2. The method of claim 1, wherein the carbon source for fermentation culture is glucose.
3. The method according to claim 1 or 2, wherein the initial concentration of the carbon source during the fermentation culture is 70g/L.
4. The method according to claim 1 or 2, wherein the concentration of the carbon source in the first stage is 70g/L and the concentration of the carbon source in the second and third stages is 40g/L.
5. The method according to claim 1, wherein the fermentation system is maintained at a pH of 6 during the fermentation culture by adjusting the pH of the fermentation system with citric acid.
6. The method according to claim 1, wherein the aeration ratio during fermentation culture is 1:1vvm, stirring speed is 600rpm, and dissolved oxygen is controlled to be not less than 70%.
7. The method according to claim 1, wherein the first stage is carried out for 48 hours at a fermentation temperature of 30 ℃; the second stage is 6 hours long, and the fermentation temperature is 25 ℃; the third stage is 66h, and the fermentation temperature is 22 ℃.
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