CN109810909B - A strain of Phaffia rhodozyma with high lycopene production and method for producing lycopene - Google Patents
A strain of Phaffia rhodozyma with high lycopene production and method for producing lycopene Download PDFInfo
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Abstract
The invention discloses a phaffia rhodozyma strain with a preservation number of CCTCC M2019083; a method for producing lycopene, comprising: 1) taking Phaffia rhodozyma, activating, inoculating to a fermentation medium, and performing shake culture to obtain a fermentation liquid; 2) centrifuging the fermentation liquor, collecting the precipitate to obtain thalli, washing with water, centrifuging, discarding the supernatant, adding 3mol/L hydrochloric acid solution, shaking uniformly, soaking, boiling water bath, cooling, centrifuging, collecting the precipitate, washing with water, centrifuging, discarding the supernatant to obtain cell debris; 3) adding acetone, extracting under shaking in dark condition, centrifuging, and collecting supernatant; 4) evaporating to dryness under reduced pressure, adding methanol to obtain methanol lycopene solution, transferring to chromatographic column, eluting with methanol, collecting eluate, evaporating to dryness under reduced pressure to obtain lycopene; the content of lycopene is 30.32mg/g dry thallus.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a high-lycopene-yield Phaffia rhodozyma strain and a lycopene production method.
Background
Lycopene, also known as psi-carotene, has a molecular formula C40H56The molecular weight is 536.8, and the conjugated double bond is composed of 11 conjugated double bonds and 2 non-conjugated double bonds. In the lower concentration solution, the lycopene is orange yellow, usually dark red powder or oily liquid, and the pure lycopene is needle-shaped dark red, insoluble in water, insoluble in polar organic solvent, soluble in organic solvent with low polarity, so that the lycopene is usually extracted by using organic solvent with low polarity. Lycopene is a terpenoid substance with strong antioxidant activity, has multiple physiological functional activities of quenching singlet oxygen, scavenging free radicals, inhibiting lipid peroxidation and the like, and has the ability of quenching singlet oxygen of beta-Carotene 2 times and vitamin E100 times, and is rated as a type A nutrient by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the United nations. Lycopene is separated from berry fruit as early as 1873, and is crystalline dark red pigment; millardet extracted lycopene from tomato in 1875, and named lycopene; later 1903, Schunck renamed it as lycopene and used it to date.
The structure of the lycopene conjugated diene not only determines the absorption peak and special red color of the lycopene conjugated diene in an organic solvent, but also endows the lycopene conjugated diene with wide physiological functions, such as antioxidation, anti-aging, heart disease prevention, atherosclerosis alleviation, multiple cancers prevention and the like. Lycopene can effectively inhibit the growth of cancer cells. The lycopene can effectively protect skin and reduce the damage of ultraviolet rays, and simultaneously has an effect of improving the immunity of an organism, and the lycopene promotes the proliferation of immune cells and plays a role in protecting phagocytes. It is known as "21 st century health product" because of its name of gold. The current production methods of lycopene mainly comprise a natural extraction method, a chemical synthesis method and a microbial fermentation method. The lycopene obtained by the natural extraction method has low yield and high cost and is limited by the source of raw materials; the chemical synthesis method has potential safety hazard; the microbial fermentation method has the advantages of simple process, high production efficiency, no influence of climate and season, and the like, and is more and more concerned. The microorganisms capable of producing lycopene comprise Blakeslea trispora, Dunaliella salina, bacteria, yeast and genetically engineered bacteria. Wherein, Escherichia coli (E. coli) producing lycopeneEscherichia coli) And Pichia pastoris (Pichia pastoris) Engineering bacteria have been successfully constructed. However, the engineering bacteria have low stability coefficient, may hide potential safety hazard, and have a lot of problems to be solved from industrial production. At present, the main strain for industrially producing lycopene is Blakeslea trispora, but most of products are mixed carotenoids, the operation process is complex and the like. The pigment producing characteristic of rhodotorula is probably similar to that of blakeslea trispora, the pigment yield of the currently obtained strain is relatively low, but the rhodotorula has the advantages of simple and extensive nutrition requirement, short growth period, no toxicity of thalli, rich nutrition and the like, and the method for producing the rhodotorula has the advantages of simple and extensive nutrition requirement, no toxicity of thalli, rich nutrition and the likeThe application has wide prospect. The excellent performance of the lycopene is widely concerned at home and abroad, while the potential value of the lycopene is not completely developed, but the lycopene cannot be synthesized by human body and can only be extracted from other substances, so that a new resource is particularly important for developing and utilizing the lycopene.
Qiuhuang and the like are subjected to ultraviolet mutation breeding to obtain a rhodotorula mutant Zk1-1 with excellent high-yield lycopene, and the yield of the lycopene reaches 20.25mg/L by optimizing culture conditions; lijie and the like perform condition optimization on key steps in breeding of the lycopene-producing red yeast by utilizing a genome rearrangement technology, and the yield of the lycopene can reach 103.47 mu g/mL; the method comprises the following steps of (1) selecting rhodotorula as a fermentation strain to produce lycopene, optimizing to obtain the optimal extraction condition, wherein the yield of the lycopene is 1.031 mug/mL; the extraction process of the lycopene by the Rhodotorula glutinis is optimized by Wanghainessmen and the like to obtain the content of the lycopene of 4.55 mg/g; wanghainessman and the like adopt metabolic regulators to regulate and control the Rhodotorula glutinis, and the lycopene accumulation reaches 176.97 mg/L; tangfeng et al added ergosterol synthesis inhibitor to make lycopene biosynthesis amount up to 24.23 mg/L.
Disclosure of Invention
The invention aims to provide a phaffia rhodozyma strain with high lycopene yield and a lycopene production method.
A strain of red-hair Fulvia yeast, the preservation number of which is CCTCC NO: M2019083.
A method for producing lycopene, comprising:
1) taking Phaffia rhodozyma, activating, inoculating the Phaffia rhodozyma to a fermentation medium according to the inoculation amount of 1-15%, performing shake culture at 18-28 ℃ at 120-300r/min, and fermenting for 48-96h to obtain fermentation liquor;
2) centrifuging the fermentation liquor at 4000-6000 r/min and 0-4 ℃ for 8-15 min, collecting precipitates to obtain thalli, washing with water, centrifuging, discarding supernate to obtain yeast mud, adding a 3mol/L hydrochloric acid solution with the volume of 20-30% of the fermentation liquor, uniformly shaking, soaking for 25-35 min, carrying out boiling water bath for 4-5 min, and cooling in an ice bath; centrifuging at 4000-6000 r/min for 8-15 min to remove hydrochloric acid solution, collecting precipitate, washing with water, centrifuging, and discarding supernatant to obtain cell debris;
3) adding acetone into the cell fragments, performing shaking extraction for 1-3 min under the condition of keeping out of the sun, centrifuging for 8-15 min at 4000-8000 r/min, and collecting supernatant; repeating the step of adding acetone until the supernatant is colorless;
4) evaporating the supernatant obtained in the step 3) at 40-50 ℃ under reduced pressure, adding methanol, transferring to a chromatographic column, eluting with methanol, collecting the eluent, and evaporating at 40-50 ℃ under reduced pressure to obtain lycopene;
the preservation number of the phaffia rhodozyma of the step 1) is CCTCC NO: M2019083;
the fermentation medium in the step 1) comprises the following components in parts by weight: 1-3 kg of yeast extract powder, 1-4 kg of malt extract powder, 2-12 kg of corn steep liquor, 2-8 kg of peptone, 5-15 kg of glucose and 1000kg of water;
the fermentation medium further comprises: h2O22~6g；
Said H2O2Is 4 g;
shake culturing for 72h at 22 ℃ and 180r/min as described in step 1);
the chromatographic column in the step 4) is a C18 solid phase extraction column.
The invention provides a phaffia rhodozyma strain with a preservation number of CCTCC NO: M2019083; a method for producing lycopene, comprising: 1) taking Phaffia rhodozyma, activating, inoculating to a fermentation medium, and performing shake culture to obtain a fermentation liquid; 2) centrifuging the fermentation liquor, collecting the precipitate to obtain thalli, washing with water, centrifuging, discarding the supernatant, adding 3mol/L hydrochloric acid solution, shaking uniformly, soaking, boiling water bath, cooling, centrifuging, collecting the precipitate, washing with water, centrifuging, discarding the supernatant to obtain cell debris; 3) adding acetone, extracting under shaking in dark condition, centrifuging, and collecting supernatant; 4) evaporating to dryness under reduced pressure, adding methanol to obtain methanol lycopene solution, transferring to chromatographic column, eluting with methanol, collecting eluate, evaporating to dryness under reduced pressure to obtain lycopene; the content of lycopene is 30.32mg/g dry thallus.
Drawings
FIG. 1 is a phylogenetic tree of the 26s D1/D2 region of Phaffia rhodozyma (CCTCC NO: M2019083);
FIG. 2 shows the comparison of the lycopene production by Phaffia rhodozyma.
Detailed Description
EXAMPLE 1 preparation of Phaffia rhodozyma species
Collecting orchard soil of Jilin agriculture university, inoculating the orchard soil to a YPD culture medium plate, culturing at a constant temperature of 20-28 ℃ for 48-96h, selecting red colonies to be inoculated in a YEPD culture medium, performing constant temperature shaking table 150-270r/min culture at a temperature of 20-28 ℃ for 48-96h, performing lycopene yield determination to obtain a strain with higher lycopene yield, namely PR106, performing bacterial morphology, physiological and biochemical analysis and 26s rRNA identification, wherein the length of 26s rRNA is 522bp, and comparing the strain with phaffia rhodozyma through a phylogenetic tree (Phaffia rhodozyma) ((26Phaffia rhodozyma) The yeast is named as Phaffia rhodozyma PR106, is preserved in China center for type culture Collection, has the preservation time of 2019, 1 month and 24 days, addresses Wuhan university in Wuhan city, Hubei province, and has the preservation number of CCTCC NO: m2019083.
EXAMPLE 2 Strain culture
Activating strains: taking Phaffia rhodozyma PR106, carrying out shake-flask culture, carrying out streak culture, culturing at 20-28 ℃ in a biochemical incubator, selecting a large and dark single colony to be inoculated into a liquid culture medium after 7 days, sterilizing for 15min at 121 ℃ in the liquid culture medium, culturing the strain for 48-96h at 18-28 ℃ and 270r/min, and carrying out third generation according to the steps;
seed culture: the activated strain after subculture uses YEPD liquid culture medium, the inoculation amount is 1-10%, and the activated strain is cultured for 48-96h under the conditions of 18-28 ℃ and 120-300r/min in a shaking table.
Example 3 extraction and purification of lycopene
Inoculating Phaffia rhodozyma PR106 into a 100mL Erlenmeyer flask filled with 30mL of fermentation medium according to the inoculation amount of 10%, and carrying out shake culture at 20 ℃ and 180r/min for 72 h; dissolved oxygen of 40-90%, controlling by adding alkali liquor, and pH6.0-7.5; the fermentation medium can be replaced by YEPD medium, but the production cost and the fermentation period are comprehensively considered, and the fermentation medium is most suitable;
then cell disruption is carried out, 20 mL of fermentation liquor is put into a 50 mL centrifuge tube, centrifugation is carried out for 10min at 5000 r/min and 4 ℃ to collect precipitation to obtain thalli, distilled water is used for washing and centrifugation to remove supernatant for three times to obtain yeast paste, 5mL of pre-prepared 3mol/L hydrochloric acid solution is added, the yeast paste is soaked for 30min after being vibrated uniformly to acidify the cell wall of yeast, boiling water bath is carried out for 4-5 min, and then the yeast paste is immediately put into ice water for cooling. Centrifuging the wall-broken cell paste at 5000 r/min for 10min to remove hydrochloric acid, collecting precipitate, repeatedly centrifuging and washing with water for 3 times, and discarding supernatant to obtain cell debris. Storing the precipitate at 4 deg.C for later determination;
extracting lycopene with acetone. Taking 20 mL of fermentation liquor, breaking cells, adding 5mL of acetone, extracting for 1 min by shaking under the condition of keeping out of the sun, centrifuging for 10min at 5000 r/min, collecting supernatant to obtain pigment acetone extract, and adding acetone for extracting again if the pigment acetone extract is not completely extracted until the cells are colorless.
Adding 1mL of extracting solution into each 1.5mL of centrifuge tube, adding 100 muL of methanol to dissolve a crude product after decompression and evaporation at 45 ℃, taking 5mL of methanol to activate a CNW C18 solid phase extraction column, and then transferring 2mL of methanol lycopene solution to a C18 column. Eluting with 10 mL of methanol, adding into the column for 4 times, collecting all eluates, evaporating to dryness at 45 deg.C under reduced pressure, adding 46 μ L of methanol into each 1.5mL centrifuge tube for redissolving, and filtering with 0.22 nm organic filter membrane for use.
Example 4 high performance liquid chromatography analysis of lycopene
Measuring the content of lycopene in Phaffia rhodozyma PR106 by adopting an ultraviolet detector high performance liquid chromatography; the chromatographic conditions are as follows:
1) a chromatographic column: a C18 column (4.6 mm. times.250 mm, 5 μm), or a column of equivalent performance;
2) mobile phase: mobile phase: acetonitrile: dichloromethane = 10: 90, filtered through 0.45 μm membrane for use;
3) flow rate: 1.0 mL/min;
4) detection wavelength: 472 nm;
5) column temperature: room temperature;
6) sample introduction volume: 20 mu L, isocratic elution and constant concentration;
analyzing the lycopene content in the phaffia rhodozyma PR106 by using a high performance liquid chromatography; setting a control group, wherein the control group is CICC33064 Phaffia rhodozyma from China center for culture collection management of industrial microorganisms; as shown in FIG. 2, the lycopene content in Phaffia rhodozyma PR106 was 27.00mg/g dry cell (117.46 mg/L fermentation broth), and the lycopene content in the control group was only 9.05 mg/g; the high yield of lycopene by the phaffia rhodozyma PR106 is proved.
Example 5 Effect of addition of Metabolic Modulator on lycopene production
Inoculating into 30mL fermentation medium containing 2% lycopene, adding different H2O2The amounts of (A) are respectively 0%, 0.2%, 0.4% and 0.6%; placing the triangular flask into a shaking table for culturing under the conditions of rotation speed of 180r/min and temperature of 20 ℃, and measuring the content of lycopene after 72 hours; the results show that when H is2O2When the amount of the lycopene is 0.4%, the content of lycopene produced by Phaffia rhodozyma PR106 is 30.32mg/g dry cell (144.9 mg/L fermentation broth), and it is proved that a proper amount of H is added to the fermentation medium2O2Is more beneficial to improving the content of lycopene.
EXAMPLE 6 preparation of the culture Medium
YPD, YEPD and fermentation media described in examples 1-5, whose compositions were:
1) YPD medium: 2.0 g of yeast extract powder, 3.0 g of malt extract powder, 5.0 g of peptone, 10.0 g of glucose, 20.0 g of agar and 1.0L of tap water;
2) YEPD medium: 2.0 g of yeast extract powder, 3.0 g of malt extract powder, 5.0 g of peptone, 10.0 g of glucose and 1.0L of tap water;
3) fermentation medium: 2.0 g of yeast extract powder, 3.0 g of malt extract powder, 8.0kg of corn steep liquor, 5.0 g of peptone, 10.0kg of glucose and 1000.0L of tap water.
The preparation method of the three culture media is prepared according to a conventional method.
Sequence listing
<110> Jilin university of agriculture
<120> high-lycopene-yield Phaffia rhodozyma strain and lycopene production method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 522
<212> DNA
<213> rRNA RNA
<400> 1
tagtacggcg agtgaagcgg gatgagctca aatttgaaat ctggcagcct ccggttgtcc 60
gagttgtaaa ctagagaagc gttttccgtg ccggcctgtg tacaagtccc ttggaatagg 120
gcgtcataga gggtgagaat cccgtccttg acacagacca ccggtgctat gtgatacgct 180
ctcgacgagt cgagttgttt gggaatgcag ctcaaattgg gtggtaaatt ccatctaagg 240
ctaaatattg gcgagagacc gatagcgaac aagtaccgtg agggaaagat gaaaagcact 300
ttggaaagag agttaaacag tacgtgaaat tgttgaaagg gaaacgattg aagtcagtca 360
tgcgtgctcg gactcagctg ggttcgtccc agtctatttc cgggtgccgc aggtcagcat 420
cagtttcggg cggtggaaaa cgggcggggg aaggtggcat ctccggatgt gttatagccc 480
ccgtttggat gcatcgcgtg ggactgagga acgcagcgcg cc 522
Claims (7)
1. Phaffia rhodozyma strain (A)Phaffia rhodozyma) The preservation number is CCTCC NO: m2019083.
2. A method for producing lycopene, comprising:
1) taking the phaffia rhodozyma of claim 1, activating, inoculating to a fermentation medium according to the inoculation amount of 1-15%, performing shake culture at 18-28 ℃ and 120-300r/min, and fermenting for 48-96 hours to obtain a fermentation liquid;
2) centrifuging the fermentation liquor at 4000-6000 r/min and 0-4 ℃ for 8-15 min, collecting precipitates to obtain thalli, washing with water, centrifuging, discarding supernate to obtain yeast mud, adding a 3mol/L hydrochloric acid solution with the volume of 20-30% of the fermentation liquor, uniformly shaking, soaking for 25-35 min, carrying out boiling water bath for 4-5 min, and cooling in an ice bath; centrifuging at 4000-6000 r/min for 8-15 min to remove hydrochloric acid solution, collecting precipitate, washing with water, centrifuging, and discarding supernatant to obtain cell debris;
3) adding acetone into the cell fragments, performing shaking extraction for 1-3 min under the condition of keeping out of the sun, centrifuging for 8-15 min at 4000-8000 r/min, and collecting supernatant; repeating the step of adding acetone until the supernatant is colorless;
4) and 3) evaporating the supernatant obtained in the step 3) to dryness at 40-50 ℃ under reduced pressure, adding methanol, transferring to a chromatographic column, eluting with methanol, collecting the eluent, and evaporating to dryness at 40-50 ℃ under reduced pressure to obtain the lycopene.
3. A lycopene production method according to claim 2, wherein: the fermentation medium in the step 1) comprises the following components in parts by weight: 1-3 kg of yeast extract powder, 1-4 kg of malt extract powder, 2-12 kg of corn steep liquor, 2-8 kg of peptone, 5-15 kg of glucose and 1000kg of water.
4. A lycopene production method according to claim 3, wherein: the fermentation medium further comprises: h2O2 2~6g。
5. A lycopene production method according to claim 4, wherein: said H2O2The amount used was 4 g.
6. A lycopene production method according to claim 5, wherein: the fermentation in the step 1) is carried out for 72 hours at 22 ℃ and 180r/min by shaking culture.
7. A lycopene production method according to claim 6, wherein: the chromatographic column in the step 4) is a C18 solid phase extraction column.
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| CN111205991B (en) * | 2020-02-26 | 2022-08-16 | 吉林农业大学 | Method for producing levo-astaxanthin through fermentation |
| CN112779295B (en) * | 2020-12-31 | 2022-12-13 | 广东博沃特生物科技有限公司 | High-density fermentation medium for producing lycopene saccharomyces cerevisiae |
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| US5691190A (en) * | 1993-01-19 | 1997-11-25 | Pernod Ricard | Phaffia rhodozyma mutants, process for producing β-carotene and use of β-carotene rich biomass |
| KR19990048085A (en) * | 1997-12-08 | 1999-07-05 | 심광경 | Astaxanthin-producing yeast mutants and methods for their production |
| WO2003038064A3 (en) * | 2001-10-29 | 2003-09-25 | Dsm Ip Assets Bv | Blakeslea trispora producing high yield of lycopene in a suitable medium in the absence of an exogenous carotenogenesis inhibitor |
| CN101705193A (en) * | 2009-11-20 | 2010-05-12 | 中国水产科学研究院黄海水产研究所 | Astaxanthin-producing ocean rhodotorula YS-185 and method for producing astaxanthin thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5691190A (en) * | 1993-01-19 | 1997-11-25 | Pernod Ricard | Phaffia rhodozyma mutants, process for producing β-carotene and use of β-carotene rich biomass |
| KR19990048085A (en) * | 1997-12-08 | 1999-07-05 | 심광경 | Astaxanthin-producing yeast mutants and methods for their production |
| WO2003038064A3 (en) * | 2001-10-29 | 2003-09-25 | Dsm Ip Assets Bv | Blakeslea trispora producing high yield of lycopene in a suitable medium in the absence of an exogenous carotenogenesis inhibitor |
| CN101705193A (en) * | 2009-11-20 | 2010-05-12 | 中国水产科学研究院黄海水产研究所 | Astaxanthin-producing ocean rhodotorula YS-185 and method for producing astaxanthin thereof |
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