CN109810909A - The phaffiafhodozyma bacterial strain and lycopene production process of one plant height production lycopene - Google Patents
The phaffiafhodozyma bacterial strain and lycopene production process of one plant height production lycopene Download PDFInfo
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Abstract
The invention discloses one plant of phaffiafhodozyma bacterium, its deposit number is CCTCC M 2019083；A kind of lycopene production process, it includes: 1) to take phaffiafhodozyma bacterium, and activation is inoculated into fermentation medium, shaking table culture obtains fermentation liquid；2) fermentation liquid is centrifuged, collects precipitating and obtain thallus, washes, supernatant is abandoned in centrifugation, and 3 mol/L hydrochloric acid solutions are added, and concussion uniformly, impregnates, boiling water bath, cools down, centrifugation, collects precipitating, and washing, centrifugation abandon supernatant, obtain cell fragment；3) acetone is added, extraction is shaken under the conditions of being protected from light, supernatant is collected in centrifugation；4) evaporated under reduced pressure is added methanol, obtains methanol lycopene solution, be transferred to chromatographic column, eluted with methanol, collects eluent, and evaporated under reduced pressure obtains lycopene；The lycopene content of preparation is 30.32mg/g dry mycelium.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to the phaffiafhodozyma bacterial strain of plant height production lycopene
And lycopene production process.
Background technique
Lycopene is also known as ψ-carrotene, molecular formula C40H56, relative molecular mass 536.8, by 11 conjugated double bonds
It is formed with 2 unconjugated double bonds.In orange-yellow in lower concentration solutions, usually dark red powder or oily liquids, pure
Lycopene is then needle-shaped peony, not soluble in water, is insoluble in polar organic solvent, dissolves in the low organic solvent of polarity, because
This extracts lycopene and usually uses the lesser organic solvent of polarity.Lycopene is a kind of very strong terpene object of antioxidant activity
Matter has quenching singlet oxygen, removes the different physiological roles such as free radical, anti-lipid peroxidation activity, quenches singlet
The ability of oxygen is 2 times of beta carotene, 100 times of vitamin E, by the World Health Organization (WHO) and FAO (Food and Agriculture Organization of the United Nation)
(FAO) it is assessed as A class nutrient.Lycopene was separated from certain kind of berries fruit early in 1873, was crystallinity peony pigment；
Millardet in 1875 extracts the crude extract containing lycopene from tomato, and lycopene is named as
Solanorubin ；Afterwards in 1903, Schunck is renamed as lycopene and is used till today.
Lycopene conjugated diene structure not only determines its absorption peak in organic solvent and special red, also assigns
Its extensive physiological function is given, such as anti-oxidant, anti-aging prevents heart disease, slows down atherosclerosis, prevention kinds cancer
Deng.Lycopene can effectively inhibit the growth of cancer cell.Lycopene can be reduced with effective protection skin by ultraviolet light
Injury, while effect is improved to the immunocompetence of body, it is thin by protection phagocytosis that lycopene, which promotes immune cell propagation,
What born of the same parents played a role.There is the title of plant gold, is known as " new lover of 21 century health care product ".The production method of lycopene at present
It mainly include natural extraction method, chemical synthesis and microbe fermentation method.The yield of lycopene that natural extraction method obtains is not high,
It is at high cost and be limited by raw material sources；There are security risks for chemical synthesis；And microbe fermentation method has simple process, production
It is high-efficient, not climate and the advantages that seasonal effect, more and more attention has been paid to.The microorganism that lycopene can be produced at present has three
Spore Bradley is mould, Dunaliella salina, bacterium, yeast and genetic engineering bacterium.Wherein, the Escherichia coli of lycopene are produced
(Escherichia coli) and Pichia pastoris (Pichia pastoris) engineering bacteria successfully constructed.But engineering bacteria
The coefficient of stability is low, may also under cover security risk, apart from industrialized production, there are also many problems to be solved.Industry is raw at present
The main bacterial strain for producing lycopene is Blakeslea trispora, but product is mostly mixed type carotenoid, operating process complexity etc..It is red
The production Pigment Properties of yeast may be similar to Blakeslea trispora, and obtained bacterial strain pigment production is relatively low at present, but rhodotorula has
It is nutritious to require the advantages that simple extensive, growth cycle is short and thallus is nontoxic, full of nutrition, development and utilization have it is wide before
Scape.The excellent performance that lycopene shows is by extensive concern both at home and abroad, and the potential value of lycopene is complete not yet
Standard-sized sheet hair, but human body itself can not synthesize this substance, can only extract from other substances, thus a kind of new resource for kind
The development and utilization of Lycopene are particularly important.
Qiu Lianfang etc. obtains the rhodotorula mutant strain Zk1-1 of excellent high yield lycopene by ultraviolet mutagenesis breeding, through training
Condition optimizing is supported, yield of lycopene reaches 20.25mg/L；Li Jie etc. is using genome rearrangement technology to production lycopene rhodotorula
Committed step in breeding carries out condition optimizing, and yield of lycopene is up to 103.47 μ g/mL；Luo Bing selects rhodotorula as hair
Yeast-like fungi strain produces lycopene, and optimization obtains optimum extraction condition, yield of lycopene is 1.031 μ g/mL；Wang Haibing etc. pairs
Glutinous rhodotorula Extracting of Lycopene is optimized, and obtains lycopene content 4.55mg/g；Wang Haibing et al. uses generation
It thanks to modulator to regulate and control glutinous rhodotorula, lycopene cumulant reaches 176.97mg/L；Tang Feng etc. passes through addition etembonate
Alcohol synthetic inhibitor makes lycopene biosynthesis amount be up to 24.23mg/L.
Summary of the invention
The object of the present invention is to provide phaffiafhodozyma bacterial strains and lycopene production process that a plant height produces lycopene.
One plant of phaffiafhodozyma bacterium, its deposit number are CCTCC M 2019083.
A kind of lycopene production process, it includes:
1) phaffiafhodozyma bacterium is taken, activates, fermentation medium is inoculated by 1 ~ 15% bacterium amount that connects, in 18 ~ 28 DEG C, 120 ~ 300r/
Shaking table culture under min, ferment 48 ~ 96h, obtains fermentation liquid；
2) fermentation liquid is centrifuged to 8 ~ 15min at 4000 ~ 6000r/min, 0 ~ 4 DEG C, precipitating is collected and obtains thallus, wash, centrifugation,
It abandons supernatant and obtains yeast paste, the 3mol/L hydrochloric acid solution of 20 ~ 30% volumes of the fermentation liquid is added, concussion is uniform, immersion 25 ~
35 min, boiling water bath 4 ~ 5 minutes, ice bath cooling；It is centrifuged 8 ~ 15 min at 4000 ~ 6000 r/min and removes hydrochloric acid solution,
Precipitating is collected, washing, centrifugation abandon supernatant, obtain cell fragment；
3) cell fragment be added acetone, under the conditions of being protected from light concussion extraction 1 ~ 3min, at 4000 ~ 8000r/min be centrifuged 8 ~
15min collects supernatant；The step of repeating above-mentioned addition acetone, until supernatant is colourless；
4) by the supernatant of step 3), the evaporated under reduced pressure at 40 ~ 50 DEG C is added methanol, and is transferred to chromatographic column, is washed with methanol
It is de-, eluent is collected, 40 ~ 50 DEG C of evaporated under reduced pressure obtain lycopene；
Phaffiafhodozyma bacterium described in step 1), its deposit number are CCTCC M 2019083；
Fermentation medium described in step 1), its components by weight percent include: 1 ~ 3kg of yeast extract, 1 ~ 4kg of fructus hordei germinatus leaching powder, corn
Starch 2 ~ 12kg, 2 ~ 8kg of peptone, 5 ~ 15kg of glucose, water 1000kg；
The fermentation medium, further includes: H2O22~6g；
The H2O2For 4g；
Described in step 1) at 22 DEG C, 180 r/min 72 h of shaking table culture；
Chromatographic column described in step 4) is C18 solid-phase extraction column.
The present invention provides one plant of phaffiafhodozyma bacterium, its deposit number is CCTCC M 2019083；A kind of tomato red
Plain production method, it includes: 1) to take phaffiafhodozyma bacterium, and activation is inoculated into fermentation medium, shaking table culture obtains fermentation liquid；2)
Fermentation liquid is centrifuged, precipitating is collected and obtains thallus, supernatant is abandoned in washing, centrifugation, and 3 mol/L hydrochloric acid solutions are added, and concussion is uniform,
It impregnates, boiling water bath, cools down, precipitating is collected in centrifugation, and washing, centrifugation abandon supernatant, obtain cell fragment；3) acetone is added, is being protected from light
Under the conditions of shake extraction, centrifugation, collect supernatant；4) evaporated under reduced pressure is added methanol, obtains methanol lycopene solution, be transferred to
Chromatographic column is eluted with methanol, collects eluent, and evaporated under reduced pressure obtains lycopene；The lycopene content of preparation is
30.32mg/g dry mycelium.
Detailed description of the invention
The phylogenetic tree in the area Fig. 1 phaffiafhodozyma (CCTCC M 2019083) 26s D1/D2；
The comparision contents result of Fig. 2 difference Phaffia rhodozyma lycopene.
Specific embodiment
The preparation of 1 phaffiafhodozyma strain of embodiment
Jilin Agriculture University's orchard soil is acquired, which is inoculated in YPD culture medium flat plate, 20-28 DEG C of constant temperature incubation
48-96 h selects red colonies to be inoculated in YEPD culture medium, carries out 20-28 DEG C of constant-temperature table 150-270r/min and cultivates 48-
96h carries out yield of lycopene measurement, and obtaining the higher bacterial strain of yield of lycopene is PR106, then carries out thallus shape
State, Physiology and biochemistry and 26s rRNA identification, 26s rRNA RNA length are 522bp, and comparing the bacterial strain by phylogenetic tree is
Phaffiafhodozyma (Phaffia rhodozyma), it is named as phaffiafhodozyma PR106, is preserved in China typical culture collection
Center, January 24 2019 preservation time, address Wuhan City, Hubei Province Wuhan University, deposit number are CCTCC M
2019083。
2 Spawn incubation of embodiment
Actication of culture: taking phaffiafhodozyma PR106, after shaking flask culture, scribing line culture is carried out, 20-28 DEG C in biochemical cultivation case
Under the conditions of cultivate, chosen after 7 days in big and saturate single colonie access fluid nutrient medium, under the conditions of 121 DEG C of fluid nutrient medium
Sterilize 15min, and strain cultivates 48-96h in 18-28 DEG C, 150-270r/min, passes three generations by this step；
Seed culture: the activated spawn after secondary culture meets bacterium amount 1-10% using YEPD fluid nutrient medium, the 18- in shaking table
28 DEG C, cultivate 48-96h under conditions of 120-300r/min.
The extraction purification of 3 lycopene of embodiment
Phaffiafhodozyma PR106 connects bacterium amount by 10%, is inoculated into the 100mL conical flask equipped with 30mL fermentation medium, 20
DEG C, 180r/min shaking table culture 72h；Dissolved oxygen 40-90%, stream plus lye control, pH6.0-7.5；The fermentation medium can also
To replace with YEPD culture medium, but comprehensively consider production cost and fermentation period, it is the most suitable with fermentation medium；
Then clasmatosis is carried out, 20 ml fermentation liquids is taken to be fitted into 50 ml centrifuge tubes, 5000r/ min, 4 DEG C of centrifugation 10min receipts
Collection precipitating obtains thallus, washes centrifugation abandoning supernatant with distilled water and obtains yeast paste afterwards three times, is added the prewired 3mol/L's of 5 mL
Hydrochloric acid solution impregnates 30min to be acidified yeast cell wall, boiling water bath 4-5 minutes, is put into ice water immediately after after shaking uniformly
Cooling.Broken cells are starched, demineralizing acid is gone with 5000 r/min centrifugation 10min, collected precipitating repeated centrifugation washing 3 times and abandon
Clearly, cell fragment is obtained.It is deposited at 4 DEG C and stores, the measurement for the later period；
Lycopene is extracted using acetone extract method.5 mL acetone are added after taking 20 mL fermentation liquids to carry out clasmatosis, are keeping away
Concussion extraction 1 min, 5000 r/min are centrifuged 10 min under the conditions of light, collect supernatant, obtain pigment acetone leaching liquor, if
It does not extract completely, acetone is added and is extracted again, until cell is in colourless.
The extracting solution of 1mL is added in every 1.5mL centrifuge tube, the dissolution of 100 μ L methanol is added after 45 DEG C of evaporated under reduced pressure and slightly mentions product,
It takes 5mL methanol to activate CNW C18 solid-phase extraction column, then shifts 2mL methanol lycopene solution into C18 column.With 10 mL first
Alcohol elution, divides 4 times and is added in column, collects whole eluents, 45 DEG C of evaporated under reduced pressure, and every 1.5 mL centrifuge tube adds 46 μ L methanol
It redissolves, crosses organic filter membrane of 0.22 nm, it is spare.
4 high-efficient liquid phase chromatogram technique analysis lycopene of embodiment
Using the content of lycopene in UV detector high effective liquid chromatography for measuring phaffiafhodozyma PR106；Chromatographic condition
Are as follows:
1) chromatographic column: C18 column (4.6 mm × 250 mm, 5 μm), or the chromatographic column of tool equal performance；
2) mobile phase: mobile phase: acetonitrile: methylene chloride=10: 90, it is spare after 0.45 μm of film filters；
3) flow velocity: 1.0mL/min；
4) Detection wavelength: 472nm；
5) column temperature: room temperature；
6) sampling volume: 20 μ L, isocratic elution, concentration are constant；
Lycopene content in high-efficient liquid phase chromatogram technique analysis phaffiafhodozyma PR106；Control group is set, and control group is source
In the red phaffia rhodozyma of the CICC33064 of Chinese industrial Microbiological Culture Collection administrative center；As a result as shown in Fig. 2, red hair husband's ferment
Lycopene content in female PR106 is 27.00mg/g dry mycelium (117.46mg/L fermentation liquid), control group lycopene content
Only 9.05mg/g；Prove that phaffiafhodozyma PR106 can be with high yield lycopene.
Embodiment 5 adds influence of the metabolic regulation object to yield of lycopene
Bacterium amount is connect by lycopene 2%, is inoculated into equipped in 30mL fermentation medium, different H is added2O2Amount, respectively
0%, 0.2%, 0.4%, 0.6%；Triangular flask is put into shaking table and is cultivated, condition of culture is 180 r/min of revolving speed, and temperature is 20 DEG C,
Lycopene content is measured after 72h；The result shows that working as H2O2Amount be 0.4% when phaffiafhodozyma PR106 generate lycopene
Content be 30.32mg/g dry mycelium (144.9mg/L fermentation liquid), it was demonstrated that add suitable H in the fermentation medium2O2More have
Conducive to raising lycopene content.
The preparation of 6 culture medium of embodiment
YPD, YEPD described in embodiment 1-5 and fermentation medium, component are respectively as follows:
1) YPD culture medium: 2.0 g of yeast extract, 3.0 g of fructus hordei germinatus leaching powder, 5.0 g of peptone, 10.0 g of glucose, agar 20.0
G, 1.0 L of tap water；
2) YEPD culture medium: 2.0 g of yeast extract, 3.0 g of fructus hordei germinatus leaching powder, 5.0 g of peptone, 10.0 g of glucose, tap water
1.0 L；
3) fermentation medium: 2.0 g of yeast extract, fructus hordei germinatus leaching powder 3.0 g, corn pulp 8.0kg, 5.0 g of peptone, glucose
10.0kg, tap water 1000.0L.
The preparation method of three kinds of culture mediums is prepared according to a conventional method.
Sequence table
<110>Jilin Agriculture University
The phaffiafhodozyma bacterial strain and lycopene production process of<120>one plant heights production lycopene
<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 (8)
1. one plant of phaffiafhodozyma bacterium, its deposit number is CCTCC M 2019083.
2. a kind of lycopene production process, it includes:
1) phaffiafhodozyma bacterium is taken, activates, fermentation medium is inoculated by 1 ~ 15% bacterium amount that connects, in 18 ~ 28 DEG C, 120 ~ 300r/
Shaking table culture under min, ferment 48 ~ 96h, obtains fermentation liquid；
2) fermentation liquid is centrifuged to 8 ~ 15min at 4000 ~ 6000r/min, 0 ~ 4 DEG C, precipitating is collected and obtains thallus, wash, centrifugation,
It abandons supernatant and obtains yeast paste, the 3mol/L hydrochloric acid solution of 20 ~ 30% volumes of the fermentation liquid is added, concussion is uniform, immersion 25 ~
35 min, boiling water bath 4 ~ 5 minutes, ice bath cooling；It is centrifuged 8 ~ 15 min at 4000 ~ 6000 r/min and removes hydrochloric acid solution,
Precipitating is collected, washing, centrifugation abandon supernatant, obtain cell fragment；
3) cell fragment be added acetone, under the conditions of being protected from light concussion extraction 1 ~ 3min, at 4000 ~ 8000r/min be centrifuged 8 ~
15min collects supernatant；The step of repeating above-mentioned addition acetone, until supernatant is colourless；
4) by the supernatant of step 3), the evaporated under reduced pressure at 40 ~ 50 DEG C is added methanol, and is transferred to chromatographic column, is washed with methanol
It is de-, eluent is collected, 40 ~ 50 DEG C of evaporated under reduced pressure obtain lycopene.
3. a kind of lycopene production process according to claim 2, it is characterised in that: red hair husband described in step 1)
Saccharomycete, its deposit number are CCTCC M 2019083.
4. a kind of lycopene production process according to claim 2 or 3, it is characterised in that: fermentation described in step 1)
Culture medium, its components by weight percent include: 1 ~ 3kg of yeast extract, 1 ~ 4kg of fructus hordei germinatus leaching powder, 2 ~ 12kg of corn pulp, 2 ~ 8kg of peptone,
5 ~ 15kg of glucose, water 1000kg.
5. a kind of lycopene production process according to claim 4, it is characterised in that: the fermentation medium, also
It include: H2O2 2~6g。
6. a kind of lycopene production process according to claim 5, it is characterised in that: the H2O2For 4g.
7. a kind of lycopene production process according to claim 6, it is characterised in that: described in step 1) 22 DEG C,
72 h of shaking table culture under 180 r/min.
8. a kind of lycopene production process according to claim 7, it is characterised in that: chromatographic column described in step 4) is
C18 solid-phase extraction column.
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Cited By (3)
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| CN110824065A (en) * | 2019-12-04 | 2020-02-21 | 南京中科药业有限公司 | Method for detecting embedded lycopene |
| CN111205991A (en) * | 2020-02-26 | 2020-05-29 | 吉林农业大学 | Method for producing levo-astaxanthin through fermentation |
| CN112779295A (en) * | 2020-12-31 | 2021-05-11 | 广东博沃特生物科技有限公司 | High-density fermentation culture medium for producing lycopene saccharomyces cerevisiae |
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| 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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