CN104560731A - Yeast-like fungi in high yield of squalene and application of yeast-like fungi - Google Patents
Yeast-like fungi in high yield of squalene and application of yeast-like fungi Download PDFInfo
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Abstract
The invention relates to application technologies of marine microorganism, in particular to a marine yeast-like fungi SD301 strain and a method producing squalene by utilizing the high-density fermentation of the strain. The yeast-like fungi is classified and named Pseudozyma sp., and is stored in the common microorganism center of the China General Microbiological Culture Collection Center located in 3rd building, 1st yard, Beichen west road, Chaoyang district, Beijing; the storing number is CGMCC No: 9687; the storing date is September 19th, 2014. The yeast-like fungi is applied in fermentation production of the squalene. The purity of the squalene obtained from the method reaches above 90%; the whole technology is convenient to operate, reduces the cost, and is suitable for industrialized production.
Description
Technical field
The present invention relates to the utilisation technology of marine microorganism, be specifically related to a kind of ocean yeast-like fungi SD301 bacterial strain and utilize its high density fermentation to produce the method for squalene.
Background technology
Squalene (squalene) is a kind of lipid unsaponifiables, its chemical name is 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosa carbon six alkene, belong to open chain triterpene, have and improve superoxide-dismutase (SOD) activity in body, enhancing body immunological competence, improvement function, anti-ageing, antifatigue, the different physiological roles such as antitumor, be a kind of avirulent marine biomaterial of preventing and curing diseases and acting on that has, and be widely used in the every field such as medicine, beauty treatment, makeup, food.The source mainly fish oil of current squalene, especially shark liver oil.But along with the exhaustion day by day of fishing resources and becoming better and approaching perfection day by day of relevant laws and regulations, people are impelled to strive to find other squalene source, as from sweet oil, plam oil, other oil crops, or from Amaranthus, seed, rice bran, wheatgerm, carry out extraction and isolation, but squalene content in plant is extremely low, accounting is 0.1 to 0.7% approximately by weight.
Another kind of replacement scheme utilizes the method for fermentable to produce squalene.The mass percentage disclosing squalene in its dry mycelium of yeast-like fungi strain of a strain product squalene as patent CN102787074 A, CN 103748212 A is 1%; Patent CN 102666837 A utilizes Yarrowia lipolytica to produce, and squalene mass content accounts for TL in born of the same parents 2%; Patent CN 103266137 B then utilizes transgenic escherichia coli to produce squalene.But the common defect of these patents is that the output of squalene in unit fermentation volume is all far below 1g/L.In bibliographical information, utilize the productive rate of fermentable production squalene then lower, as thraustochytriale ACEM 6063 bacterial strain squalene output be approximately 0.1 milligram of/gram of biomass (see the people such as Le Weisi " true tumor technology ", calendar year 2001,439-447 page.), and the output of Schizochytrium mangrovei FB1 bacterial strain is approximately 0.162 milligram of/gram of biomass (see Jiang Dengren, " agricultural and food chemistry magazine ", 2004, 52nd phase, number of pages 1196 to 1200), and the output of Aurantiochytrium BR-MP4-A1 bacterial strain is approximately 0.18 milligram of/gram of biomass (see Li Dengren, " agricultural and food chemistry magazine ", 2009, 57th phase, number of pages 4267 to 4272), and normal its ability of producing squalene of yeast-like fungi Pseudozyma sp.JCC 207 bacterial strain waiting people to be separated to is the highest, be only 340.52 mg/litre fermented liquids.Therefore, the microorganism screening high squalene output solves the effective means that current biological fermentation process produces squalene industry critical bottleneck.
Summary of the invention
The object of this invention is to provide a kind of yeast-like fungi and application thereof of high yield squalene.
For achieving the above object, the present invention adopts technical scheme to be:
A kind of yeast-like fungi of high yield squalene, yeast-like fungi Classification And Nomenclature is yeast-like fungi (Pseudozyma sp.), be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, its deposit number is CGMCC No:9687, and preservation date is on September 19th, 2014.
An application for the yeast-like fungi of high yield squalene, the application of described yeast-like fungi in fermentative production squalene.
High density fermentation produces a method for squalene, and with CGMCC No:9687 bacterial strain for starting strain, the fermentation of liquid medium within middle-high density, is separated and obtains somatic cells, by somatic cells through broken, extraction, refining acquisition squalene.
Be specially:
1. will be kept at the inoculation of glycerine pipe in seed culture medium, inoculum size 3-5% (v/v), at 20-30 DEG C, with the rotating speed of 150-200rpm, cultivate 24-48h, obtain first order seed;
2. first order seed is accessed in seed culture medium, inoculum size 3-5% (v/v), at 20-30 DEG C, with the rotating speed of 150-200rpm, cultivate 24-48h, obtain secondary seed;
3. secondary seed solution is accessed in fermention medium, inoculum size 2-10% (v/v), air flow 0.2-2vvm, mixing speed 200-800rpm, tank temperature 15-37 DEG C, pH 4-9, fermentation 60-120h, fermentative production squalene.
Obtaining biomass after above-mentioned fermentation is 150-180g/L, and squalene output is 9-15g/L.
In described seed culture medium, carbon source content is 30-60g/L, and nitrogenous source content is 10-20g/L, and solvent is the seawater of 1:1 (mass ratio) and the mixture of distilled water.
Described carbon source is glucose, glycerine, fructose, wood sugar, sucrose, maltose, molasses, starch saccharificating liquid or lignocellulose saccharified liquid; Described nitrogenous source is organic nitrogen source or inorganic nitrogen-sourced.
Described organic nitrogen source is yeast extract, peptone, Tryptones, corn steep liquor, beef extract, soybean protein, Sodium Glutamate or urea, described inorganic nitrogen-sourced be ammonium chloride, ammonium sulfate, ammonium nitrate, SODIUMNITRATE, ammoniacal liquor or saltpetre.
Following component is comprised: glucose 20-120g/L in described fermention medium, yeast extract 0.2-30g/L, peptone 0.2-20g/L, potassium primary phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 5-30g/L, vitamin B13-200mg/L, vitamin B6 3-200mg/L, vitamin B12 1-50mg/L, vitamin H 1-50mg/L.
The mode of described secondary seed fermentation culture is batch fermentation, fed-batch fermentation, continuously ferments or semicontinuous fermentation.
Described separation method be centrifugal, filter or flocculation; Described breaking method is extrusion wall-breaking or enzymolysis process broken wall; Described extraction, purification step are utilize non-polar solvent to reclaim the thick oil containing squalene, and described thick oil molecule distillation is obtained squalene.
Described non-polar solvent is the mixed solvent of normal hexane or normal hexane-ethanol; Described molecular distillation for steaming main distillate fraction squalene under 190 ~ 210 DEG C and pressure 5 ~ 20Pa condition.
Be further, by whizzer (centrifugal force 4500g) separate fermentation liquid, be that the wet thallus of 65:1 mixes with helicase and carries out broken wall by mass ratio, add isopyknic normal hexane and isopyknic ethanol with wet thallus afterwards again, mix and leave standstill 4 hours afterwards.Upper organic phase is reclaimed organic solvent, and residue obtains squalene crude product.
Compared with the prior art the present invention has following distinguishing feature and positively effect:
(1) obtain from the screening of mangrove forest waters, Zhanjiang, Guangdong Province the yeast-like fungi bacterial strain that a plant height produces squalene, this bacterial strain can grow fast under 15 DEG C of-37 DEG C of conditions, and accumulates squalene in a large number.
(2) fast growth, fermentation time is short, and squalene output is large.Under above-mentioned fermentation condition, within 80-100 hour, can obtain maximum production, acquisition biomass is 150-180g/L, and squalene output is 9-15g/L.
(3) product purity is high.Under said extracted and molecular distillation condition, the squalene purity of acquisition is greater than 90%.
Accompanying drawing explanation
Fig. 1 is the yeast-like fungi SD301 bacterial strain form under the microscope that the embodiment of the present invention provides, and a is optical microphotograph Microscopic observation, and b is scanning electron microscopic observation;
Fig. 2 is the gas chromatogram of the yeast-like fungi SD301 bacterial strain squalene content analysis that the embodiment of the present invention provides;
Fig. 3 is the evolutionary tree of the yeast-like fungi SD301 bacterial strain ITS sequence utilizing MEGA5 software to obtain that the embodiment of the present invention provides.
Embodiment
Gained one of the present invention strain can yeast-like fungi Pseudozyma sp.SD 301 bacterial strain of high yield squalene, has good industrial application value.
Embodiment 1: yeast-like fungi is to the utilization of carbon source
Yeast-like fungi is screened and obtains from mangrove forest waters, Zhanjiang, Guangdong Province, its Classification And Nomenclature is yeast-like fungi (Pseudozyma sp.), laboratory called after yeast-like fungi SD301 bacterial strain, be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, its deposit number is CGMCC No:9687, preservation date on September 19th, 2014.
This yeast-like fungi SD301 bacterial strain is cylindrical yeast shape vegetal cell, and thalline is about 3-6um, and fusiform spore is growing at thalline one end (Fig. 1).Have the accumulation of a large amount of squalene in its cell, the mass ratio that squalene content accounts for dry cell weight is greater than 5% (Fig. 2).Compare (Fig. 3) to its ITS regional sequence with molecular biology method, find that the homology of the ITS sequence that this bacterial strain and Pseudozyma belong to is greater than 99%, therefore judge that this bacterial strain belongs to yeast-like fungi, this sequence is denoted as SEQ ID NO.1
。
Yeast-like fungi is to the utilization of carbon source
In the triangle shaking flask of 250ml, configuration 50ml substratum, nitrogenous source is yeast extract 20g/L, and salinity is 15, adds different carbon sources (glucose, glycerine, fructose, wood sugar, sucrose, maltose, molasses, starch saccharificating liquid or lignocellulose saccharified liquid) respectively, concentration is 60g/L, adjust pH is 6.0, after autoclaving, the pre-incubated bacterial classification seed liquor of access 5ml, 28 DEG C of shaking culture 5 days on airbath vibrator, vibration rotating speed is 180rpm.Collected by centrifugation thalline, lyophilize, to constant weight, surveys its dry weight; Get part thalline, measured the percentage composition of squalene in thalline by GC-MS.The results are shown in Table 1.
Table 1 yeast-like fungi SD301 bacterial strain is to the utilization of carbon source
Yeast-like fungi is to the utilization of nitrogenous source
In the triangle shaking flask of 250ml, configuration 50m substratum, employing glucose is carbon source, concentration 60g/L, salinity is 15, add the organic nitrogen source (yeast extract that concentration is 20g/L respectively, peptone, Tryptones, corn steep liquor, beef extract, soybean protein, Sodium Glutamate or urea) and the inorganic nitrogen-sourced (ammonium chloride of 5g/L, ammonium sulfate, ammonium nitrate, SODIUMNITRATE, ammoniacal liquor or saltpetre), adjust ph is 6.0, after autoclaving, the pre-incubated bacterial classification seed liquor of access 5ml, 28 DEG C of shaking culture 5 days on airbath vibrator, vibration rotating speed is 180rpm.Analytical procedure is the same, and experimental result is in table 2.
Table 2 yeast-like fungi SD301 bacterial strain is to the utilization of nitrogenous source
Embodiment 2 3L fermentor tank is tested
The bacterial strain access being kept at glycerine pipe is equipped with in the 250ml shaking flask of 50ml seed culture medium, in the shaking table of 20 DEG C, with the rotating speed of 150rpm, cultivates 48h, obtain first order seed; First order seed access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in the shaking table of 20 DEG C, with the rotating speed of 150rpm, cultivates 48h, obtain secondary seed; 1.2L fermention medium is added, the secondary seed solution of access activation in 3L bio-reactor.In fermenting process, air flow 0.2vvm, mixing speed 200rpm, tank temperature 15 DEG C, pH 4, fermentation time 120 hours.Each when glucose concn is down to about 10g/L, flow feeding liquid, makes sugared concentration return to about 60g/L.In fermenting process, feed supplement 4 times altogether.
Wherein, in seed culture medium, glucose is 30g/L, and yeast powder content is 10g/L, and solvent is the seawater of 1:1 (mass ratio) and the mixture of distilled water.
Following component is comprised: glucose 120g/L, yeast extract 30g/L, peptone 0.2g/L, potassium primary phosphate 8g/L in fermention medium, magnesium sulfate 5g/L, sea crystal 30g/L, vitamin B12 00mg/L, vitamin B6 200mg/L, vitamin B12 50mg/L, vitamin H 50mg/L.
Feed supplement fluid component is: glucose 800g/L, yeast extract 50g/L.
Biomass calculates:
Get fermented liquid 30mL, the centrifugal 10min of 4000g, collect thalline, take its dry mycelium quality after drying, and be converted to biological yield, in gram often liter of fermented liquid (g/L).
The preparation of squalene
By whizzer (centrifugal force 4500g) separate fermentation liquid, collect wet thallus, be that the wet thallus of 65:1 mixes with helicase and carries out broken wall by mass ratio, add isopyknic normal hexane and isopyknic ethanol with wet thallus afterwards again, mix and leave standstill 4 hours afterwards.Upper organic phase is reclaimed organic solvent, and residue obtains squalene crude product.
Squalene detection method
Adopt Agilent 7890B gas-chromatography, chromatographic column model is HP-5 post (30m × 0.25mm × 0.25 μm); Carrier gas is high pure nitrogen, constant current mode, flow velocity 1.0mL/min; Sampling volume is 1 μ L, and detector is hydrogen ion flame detector.Heating schedule is: initial temperature 100 DEG C keeps 1min, rises to 300 DEG C with 15 DEG C/min, keeps 6min.Through the test of squalene standard substance, the chromatographic peak that under this condition, about 12.86min occurs is the peak of squalene material.
After fermentation ends, the output that 3L fermentor tank obtains class yeast is 162.39g/L, and squalene output is 11.69g/L.
Embodiment 3 10L fermentor tank is tested
The bacterial strain access being kept at glycerine pipe is equipped with in the 250ml shaking flask of 50ml seed culture medium, in the shaking table of 30 DEG C, with the rotating speed of 200rpm, cultivates 24h, obtain first order seed; First order seed access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in the shaking table of 30 DEG C, with the rotating speed of 200rpm, cultivates 24h, obtain secondary seed; 6L fermention medium is added, the secondary seed solution of access activation in 10L bio-reactor.In fermenting process, air flow 2vvm, mixing speed 800rpm, tank temperature 37 DEG C, pH 9, fermentation time 60 hours.Each when glucose concn is down to about 10g/L, flow feeding liquid, makes sugared concentration return to about 60g/L.In fermenting process, feed supplement 4 times altogether.
Wherein, in seed culture medium, glucose is 60g/L, and yeast powder content is 20g/L, and solvent is the seawater of 1:1 (mass ratio) and the mixture of distilled water.
Following component is comprised: glucose 20g/L, yeast extract 0.2g/L, peptone 20g/L, potassium primary phosphate 0.5g/L in fermention medium, magnesium sulfate 0.5g/L, sea crystal 5g/L, vitamin B13 mg/L, vitamin B6 3mg/L, vitamin B12 1mg/L, vitamin H 1mg/L.
Feed supplement fluid component is: glucose 800g/L, yeast extract 50g/L.
After fermentation ends, the output that 10L fermentor tank obtains class yeast is 150.43g/L, and squalene output is 9.03g/L.
Embodiment 4 100L fermentor tank is tested
The bacterial strain access being kept at glycerine pipe is equipped with in the 250ml shaking flask of 50ml seed culture medium, in the shaking table of 28 DEG C, with the rotating speed of 200rpm, cultivates 24h, obtain first order seed; First order seed access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in the shaking table of 28 DEG C, with the rotating speed of 200rpm, cultivates 24h, obtain secondary seed; 65L fermention medium is added, the secondary seed solution of access activation in 100L bio-reactor.In fermenting process, air flow 1.7vvm, mixing speed 600rpm, tank temperature 28 DEG C, pH7, fermentation time 120 hours.Each when glucose concn is down to about 10g/L, flow feeding liquid, makes sugared concentration return to about 60g/L.In fermenting process, feed supplement 5 times altogether.
Wherein, in seed culture medium, glucose is 60g/L, and yeast powder content is 20g/L, and solvent is the seawater of 1:1 (mass ratio) and the mixture of distilled water.
Following component is comprised: glucose 120g/L, yeast extract 30g/L, peptone 10g/L, potassium primary phosphate 8g/L in fermention medium, magnesium sulfate 5g/L, sea crystal 30g/L, vitamin B12 00mg/L, vitamin B6 200mg/L, vitamin B12 50mg/L, vitamin H 50mg/L.
Feed supplement fluid component is: glucose 800g/L, yeast extract 50g/L.
After fermentation ends, the output that 100L fermentor tank obtains class yeast is 182.25g/L, and squalene output is 15.49g/L.
The preparation of embodiment 5 squalene fine work
The fermented liquid of 2.5 tons of above-described embodiments acquisitions is separated by disc centrifuge (centrifugal force 4500g), collect wet thallus 1 ton altogether, 1.5% helicase weighed adding wet thallus quality carries out broken wall, adds 1 ton of normal hexane afterwards again, and 1 ton of ethanol mixes latter standing 4 hours.Get upper organic phase evaporation and reclaim organic solvent, obtain squalene crude product.Transferred to by crude product in water distilling apparatus, at 190 DEG C, pressure is carry out distillation under 5Pa condition to obtain squalene fine work 26.8Kg, and purity is greater than 93% after testing.The preparation of embodiment 7 squalene fine work
The fermented liquid of 10 tons of above-described embodiments acquisitions is separated by disc centrifuge (centrifugal force 4500g), collect wet thallus about 4.3 tons altogether, 1.5% helicase weighed adding wet thallus quality carries out broken wall, adds 4 tons of normal hexanes afterwards again, mixes latter standing 4 hours.Get upper organic phase evaporation and reclaim organic solvent, obtain squalene crude product.Transferred to by crude product in water distilling apparatus, at 210 DEG C, pressure is carry out distillation under 20Pa condition to obtain squalene fine work and be about 128Kg, and purity is greater than 90% after testing.
Claims (7)
1. the yeast-like fungi of a high yield squalene, it is characterized in that: yeast-like fungi Classification And Nomenclature is yeast-like fungi (Pseudozyma sp.), be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, its deposit number is CGMCC No:9687, and preservation date is on September 19th, 2014.
2. an application for the yeast-like fungi of high yield squalene according to claim 1, is characterized in that: the application of described yeast-like fungi in fermentative production squalene.
3. high density fermentation produces a method for squalene, it is characterized in that: with CGMCC No:9687 bacterial strain for starting strain, and the fermentation of liquid medium within middle-high density, is separated and obtains somatic cells, by somatic cells through broken, extraction, refining acquisition squalene.
4. produce the method for squalene by high density fermentation according to claim 3, it is characterized in that:
1. will be kept at the inoculation of glycerine pipe in seed culture medium, inoculum size 3-5% (v/v), at 20-30 DEG C, with the rotating speed of 150-200rpm, cultivate 24-48h, obtain first order seed;
2. first order seed is accessed in seed culture medium, inoculum size 3-5% (v/v), at 20-30 DEG C, with the rotating speed of 150-200rpm, cultivate 24-48h, obtain secondary seed;
3. secondary seed solution is accessed in fermention medium, inoculum size 2-10% (v/v), air flow 0.2-2vvm, mixing speed 200-800rpm, tank temperature 15-37 DEG C, pH 4-9, fermentation 60-120h, fermentative production squalene.
5. the method for squalene is produced by high density fermentation according to claim 4, it is characterized in that: in described seed culture medium, carbon source content is 30-60g/L, and nitrogenous source content is 10-20g/L, and solvent is the seawater of 1:1 (mass ratio) and the mixture of distilled water.
6. produce the method for squalene by high density fermentation according to claim 5, it is characterized in that: described carbon source is glucose, glycerine, fructose, wood sugar, sucrose, maltose, molasses, starch saccharificating liquid or lignocellulose saccharified liquid; Described nitrogenous source is organic nitrogen source or inorganic nitrogen-sourced.
7. the method for squalene is produced by high density fermentation according to claim 4, it is characterized in that: in described fermention medium, comprise following component: glucose 20-120g/L, yeast extract 0.2-30g/L, peptone 0.2-20g/L, potassium primary phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 5-30g/L, vitamin B13-200mg/L, vitamin B6 3-200mg/L, vitamin B12 1-50mg/L, vitamin H 1-50mg/L.
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| CN107574185A (en) * | 2017-09-11 | 2018-01-12 | 天津大学 | The thraustochytriale fermentation medium and its cultural method of a kind of high yield squalene |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105920580A (en) * | 2016-04-28 | 2016-09-07 | 中山大学 | Applications of compound P-3 derived from marine microorganisms in preparing anti-aging medicines |
| CN107574185A (en) * | 2017-09-11 | 2018-01-12 | 天津大学 | The thraustochytriale fermentation medium and its cultural method of a kind of high yield squalene |
| CN108866112A (en) * | 2018-08-17 | 2018-11-23 | 青岛中科潮生生物技术有限公司 | The method that squalene is prepared using lignocellulosic |
| CN108866112B (en) * | 2018-08-17 | 2021-04-27 | 青岛中科潮生生物技术有限公司 | Method for preparing squalene by adopting lignocellulose |
| CN114854608A (en) * | 2022-04-18 | 2022-08-05 | 自然资源部第三海洋研究所 | Degradable polyurethane yeast fungus strain, identification method and application |
| CN114854608B (en) * | 2022-04-18 | 2024-05-07 | 自然资源部第三海洋研究所 | Degradable polyurethane yeast fungus strain, identification method and application |
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