CN101555490A - Method for improving the yield of lycopene by enhancing growth of Blakeslea trispora - Google Patents
Method for improving the yield of lycopene by enhancing growth of Blakeslea trispora Download PDFInfo
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- CN101555490A CN101555490A CNA2009100843730A CN200910084373A CN101555490A CN 101555490 A CN101555490 A CN 101555490A CN A2009100843730 A CNA2009100843730 A CN A2009100843730A CN 200910084373 A CN200910084373 A CN 200910084373A CN 101555490 A CN101555490 A CN 101555490A
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- lycopene
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- blakeslea trispora
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Abstract
The invention relates to a method for improving the yield of lycopene by enhancing the growth of Blakeslea trispora, belonging to the field of improving liquid-submerged fermentation of aerobic microorganisms to produce secondary metabolite, in particular to a method for adding active carbon to enhance Blakeslea trispora to produce lycopene. The method comprises the following steps of: adding active carbon in a fermentation liquor, and utilizing the characteristic that active carbon absorbs gas to increase the concentration of dissolved oxygen in the fermentation liquor, improve the viscosity of the fermentation liquor, enhance the growth of Blakeslea trispora and improve the yield of lycopene by more than 150 percent than the original yield. The method has low cost, convenient operation and obvious effect, can replace liquid alkane and hydrogen peroxide as an oxygen-carrying agent, avoids toxicity of chemical substances to the strains, simplifies post purification process of the lycopene and provides good basis for industrial production.
Description
Technical field
The invention belongs to the field of improving liquid submerged fermentation aerobic microbiological secondary metabolite, specially refer to and add the method that gac improves yield of lycopene by enhancing growth of Blakeslea trispora.
Background technology
Lyeopene is a kind of important carotenoid; has very strong antioxygenation; be the strongest carotenoid of oxidation-resistance; has very strong removing singlet oxygen function; the protection body cell is avoided oxidative damage; realize its physiologically active, can prevent and treat prostate cancer, cancer of the stomach, skin carcinoma etc. effectively, the restraining effect of uterus carcinoma, lung carcinoma cell is significantly higher than β-Hu Luobusu, alpha-carotene.
Lyeopene can be synthesized by chemical process by extracting in the tomato skin, also can produce through microbial fermentation.Consider that from the product quality and production technology, the resources costs aspect that obtain utilizing microbial technique to produce Lyeopene is the direction of future development.Wherein, trispore Bruce mould (Blakeslea trispora) (+) (-) bacterial strain can synthesize β-Hu Luobusu when cultivating simultaneously in a large number, realized suitability for industrialized production in eighties of last century the fifties later stage, in this mold fermentation process, add the nitrogen heterocyclic ring compounds that is fit to, can block the process that cyclization of lycopene forms β-Hu Luobusu, cause the accumulation of Lyeopene.This method has been proposed, patent documentation US 3,369,974 afterwards in patent US 3097146, JP 48016189, and JP 48016190, JP73016189, JP73016190, RU2102416, JP09313167 has all reported the correlative study achievement among the US3369974.In the last few years, also there were the correlative study report in domestic each research department and colleges and universities.CN1528906A has described a kind of with blakeslea trispora fermentative production lycopene method, and CN1582328A has invented the blakeslea trispora that can produce the 0.3g/L Lyeopene in a kind of appropriate media that does not have an external source carotene formation inhibitor.Because trispore Bruce mould is a kind of high oxygen bacterium of having a liking for, demand to oxygen in the fermenting process is very big, the production substratum is the farinaceous size after the gelatinization, viscosity is very big, having brought great difficulty for the oxygen transmission in the fermenting process, be to satisfy oxygen requirement in the growth process by increasing air blast and stir speed (S.S.) among the domestic patent CN1582328A, but this method need expend a large amount of energy, production cost is improved, suitability for industrialized production is caused difficulty.Report that adding liquid alkane in fermented liquid can increase dissolved oxygen content, but interpolation concentration is as far as possible little, avoids thalline is damaged, later stage product separation and Extraction will be passed through detoxification treatment, complex operation.Therefore, remain to be occurred a kind of safe, nontoxic, cheap oxygen agent of taking on the market and solve this problem.
Gac is a kind of black powdery, and granulous is amorphous to have porous carbon, and main component is a carbon, also contains a small amount of oxygen, hydrogen, sulphur, nitrogen, chlorine, has (500～1000 meters of bigger surface-area
2/ gram), very strong absorption property is arranged, can adsorbed gas on its surface, liquid or colloidal solids.Utilize this character it can be added in the fermented liquid, discharge the oxygen that it carries, optimize the fluid property of high viscosity fermentation liquid, reduce fermentation broth viscosity, improve dissolved oxygen content, satisfy trispore Bruce mould growth requisite oxygen, improve yield of lycopene.
Summary of the invention
Main purpose of the present invention is exactly to improve the output that the trispore Bruce mould liquid submerged fermentation is produced Lyeopene.Specific implementation method is to add granular active carbon in fermented liquid, bans liquid state and takes the oxygen agent, and the optimization production flow process reduces production costs, and improves yield of lycopene.
Gac does not have any toxic action to thalline, and solid granule can increase the fermented liquid fluid and flow, and accelerates the dissolving of oxygen in liquid, and it is good that height is had a liking for the oxygen bacteria growing.When fermentation ends, solid granule has improved the filtration velocity of thalline, has improved working efficiency.The also adsorbable Lyeopene that remains in the liquid of gac is reduced the loss.
The method that a kind of yield of lycopene by enhancing growth of Blakeslea trispora is provided of the present invention is characterized in that: before fermentation beginning or among in fermented liquid, add gac.
Further, the particle diameter of the gac of adding is 60 orders～200 orders.
Further, the mass concentration of the gac of adding is 0.5～2.0g/L starting fermentation liquid.
Further, the time of adding gac is fermentation the 0th～2 day.
Concrete technological line of the present invention is as follows:
1) the dull and stereotyped cultivation: remove skin potato 20g, add the 200ml deionized water and boil, filter potato after the cooling, clear liquid adds 2g glucose, 2g agar, 115 ℃ of sterilizations in 30 minutes of dissolving back packing, preparation PDA substratum.Get trispore Bruce mould (+) (-) bacterial strain spore suspension separate application in the flat board that contains the PDA substratum, cultivated 3 days, cultivated 2 days for 22 ℃ in 28 ℃ of lucifuges.
2) gac is handled through hydrochloric acid activation, and the oven dry back is standby.
3) seed culture: substratum is boiled by starch, glucose, corn steep liquor and forms, add dissolved sylvite and magnesium salts, pH regulator to 6.4, with the cultured trispore Bruce mould of flat board (+) (-) bacterial strain, getting the mycoderma access respectively is equipped with in the 250ml Erlenmeyer flask of seed culture medium, under 26～28 ℃ 180～200 rev/mins condition, the lucifuge concussion was cultivated 36～40 hours.
4) fermentation culture: fermention medium is boiled by starch, soybean cake powder, corn steep liquor and forms, add dissolved sylvite and magnesium salts, pH regulator to 6.7, with cultured (+), 1: 4 by volume mixed of (-) bacterium seed liquor, inoculum size access with volume ratio 10% is equipped with in the 500ml Erlenmeyer flask of fermention medium, and the gac after the activation treatment adds fermention medium according to requirement of experiment.Under 26～28 ℃, 220～250 rev/mins condition, the lucifuge concussion was cultivated 120 hours.Add nicotine or the agent of pyridines metabolic block in the time of 48 hours in fermentation.
5) microorganism collection: after the fermentation ends, collect wet thallus with the filtered through gauze fermented liquid, 45 ℃ of 0.08MP vacuum-dryings obtain dry mycelium.
6) content of lycopene analysis: pulverize dry mycelium, accurately take by weighing 0.03～0.05g dry bacterial powder, petroleum ether extraction is used the high effective liquid chromatography for measuring content of lycopene.
Chromatographic condition:
Separator column: Diamonsil C
18, ID5 μ m, 250 * 4.6mm;
Moving phase: acetonitrile: methylene dichloride (75: 25, V/V)
Testing conditions: wavelength 450nm; 28 ℃ of column temperatures; Flow velocity 1.5mL/min.
The retention time of Lyeopene and Pure Lycopene is more definite, and assay is determined by external standard method.
Description of drawings
The blank fungus extract color atlas (climax is the Lyeopene detected peaks) of Fig. 1
Thalline extract color atlas (climax is the Lyeopene detected peaks) when the gac addition is 1.0g/L among Fig. 2 embodiment 2
Embodiment
Embodiment 1: 1. dull and stereotyped the cultivation: remove skin potato 20g, add the 200ml deionized water and boil, filter potato after the cooling, clear liquid adds 2g glucose, 2g agar, 115 ℃ of sterilizations in 30 minutes of dissolving back packing, preparation PDA substratum.Get blakeslea trispora (+) (-) bacterial strain spore suspension separate application in the flat board that contains the PDA substratum, cultivated 3 days, cultivated 2 days for 22 ℃ in 28 ℃ of lucifuges.2. Activated Carbon Pretreatment: gac is filtered through 200 orders, 100 orders, 60 orders, 16 orders, 5 mesh sieves, sift out the gac of different-grain diameter, be immersed in mass percent concentration respectively and be in 0.5% the hydrochloric acid 2 hours, suction filtration, deionized water is washed till neutrality, 40 ℃ of dry for standby.3. seed culture medium preparation: take by weighing starch 40g, glucose 20g, corn steep liquor 50g, KH
2PO
41.5g, MgSO
40.1g, being dissolved in 1 liter of deionized water, the heated and stirred gelatinization is thick to becoming sticky, and regulates pH to 6.4.Be sub-packed in the 250ml Erlenmeyer flask 115 ℃ of sterilizations in 30 minutes.4. fermention medium preparation: take by weighing starch 20g, soybean cake powder 40g, corn steep liquor 30gKH
2PO
41g, MgSO
40.1g, being dissolved in 1 liter of deionized water, the heated and stirred gelatinization is thick to becoming sticky, and regulates pH to 6.7.Accurately take by weighing each 0.05g of different-grain diameter gac respectively in the 500ml Erlenmeyer flask, the fermentation culture base unit weight that gelatinization is good is respectively got 50ml and is poured in the Erlenmeyer flask, mixing, and with eight layers of good bottleneck of gauze plug, the outside bandages with kraft paper, 121 ℃ of sterilizations in 20 minutes.5. seed culture: trispore Bruce mould (+), (-) bacterial strain that the plate of making even is cultivated are inoculated in respectively in the seed culture medium, and label is cultivated in 28 ℃ of 180 rev/mins of lucifuges and to be obtained (+), (-) bacterium seed liquor.6. fermentation: with (+) bacterium seed liquor: (-) bacterium seed liquor is that 1: 4 volume ratio is inoculated in the fermention medium according to the inoculum size of volume ratio 10%, 28 ℃ of 220 rev/mins of lucifuges are cultivated, cultivating the 48th hour adding blocker, ferment and collect thalline after 120 hours, 45 ℃ of 0.08MP vacuum-dryings obtain dry mycelium.7. assay determination: pulverize dry mycelium, accurately take by weighing 0.03g, add petroleum ether extraction, use the high-efficient liquid phase chromatogram technique analysis content of lycopene to colourless.
The gained result is as follows:
Add 200 order gacs, yield of lycopene is the 733mg/L fermented liquid, improves 64% than blank.
Add 100 order gacs, yield of lycopene is the 799mg/L fermented liquid, improves 79% than blank.
Add 60 order gacs, yield of lycopene is the 677mg/L fermented liquid, improves 52% than blank.
Embodiment 2: 1. dull and stereotyped the cultivation: remove skin potato 20g, add the 200ml deionized water and boil, filter potato after the cooling, clear liquid adds 2g glucose, 2g agar, 115 ℃ of sterilizations in 30 minutes of dissolving back packing, preparation PDA substratum.Get trispore Bruce mould (+) (-) bacterial strain spore suspension separate application in the flat board that contains the PDA substratum, cultivated 3 days, cultivated 2 days for 22 ℃ in 28 ℃ of lucifuges.2. Activated Carbon Pretreatment: it is in 0.7% the hydrochloric acid 2 hours that 100 order gacs are immersed in mass percent concentration, suction filtration, and deionized water is washed till neutrality, 40 ℃ of dry for standby.3. seed culture medium preparation: take by weighing starch 40g, glucose 20g, corn steep liquor 50g, KH
2PO
41.5g, MgSO
40.1g,, being dissolved in 1 liter of deionized water, the heated and stirred gelatinization is thick to becoming sticky, and regulates pH value to 6.4.Be sub-packed in the 250ml Erlenmeyer flask 115 ℃ of sterilizations in 30 minutes.4. fermention medium preparation: take by weighing starch 20g, soybean cake powder 40g, corn steep liquor 30g, KH
2PO
41g, MgSO
40.1g, being dissolved in 1 liter of deionized water dissolving, the heated and stirred gelatinization is thick to becoming sticky, and regulates pH value to 6.7.The gac that accurately take by weighing 0.005,0.025,0.05,0.1,0.15,0.5g activates is in the 500ml Erlenmeyer flask, the fermention medium that gelatinization is good is measured 50ml and is poured into wherein, and mixing is with eight layers of good bottleneck of gauze plug, the outside bandages with kraft paper, 121 ℃ of sterilizations in 20 minutes.5. seed culture: trispore Bruce mould (+), (-) bacterial strain that the plate of making even is cultivated are inoculated in respectively in the seed culture medium, and label is cultivated in 26 ℃ of 190 rev/mins of lucifuges and to be obtained (+), (-) bacterium seed liquor.6. fermentation: with (+) bacterium seed liquor: (-) bacterium seed liquor is that 1: 4 volume ratio is inoculated in the fermention medium according to the inoculum size of volume ratio 10%, 26 ℃ of 230 rev/mins of lucifuges are cultivated, add blocker when cultivating the 48th hour, ferment and collect thalline after 120 hours, 45 ℃ of 0.08MP vacuum-dryings obtain dry mycelium.7. assay determination: pulverize dry mycelium, accurately take by weighing 0.04g, add petroleum ether extraction, use the high-efficient liquid phase chromatogram technique analysis content of lycopene to colourless.
The gained result is as follows:
Adding 100 order concentration of activated carbon is 0.5g/L, and yield of lycopene is the 609mg/L fermented liquid, improves 37% than blank.
Adding 100 order concentration of activated carbon is 1.0g/L, and yield of lycopene is the 799mg/L fermented liquid, improves 79% than blank.
Adding 100 order concentration of activated carbon is 2.0g/L, and yield of lycopene is the 592mg/L fermented liquid, improves 33% than blank.
Embodiment 3: 1. dull and stereotyped the cultivation: remove skin potato 20g, add the 200ml deionized water and boil, filter potato after the cooling, clear liquid adds 2g glucose, 2g agar, 115 ℃ of sterilizations in 30 minutes of dissolving back packing, preparation PDA substratum.Get trispore Bruce mould (+) (-) bacterial strain spore suspension separate application in the flat board that contains the PDA substratum, cultivated 3 days, cultivated 2 days for 22 ℃ in 28 ℃ of lucifuges.2. Activated Carbon Pretreatment: it is in 1.0% hydrochloric acid 2 hours that 100 order gacs are immersed in mass percent concentration, suction filtration, and deionized water is washed till neutrality, 40 ℃ of dry for standby.3. seed culture medium preparation: take by weighing starch 30g, glucose 15g, corn steep liquor 45g, KH
2PO
41.5g, MgSO
40.1g, being dissolved in 1 liter of deionized water, the heated and stirred gelatinization is thick to becoming sticky, and regulating the pH value is 6.4.Be sub-packed in the 250ml Erlenmeyer flask 115 ℃ of sterilizations in 30 minutes.4. fermention medium preparation: take by weighing starch 30g, soybean cake powder 50g, corn steep liquor 40g, KH
2PO
41g, MgSO
40.1g, being dissolved in 1 liter of deionized water, the heated and stirred gelatinization is thick to becoming sticky, and regulates pH value to 6.7.Add the 0.05g gac therein in one bottle, all the other do not add, and the fermention medium that gelatinization is good is measured 50ml and poured in the 500ml Erlenmeyer flask, mixing, and with eight layers of good bottleneck of gauze plug, the outside bandages with kraft paper, sterilizes in 20 minutes for 121 ℃.5. seed culture:: trispore Bruce mould (+), (-) bacterial strain that the plate of making even is cultivated are inoculated in respectively in the seed culture medium, and label is cultivated in 27 ℃ of 200 rev/mins of lucifuges and to be obtained (+), (-) bacterium seed liquor.6. fermentation: with (+) bacterium seed liquor: (-) bacterium seed liquor is that 1: 4 volume ratio is inoculated in the fermention medium according to the inoculum size of volume ratio 10%, 27 ℃ of 250rpm cultivate, gradation adds active carbon powder 0.05g when having cultivated 24,48,72,96 hours, add blocker when having cultivated 48 hours, ferment and collect thalline after 120 hours, 45 ℃ of 0.08MP vacuum-dryings obtain dry mycelium.7. assay determination: pulverize dry mycelium, accurately take by weighing 0.05g, add petroleum ether extraction, use the high-efficient liquid phase chromatogram technique analysis content of lycopene to colourless.
The gained result is as follows:
Add 100 order gacs during the fermentation beginning, yield of lycopene is the 764mg/L fermented liquid, improves 68% than blank.
100 order gacs were added on the 1st day in fermentation beginning back, and yield of lycopene is the 654mg/L fermented liquid, improves 43% than blank.
100 order gacs were added on the 2nd day in fermentation beginning back, and yield of lycopene is the 537mg/L fermented liquid, improves 18% than blank.
Claims (4)
1, a kind of method that improves yield of lycopene by enhancing growth of Blakeslea trispora is characterized in that: before fermentation beginning or among in fermented liquid, add gac.
2, according to the method for claim 1, it is characterized in that: wherein the particle diameter of the gac of Jia Ruing is 60 orders～200 orders.
3, according to the method for claim 1, it is characterized in that: wherein the mass concentration of the gac of Jia Ruing is 0.5～2.0g/L starting fermentation liquid.
4, according to the method for claim 1, it is characterized in that: the time that wherein adds gac is fermentation the 0th～2 day.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667081A (en) * | 2013-12-10 | 2014-03-26 | 常州臻扬生物工程有限公司 | Lycopene high-yielding strain and application thereof |
| CN107475306A (en) * | 2017-09-22 | 2017-12-15 | 嘉必优生物技术（武汉）股份有限公司 | A kind of method for preparing lycopene and a kind of lycopene product |
-
2009
- 2009-05-22 CN CN2009100843730A patent/CN101555490B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667081A (en) * | 2013-12-10 | 2014-03-26 | 常州臻扬生物工程有限公司 | Lycopene high-yielding strain and application thereof |
| CN103667081B (en) * | 2013-12-10 | 2016-01-20 | 常州臻扬生物工程有限公司 | One strain Lyeopene superior strain and application thereof |
| CN107475306A (en) * | 2017-09-22 | 2017-12-15 | 嘉必优生物技术（武汉）股份有限公司 | A kind of method for preparing lycopene and a kind of lycopene product |
| CN107475306B (en) * | 2017-09-22 | 2020-08-11 | 嘉必优生物技术（武汉）股份有限公司 | Method for preparing lycopene and lycopene product |
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| CN101555490B (en) | 2011-04-20 |
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