CN107604010A - A kind of fermentation manufacturing technique of lycopene - Google Patents
A kind of fermentation manufacturing technique of lycopene Download PDFInfo
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- CN107604010A CN107604010A CN201710981395.1A CN201710981395A CN107604010A CN 107604010 A CN107604010 A CN 107604010A CN 201710981395 A CN201710981395 A CN 201710981395A CN 107604010 A CN107604010 A CN 107604010A
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Abstract
The invention discloses a kind of fermentation manufacturing technique of lycopene, it is related to field of biological pharmacy.The fermentation manufacturing technique of the lycopene of the present invention includes：Prepared by seed, respectively access positive bacterium spore and negative bacterium spore in sterilized physiological saline, and stirring obtains positive bacterium spore suspension and negative bacterium spore suspension；Seed culture, according to concentration ratio it is 2 according to positive bacterium spore suspension and negative bacterium spore suspension：35 access seed culture mediums carry out seed culture and obtain seed liquor；Fermented and cultured；Fermented and cultured, seed liquor access fermentation medium is subjected to fermented and cultured.The form of the process modification seed liquor, positive and negative spore suspension is directly seeded to culture in seed culture medium and carries out conjugation, caused thalline is minimum bacterium ball or mycelia, and agglomerating thalline does not occur, is advantageous to the absorption of oxygen and nutriment in its liquid medium within；Fermentation condition is improved, beneficial to the accumulation of lycopene, improves the yield of lycopene.
Description
Technical field
The present invention relates to field of biological pharmacy, especially a kind of fermentation manufacturing technique of lycopene.
Background technology
Lycopene is a Carotenoids, has and improves body immunity, anticancer, anti-oxidant, reducing blood lipid, protection skin
Skin and other effects, it is widely used in food, medicine and cosmetic field.Natural lycopene, is primarily present in plant of Solanaceae
In the ripening fruits of tomato.As people increasingly focus on green, health, lycopene is constantly by the extensive pass of people
Note.Mankind itself can not synthesize lycopene, need to be supplemented by modes such as meals.According to U.S. CMR（Customer account management relation）It is international
Company predicts that year is increased 35% by lycopene product sale, it is contemplated that in two or three years reaches 20,000,000,000 dollars of sales volume, the world is transnational
Medical giant and domestic medical giants march lycopene industry one after another.
At present, the production method of lycopene mainly has natural extraction, chemical synthesis, microbial fermentation.It is it has proven convenient that natural
Lycopene is more easy to absorb than the lycopene of chemical synthesis, moreover, the presence of chemical synthesis chemicals residual, therefore, in recent years
To have shifted towards the exploitation of natural prodcuts.Natural lycopene is extracted from plant material and is vulnerable to condition and yield limitation, and it is micro-
Biological fermentation process produces natural lycopene and considers to be superior to above two from factors such as quality, technology, resource, cost and environment
Method, thus more application prospect.It is disadvantageous in that microorganism not currently with Production by Microorganism Fermentation lycopene
Can high-caliber accumulation lycopene, cause that fermentation production rate is low, production cost is higher.
The content of the invention
The goal of the invention of the present invention is：For above-mentioned problem, there is provided a kind of fermenting and producing side of lycopene
Method, this method have higher expression activity by improving the seed preparation condition of microorganism, the microorganism for being used in fermentation；Together
Shi Gaijin fermentation culture conditions, the metabolic fluxes of lycopene are accumulated with enhancing, so as to improve the yield of lycopene.
The technical solution adopted by the present invention is as follows：
A kind of fermentation manufacturing technique of lycopene, it includes：
Step 1：Prepared by seed, respectively access positive bacterium spore and negative bacterium spore in sterilized physiological saline, and stirring obtains just
Bacterium spore suspension and negative bacterium spore suspension；
Step 2：Seed culture, according to concentration ratio it is 2 according to positive bacterium spore suspension and negative bacterium spore suspension：3-5 access seed trainings
Support base progress seed culture and obtain seed liquor；
Step 3：Fermented and cultured；Fermented and cultured, seed liquor access fermentation medium is subjected to fermented and cultured.
The strain used in the present invention is trispore Bruce mould（Blakeslea trispora）'s（+）（-）Bacterial strain, it is former
Beginning strain is purchased from ATCC, and numbering is respectively ATCC 14271（+）, ATCC 14272（-）.It should be noted that in the present invention
Method other most of subspecies of trispore Bruce mould are also suitable.The strain without mutagenesis is directly used in the present invention,
Reason is that the strain stability through mutagenesis is not high, is not suitable for industry's enlarging production.
Connect by adopting the above-described technical solution, positive and negative spore suspension is directly seeded into culture in seed culture medium
Symphysis is grown, and caused thalline is minimum bacterium ball or mycelia, and agglomerating thalline does not occur, is advantageous to oxygen in its liquid medium within
With the absorption of nutriment.Negative bacterium is the main producing strains of lycopene, and positive bacterium produces as pairing bacterium and negative bacterium co-fermentation
Trisporic acid, so as to promote the synthesis of lycopene.Because trispore Bruce mould needs to carry out conjugation during the fermentation
Ability expression product, to improve the yield of lycopene, needs to meet certain ratio between positive bacterium and negative bacterium, properly increases negative
The ratio of bacterium, be advantageous to lycopene accumulation, but its ratio is too high, trisporic acid growing amount very little, influences Product formation.
In preferred embodiments of the present invention, the positive bacterium spore and negative bacterium spore obtain by following culture：By strain
Setting-out accesses the plate of the culture medium containing potato, in sterile culture case, 26-30 DEG C of keeping temperature, cultivates 2-4d；Then reduce temperature
Degree stimulates spore to generate, and cultivate spore 1-3d to 16-20 DEG C.
By adopting the above-described technical solution, the 26-30 DEG C of optimum temperature for growth, grows 1- at this temperature
3d, strain are in the reduction of exponential phase, now cultivation temperature, are advantageous to stimulate the generation of spore.
In preferred embodiments of the present invention, during cultivating spore, oxygen is passed through into incubator with 2L/min speed,
Give illumination, intensity of illumination 4WLx simultaneously.
By adopting the above-described technical solution, the generation of the change of increase environment, further stimulation spore, on the other hand,
The thermophilic oxygen of trispore Bruce mould spore, light, is advantageous to spore growth.
In preferred embodiments of the present invention, the temperature of seed culture process is 26-30 DEG C, incubation time 1-3d, culture
Process stir speed (S.S.) is 200r/min, to the ventilation rate for giving 1vvm simultaneously, and 4WLx intensities of illumination.
By adopting the above-described technical solution, the positive and negative rapid conjugation of spore, produces spherical or thread thalline, does not send out
Generation group phenomenon.
In preferred embodiments of the present invention, the composition of the fermentation medium is cornstarch 4%, glucose 2%, soyabean cake
Powder 1%, soybean protein isolate 3%, corn steep liquor 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, vegetable oil 2%, VB10.5%, 2,6-
Di-tert-butyl-4-methy phenol 0.05%, and pH is adjusted to 7.3.
By adopting the above-described technical solution, cornstarch, glucose are carbon source, soybean cake powder, soybean protein isolate with
And corn steep liquor is nitrogen source, magnesium sulfate, vegetable oil, VB1For nourishing additive agent, 2,6- di-tert-butyl-4-methy phenols are anti-oxidant
Agent, potassium dihydrogen phosphate are stabilizer.The starting pH7.3 of zymotic fluid is advantageous to the accumulation of lycopene, but during the fermentation, pH
Need not constant control, no silhouette rings accumulation of the thalline using organic acid and lycopene caused by fermentation.
In preferred embodiments of the present invention, when spore suspension is accessed into fermentation medium, bacterium spore suspension and fermentation are born
The volume ratio of culture medium is 1：4-6.
Due to taking above-mentioned technical proposal, inoculative proportion is improved in right amount, is advantageous to increase and the product of thalline quantity
Accumulation, but inoculum concentration is too high, easily causes the serious bacteriolysis of deuterogenesis of fermenting.
In preferred embodiments of the present invention, fermented and cultured process lucifuge, incubation time 120h, stir speed (S.S.) 400r/
Min, ventilation rate 1vvm, fermented and cultured 0-48h, cultivation temperature are 28-30 DEG C, and 22-26 DEG C is cooled to after 48h.
The preceding 48h of fermented and cultured, based on the quantity increment of thalline, caused lycopene is less, properly increases temperature
Be advantageous to improve thalline quantity and activity.After 48h, lycopene largely generates, and the optimum temperature for accumulating lycopene is 22-26
DEG C, temperature is too low, suppresses microbial activity, the too high content that will increase beta carotene and γ carrotene of temperature, and appropriate improve turns
Speed can improve dissolved oxygen speed, but high speed of agitator forms and does to obtain shearing force, is damaged thalline, reduces bioactivity.
In preferred embodiments of the present invention, after fermentation carries out 48h, conditioning agent, the conditioning agent bag are added into zymotic fluid
Include creatinine in mass：Leucine：Abscisic acid：Penicillin=60：5：4：1, wherein concentration of the penicillin in zymotic fluid is
0.1g/L。
Due to taking above-mentioned technical proposal, creatinine is lycopene cox-2 inhibitors, can effectively suppress tomato red
The epoxidation of element and follow-up metabolic process, to accumulate lycopene.Abscisic acid is trisporic acid analogue, and trisporic acid is
The sex hormone of trispore Bruce mould, energy effective stimulus promote the generation of lycopene.Leucine is 3- hydroxy-3-methyl coenzyme
A precursor substance, 3- hydroxy-3-methyls coacetylase can form mevalonic acid, and penicillin is mevalonate kinase activator, activation
Mevalonate kinase, the synthesis of carotenoid precursors material Isoprenoid is improved, so as to improve yield of lycopene.Four
Kind material passes through different approaches：Or suppress lycopene and decompose, or promote its synthesis, act synergistically, to improve lycopene production
Amount.
In preferred embodiments of the present invention, after fermentation carries out 48h, glucose 1.0%, ammonium sulfate are added into zymotic fluid
1.0%, dipotassium hydrogen phosphate 0.1%.
By adopting the above-described technical solution, bacterial metabolism needed nutrient matter is supplemented, and it is in acidity to maintain zymotic fluid,
Promote lycopene accumulation.
In summary, by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
1. improving the form of seed liquor, positive and negative spore suspension is directly seeded into culture in seed culture medium carries out conjugation,
Caused thalline is minimum bacterium ball or mycelia, and agglomerating thalline does not occur, is advantageous to oxygen and nutrition in its liquid medium within
The absorption of material.
2. improving fermentation condition, beneficial to the accumulation of lycopene, the yield of lycopene is improved.
Embodiment
All features disclosed in this specification, or disclosed all methods or during the step of, except mutually exclusive
Feature and/or step beyond, can combine in any way.
This specification（Including any accessory claim, summary）Disclosed in any feature, unless specifically stated otherwise,
Replaced by other equivalent or with similar purpose alternative features.I.e., unless specifically stated otherwise, each feature is a series of
An example in equivalent or similar characteristics.
Embodiment 1
The present embodiment provides a kind of technique of biofermentation production lycopene, and it comprises the following steps：
Step 1：Peeled potatoes 200g is taken, 1000mL deionized waters is added and boils and keep 30min；Two layers of gauze mistake after cooling
Filter, takes filtrate, adds deionized water to 1000mL, and add 20g glucose and 10g agar；Sterilize 30min at 115 DEG C, is made
Potato culture medium（PDA）.Take ATCC 14271（+）（Hereinafter referred to as positive bacterium）With ATCC 14272（-）（Hereinafter referred to as negative bacterium）Point
It is not coated on PDA plate, is cultivated 4 days in 26 DEG C of constant incubators, then reduces temperature to 16 DEG C, stimulate spore to generate,
And oxygen is passed through into incubator with 2L/min speed, while give the illumination cultivation spore 3d that intensity is 4WLx.
Step 2：Positive bacterium spore and negative bacterium spore are respectively connected to the triangular flask equipped with 20mL sterile salines, magnetic force
30min is stirred, obtains positive bacterium spore suspension and negative bacterium spore suspension.
Step 3：By positive bacterium spore suspension and the same triangle equipped with sterilizing seed culture medium of negative bacterium spore suspension access
Bottle, and positive the ratio between bacterium spore and negative bacterium spore concentration are 2：3, it is then 26 DEG C, stir speed (S.S.) 200r/min in temperature, ventilation
Rate is 1vvm, and cultivating 3d under conditions of intensity of illumination 4WLx obtains seed liquor.The formula of seed culture medium is（In mass）：Form sediment
Powder 4%, glucose 2.5%, corn steep liquor 5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.01%, vitamin B1 0.001%, pH7.0 is adjusted,
It is dissolved to 1L.
Step 4：According to seed liquor：Fermentation medium=1：Seed liquor is seeded to sterilized fermented and cultured by 4 ratio
In base, the formula of fermentation medium is（In mass）：Cornstarch 4%, glucose 2%, soybean cake powder 1%, soybean protein isolate
3%, corn steep liquor 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, vegetable oil 2%, vitamin B10.5%, 2,6- di-t-butyl -4-
Methylphenol 0.05%, and pH is adjusted to 7.3.
Fermented and cultured continues 120h, stir speed (S.S.) 400r/min, ventilation rate 1vvm.Fermented and cultured 0-48h, culture
Temperature is 28 DEG C, and 26 DEG C are cooled to after 48h.And ferment after carrying out 48h, conditioning agent is added into zymotic fluid, to adjust generation
Xie Liu.The conditioning agent includes leucine in mass：Abscisic acid：Creatinine：Penicillin=60：5：4：1, wherein penicillin exists
Concentration in zymotic fluid is 0.1g/L.While adding conditioning agent, the addition glucose 1.0% into zymotic fluid, ammonium sulfate 1.0%,
Dipotassium hydrogen phosphate 0.1% is with the material that supplements the nutrients.
Embodiment 2
The present embodiment provides a kind of technique of biofermentation production lycopene, and it comprises the following steps：
Step 1：Peeled potatoes 200g is taken, 1000mL deionized waters is added and boils and keep 30min；Two layers of gauze mistake after cooling
Filter, takes filtrate, adds deionized water to 1000mL, and add 20g glucose and 10g agar；Sterilize 30min at 115 DEG C, is made
Potato culture medium（PDA）.Take ATCC 14271（+）（Hereinafter referred to as positive bacterium）With ATCC 14272（-）（Hereinafter referred to as negative bacterium）Point
It is not coated on PDA plate, is cultivated 3 days in 28 DEG C of constant incubators, then reduces temperature to 18 DEG C, stimulate spore to generate,
And oxygen is passed through into incubator with 2L/min speed, while give the illumination cultivation spore 2d that intensity is 4WLx.
Step 2：Positive bacterium spore and negative bacterium spore are respectively connected to the triangular flask equipped with 20mL sterile salines, magnetic force
30min is stirred, obtains positive bacterium spore suspension and negative bacterium spore suspension.
Step 3：By positive bacterium spore suspension and the same triangle equipped with sterilizing seed culture medium of negative bacterium spore suspension access
Bottle, and positive the ratio between bacterium spore and negative bacterium spore concentration are 1：2, it is then 28 DEG C, stir speed (S.S.) 200r/min in temperature, ventilation
Rate is 1vvm, and cultivating 2d under conditions of intensity of illumination 4WLx obtains seed liquor.The formula of seed culture medium is（In mass）：Form sediment
Powder 4%, glucose 2.5%, corn steep liquor 5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.01%, vitamin B1 0.001%, pH7.0 is adjusted,
It is dissolved to 1L.
Step 4：According to seed liquor：Fermentation medium=1：Seed liquor is seeded to sterilized fermented and cultured by 5 ratio
In base, the formula of fermentation medium is（In mass）：Cornstarch 4%, glucose 2%, soybean cake powder 1%, soybean protein isolate
3%, corn steep liquor 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, vegetable oil 2%, vitamin B10.5%, 2,6- di-t-butyl -4-
Methylphenol 0.05%, and pH is adjusted to 7.3.
Fermented and cultured continues 120h, stir speed (S.S.) 400r/min, ventilation rate 1vvm.Fermented and cultured 0-48h, culture
Temperature is 29 DEG C, and 25 DEG C are cooled to after 48h.And ferment after carrying out 48h, conditioning agent is added into zymotic fluid, to adjust generation
Xie Liu.The conditioning agent includes leucine in mass：Abscisic acid：Creatinine：Penicillin=60：5：4：1, wherein penicillin exists
Concentration in zymotic fluid is 0.1g/L.While adding conditioning agent, the addition glucose 1.0% into zymotic fluid, ammonium sulfate 1.0%,
Dipotassium hydrogen phosphate 0.1% is with the material that supplements the nutrients.
Embodiment 3
The present embodiment provides a kind of technique of biofermentation production lycopene, and it comprises the following steps：
Step 1：Peeled potatoes 200g is taken, 1000mL deionized waters is added and boils and keep 30min；Two layers of gauze mistake after cooling
Filter, takes filtrate, adds deionized water to 1000mL, and add 20g glucose and 10g agar；Sterilize 30min at 115 DEG C, is made
Potato culture medium（PDA）.Take ATCC 14271（+）（Hereinafter referred to as positive bacterium）With ATCC 14272（-）（Hereinafter referred to as negative bacterium）Point
It is not coated on PDA plate, is cultivated 2 days in 30 DEG C of constant incubators, then reduces temperature to 20 DEG C, stimulate spore to generate,
And oxygen is passed through into incubator with 2L/min speed, while give the illumination cultivation spore 1d that intensity is 4WLx.
Step 2：Positive bacterium spore and negative bacterium spore are respectively connected to the triangular flask equipped with 20mL sterile salines, magnetic force
30min is stirred, obtains positive bacterium spore suspension and negative bacterium spore suspension.
Step 3：By positive bacterium spore suspension and the same triangle equipped with sterilizing seed culture medium of negative bacterium spore suspension access
Bottle, and positive the ratio between bacterium spore and negative bacterium spore concentration are 2：5, it is then 30 DEG C, stir speed (S.S.) 200r/min in temperature, ventilation
Rate is 1vvm, and cultivating 1d under conditions of intensity of illumination 4WLx obtains seed liquor.The formula of seed culture medium is（In mass）：Form sediment
Powder 4%, glucose 2.5%, corn steep liquor 5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.01%, vitamin B1 0.001%, pH7.0 is adjusted,
It is dissolved to 1L.
Step 4：According to seed liquor：Fermentation medium=1：Seed liquor is seeded to sterilized fermented and cultured by 6 ratio
In base, the formula of fermentation medium is（In mass）：Cornstarch 4%, glucose 2%, soybean cake powder 1%, soybean protein isolate
3%, corn steep liquor 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, vegetable oil 2%, vitamin B10.5%, 2,6- di-t-butyl -4-
Methylphenol 0.05%, and pH is adjusted to 7.3.
Fermented and cultured continues 120h, stir speed (S.S.) 400r/min, ventilation rate 1vvm.Fermented and cultured 0-48h, culture
Temperature is 30 DEG C, and 22 DEG C are cooled to after 48h.And ferment after carrying out 48h, conditioning agent is added into zymotic fluid, to adjust generation
Xie Liu.The conditioning agent includes leucine in mass：Abscisic acid：Creatinine：Penicillin=60：5：4：1, wherein penicillin exists
Concentration in zymotic fluid is 0.1g/L.While adding conditioning agent, the addition glucose 1.0% into zymotic fluid, ammonium sulfate 1.0%,
Dipotassium hydrogen phosphate 0.1% is with the material that supplements the nutrients.
The invention is not limited in foregoing embodiment.The present invention, which expands to, any in this manual to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.
Claims (9)
1. a kind of fermentation manufacturing technique of lycopene, it is characterised in that it includes：
Step 1：Prepared by seed, respectively access positive bacterium spore and negative bacterium spore in sterilized physiological saline, and stirring obtains just
Bacterium spore suspension and negative bacterium spore suspension；
Step 2：Seed culture, according to concentration ratio it is 2 according to positive bacterium spore suspension and negative bacterium spore suspension：3-5 access seed trainings
Support base progress seed culture and obtain seed liquor；
Step 2：Fermented and cultured, seed liquor access fermentation medium is subjected to fermented and cultured.
2. fermentation manufacturing technique according to claim 1, it is characterised in that the positive bacterium spore and negative bacterium spore pass through
Following culture obtains：Strain setting-out is accessed to the plate of the culture medium containing potato, in sterile culture case, 26-30 DEG C of keeping temperature,
Cultivate 2-4d；Temperature is then reduced to 16-20 DEG C, stimulates spore to generate, and cultivate spore 1-3d.
3. fermentation manufacturing technique according to claim 2, it is characterised in that during culture spore, with 2L/min speed
Rate is passed through oxygen into incubator, while gives illumination, intensity of illumination 4WLx.
4. fermentation manufacturing technique according to claim 3, it is characterised in that the temperature of seed culture process is 26-30 DEG C,
Incubation time is 1-3d, and incubation stir speed (S.S.) is 200r/min, strong to the ventilation rate for giving 1vvm simultaneously, and 4WLx illumination
Degree.
5. according to the fermentation manufacturing technique any one of claim 1-4, it is characterised in that the fermentation medium into
It is divided into cornstarch 4%, glucose 2%, soybean cake powder 1%, soybean protein isolate 3%, corn steep liquor 2%, potassium dihydrogen phosphate 0.1%, sulphur
Sour magnesium 0.02%, vegetable oil 2%, vitamin B10.5%, 2,6- di-tert-butyl-4-methy phenols 0.05%, and pH is adjusted to 7.3.
6. fermentation manufacturing technique according to claim 5, it is characterised in that when seed liquor is accessed into fermentation medium,
Seed liquor and the volume ratio of fermentation medium are 1：4-6.
7. fermentation manufacturing technique according to claim 6, it is characterised in that fermented and cultured process lucifuge, incubation time are
120h, stir speed (S.S.) 400r/min, ventilation rate 1vvm, fermented and cultured 0-48h, cultivation temperature is 28-30 DEG C, 48h
After be cooled to 22-26 DEG C.
8. fermentation manufacturing technique according to claim 7, it is characterised in that after fermentation carries out 48h, added into zymotic fluid
Conditioning agent, the conditioning agent include leucine in mass：Abscisic acid：Creatinine：Penicillin=60：5：4：1, wherein penicillin
Concentration in zymotic fluid is 0.1g/L.
9. fermentation manufacturing technique according to claim 8, it is characterised in that after fermentation carries out 48h, added into zymotic fluid
Glucose 1.0%, ammonium sulfate 1.0%, dipotassium hydrogen phosphate 0.1%.
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| CN201710981395.1A CN107604010A (en) | 2017-10-20 | 2017-10-20 | A kind of fermentation manufacturing technique of lycopene |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277238A (en) * | 2018-04-09 | 2018-07-13 | 中国科学院合肥物质科学研究院 | A kind of method and lycopene of Blakeslea trispora fermentation production lycopene |
-
2017
- 2017-10-20 CN CN201710981395.1A patent/CN107604010A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277238A (en) * | 2018-04-09 | 2018-07-13 | 中国科学院合肥物质科学研究院 | A kind of method and lycopene of Blakeslea trispora fermentation production lycopene |
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