CN108251457A - A kind of composite extractant and the method for promoting monascorubin production using composite extractant - Google Patents
A kind of composite extractant and the method for promoting monascorubin production using composite extractant Download PDFInfo
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- CN108251457A CN108251457A CN201711410018.9A CN201711410018A CN108251457A CN 108251457 A CN108251457 A CN 108251457A CN 201711410018 A CN201711410018 A CN 201711410018A CN 108251457 A CN108251457 A CN 108251457A
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Abstract
The invention belongs to field of fermentation engineering, disclose a kind of composite extractant and promote the method for monascorubin production using composite extractant, the method for production includes：By the Monascus liquid seeds in exponential phase in the inoculum concentration access fermentation medium of volume ratio 3%~8%；Peanut oil, addition 70mL/ are added in the fermentation medium simultaneouslyL zymotic fluids~180mL/L zymotic fluids；The shaking flask culture under conditions of 27 DEG C~33 DEG C of temperature；To contain in Monascus liquid seeds, peanut oil fermentation system add nonionic surfactant Span80, additive amount 2.8g/L zymotic fluids~3.4g/L zymotic fluids, continue shake flask fermentation etc..The ultimate output that the present invention is added to red, orange, yellow three kinds of pigments in the zymotic fluid of the composite extractant has been respectively increased 76%, 85% and 89% compared to control group.
Description
Technical field
Promote red yeast rice the invention belongs to field of fermentation engineering more particularly to a kind of composite extractant and using composite extractant
The method of pigment production.
Background technology
Monascorubin is a kind of safe natural pigment, and soda acid, oxidant, reducing agent are relatively stablized, have thermostabilization
Property good, protein strong coloring force, tone it is soft, stable and anti-oxidant to pH the features such as, can partly replace colour former nitrite
Applied to colorings such as meat products, dilated food, aquatic products and seasoning class cans, therefore monascorubin is applied with huge market
Prospect.
The production method of monascorubin is mainly liquid fermentation method at present, but this traditional liquid state fermentation mode faces
Two problems：Negative feedback inhibition in fermentation process in somatic cells caused by the high concentration of product；Intracellular after fermentation
Pigment separated purifies a large amount of consumption to organic solvent.In view of the above problems, there is researcher's discovery, it is coupled and is extracted using Two Liquid Phases
It is extracellular that fermentation system can promote para chrome to be secreted into, and releases intracellular product feedback inhibition, increases pigment production.
Two Liquid Phases coupling extractive fermentation is also known as " milking fermentation ", and the system is by adding nonionic table into fermentation medium
Face activating agent, forms along with cloud point system, and fermentation system automatically forms the aggregation phase of nonionic surfactant micella enrichment
Seldom dilute phase with micella.Hydrophobicity monascorubin is extracted into extracellular and is largely enriched in aggregation phase from intracellular in fermentation process,
So as to release intracellular product feedback inhibition, increase pigment production.In addition para chrome is largely secreted into extracellular, is reduced organic
The dosage of Extraction solvent.It is found through being retrieved to existing literature technology, only relevant teams find nonionic surfactant at present
Triton X-100 have good biocompatibility and pigment extraction ability in monascorubin fermentation, (live on Hu Zhiqiang surfaces
Research [D] the South China Science ＆ Engineering University of extractive fermentation monascorubin in property agent micellar solution, 2013.) but the color of Triton X-100
Plain extracting power is limited, and the more preferably extractant that remains to be discovered carrys out the extensive yield for improving monascorubin.
In conclusion problem of the existing technology is：
In present Fermentation Condition of Monascus spp field, the nonionic surfactant quilt of several series such as only Triton X, Tween
Research, we have filtered out a kind of rush better nonionic surfactant of colouring matter secretion effect on the basis of these researchs
Span80.And the combination of Span80 and vegetable oil can further obtain better pigment production.
The existing methods such as bacterial screening, fermentation condition optimization reach bottleneck at present, it is difficult to further improve monascorubin
Yield, and then seek a kind of novel fermentation method i.e. Two Liquid Phases coupling extractive fermentation system.And nonionic surfactant
Span80 does not have been reported that also after being mixed according to a certain percentage with peanut oil as method of the extractant for monascorubin production.
Invention content
In view of the problems of the existing technology, promote the present invention provides a kind of composite extractant and using composite extractant
The method of monascorubin production.
The invention is realized in this way a kind of method for promoting monascorubin production using composite extractant, the utilization
Composite extractant promotes the method for monascorubin production to include：
Monascus liquid seeds in exponential phase are accessed into fermented and cultured with the inoculum concentration of volume ratio 3%~8%
In base；Peanut oil, addition 70mL/ are added in the fermentation medium simultaneouslyL zymotic fluids~180mL/L zymotic fluids；In temperature 27 DEG C~33
Shaking flask culture under conditions of DEG C；
To containing nonionic surfactant Span80 is added in Monascus liquid seeds, peanut oil fermentation system, add
It measures as 2.8g/L zymotic fluids~3.4g/L zymotic fluids, continue shake flask fermentation；
The initial pH of the fermentation medium is 3.0-5.0.
Further, it is described the method for monascorubin production to be promoted to specifically include using composite extractant：
Step 1, by monascus purpureus in constant incubator activated spawn, then by the inoculation activated to seed
Liquid, seed liquor are cultivated in shaking table；
Step 2, the initial pH value for adjusting fermentation medium are 3.0-5.0；
Monascus liquid seeds in exponential phase are accessed dress by step 3 with the inoculum concentration of volume ratio 3%~8%
Have in the 250mL triangular flasks of 50mL fermentation mediums, while add in peanut oil in the fermentation medium, addition is
70mL/L zymotic fluids~180mL/L zymotic fluids；It is 150r/min~250r/min in rotating speed, shaking flask is trained under conditions of 27 DEG C~33 DEG C of temperature
Support 48h~96h；
Step 4, fermentation medium add non-ionic surface active into fermentation system after shaking flask culture 48h~96h
Agent Span80, additive amount 2.8g/L zymotic fluids~3.4g/L zymotic fluids, continue shake flask fermentation 3 days~8 days；
Step 5 after fermentation, takes zymotic fluid, centrifugation, obtains exo-cell pigment in supernatant, the filter residue cell after filtering is used
Ethyl alcohol extracts, and obtains para chrome.
Further, step 1 specifically includes：By monascus purpureus in 30 DEG C of constant incubators activated spawn 3d~4d, then
By the inoculation activated to seed liquor, seed liquor is cultivated in 28 DEG C~32 DEG C, the shaking table of 160r/min~200r/min
30h~50h；
Further, step 5 specifically includes：After fermentation, zymotic fluid is taken, 10000r/min~20000r/min is centrifuged,
Exo-cell pigment is obtained in supernatant, the ethyl alcohol of 50%~90% volume fraction of filter residue cell after filtering carries at 50 DEG C~80 DEG C
1h~3h is taken, obtains para chrome.
Further, in step 1, the strain is monascus purpureus NJ1 or monascus purpureus RP2.
Another object of the present invention is to provide a kind of composite extractant by nonionic surfactant Span80 (additive amounts
For 2.8-3.4g/L zymotic fluids) and peanut oil (additive amount 70-180mL/L zymotic fluids) composition.
Nonionic surfactant Span80 of the present invention is a kind of organic solvent, and the purpose for adding this solvent is to form cloud point
System realizes extractive fermentation.And without strain.Different addition time, last pigment production are different.
Advantages of the present invention and good effect are：
The micellar solution and peanut oil of preferred nonionic surfactants Span80 of the present invention is combined as by a certain percentage
Extractant realizes the extractive fermentation and cloud point extraction of intracellular monascorubin.Intracellular monascorubin is extracted into extracellular extractant
Enrichment phase releases intracellular product feedback inhibition, increases pigment production.It is added to red, orange, yellow in the zymotic fluid of the composite extractant
The ultimate output of three kinds of pigments respectively reaches 220.70U/mL, 190.64U/mL, 192.63U/mL, compared to control group, three kinds
The yield of pigment has been respectively increased 76%, 85% and 89%.The addition of the composite extractant not only makes the dissolved oxygen amount of fermentation system
Increase 20%-30%, and cell wall and membrane passage can be improved, promote the secretion of para chrome.
The present invention can make fermentation time reduction 4-6 days, and pigment production increases 70%-90%, significantly improves economic benefit,
(fermentation time of present plant is generally 15 days, the monascorubin market price be 150 yuan/kg, the monascus yellow pigment market price
For 200 yuan/kg) there is preferable application prospect.
The present invention is added to the ultimate output of red, orange, yellow three kinds of pigments in the zymotic fluid of the composite extractant compared to right
76%, 85% and 89% has been respectively increased according to group.
Description of the drawings
Fig. 1 is the method flow diagram provided in an embodiment of the present invention for promoting monascorubin production using composite extractant.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The application principle of the present invention is described in detail below in conjunction with the accompanying drawings.
The method provided in an embodiment of the present invention for promoting monascorubin production using composite extractant, including：
Monascus liquid seeds in exponential phase are accessed into fermented and cultured with the inoculum concentration of volume ratio 3%~8%
In base；Peanut oil, addition 70mL/ are added in the fermentation medium simultaneouslyL zymotic fluids~180mL/L zymotic fluids；In temperature 27 DEG C~33
Shaking flask culture under conditions of DEG C；
To containing nonionic surfactant Span80 is added in Monascus liquid seeds, peanut oil fermentation system, add
It measures as 2.8g/L zymotic fluids~3.4g/L zymotic fluids, continue shake flask fermentation；
The initial pH of the fermentation medium is 3.0-5.0, final ph 2.0-6.0.
It is provided in an embodiment of the present invention the method for monascorubin production to be promoted to specifically include using composite extractant such as Fig. 1：
S101：By monascus purpureus in constant incubator activated spawn, then by the inoculation activated to seed liquor,
Seed liquor is cultivated in shaking table；
S102：The initial pH value for adjusting fermentation medium is 3.0-5.0；
S103：Monascus liquid seeds in exponential phase are equipped with the inoculum concentration access of volume ratio 3%~8%
In the 250mL triangular flasks of 50mL fermentation mediums, while peanut oil is added in the fermentation medium, addition 70mL/L zymotic fluids
~180mL/L zymotic fluids；It is 150r/min~250r/min in rotating speed, shaking flask culture 48h under conditions of 27 DEG C~33 DEG C of temperature~
96h；
S104：Fermentation medium adds nonionic surfactant into fermentation system after shaking flask culture 48h~96h
Span80, additive amount 2.8g/L zymotic fluids~3.4g/L zymotic fluids, continue shake flask fermentation 3 days~8 days；
S105：After fermentation, zymotic fluid is taken, is centrifuged, exo-cell pigment, the filter residue cell second after filtering are obtained in supernatant
Alcohol extracting obtains para chrome.
S101 is specifically included：By monascus purpureus in 30 DEG C of constant incubators activated spawn 3d~4d, then will activate
Inoculation to seed liquor, seed liquor cultivates 30h~50h in 28 DEG C~32 DEG C, the shaking table of 160r/min~200r/min；
S105 is specifically included：After fermentation, zymotic fluid is taken, 10000r/min~20000r/min is centrifuged, in supernatant
Exo-cell pigment, the ethyl alcohol of 50%~90% volume fraction of filter residue cell after filtering extracted at 50 DEG C~80 DEG C 1h~
3h obtains para chrome.
The basic fermentation medium contains glucose 30-60g/L, analysis for soybean powder 15-45g/L, glycerine 70-100g/L, egg
White peptone 5-20g/L, MgSO4·7H2O 0.5-1.5g/L, KH2PO41.5-3.5g/L, NaNO32-10g/L, ZnSO4·
7H2O0.5-3.5g/L。
The structure of somatic cells wall can be changed in the nonionic surfactant Span80 so that the netted connection of bacterium wall is dredged
Pine, conducive to the disengaging of the extracellular substance of intracellular, and vegetable oil can improve dissolved oxygen amount.Oily Ester in extraction phase is insoluble in water,
Hydrophobicity monascorubin in fermentation system can be enriched in extractant phase, product negative feedback inhibition is released, increase substantially fermentation
System synthesizes the efficiency of monascorubin.
The additive amount of the nonionic surfactant Span80 is 2.8-3.4g/L zymotic fluids, and the addition time is red yeast rice kind
48-96h after sub- liquid inoculation.The additive amount of peanut oil is 70-180mL/L zymotic fluids, is just added at the beginning of fermentation.
The application principle of the present invention is further described with reference to specific embodiment.
The red yeast rice strain that the present invention applies is provided by Nanjing University of Technology's microbial collection center.
Embodiment one
Slant medium：Potato dextrose agar (PDA)
Seed liquid culture medium：Glucose 20-40g/L, analysis for soybean powder 10-30g/L, glycerine 60-80g/L, peptone 5-15g/
L, MgSO4·7H2O 0.5-3g/L, KH2PO41-4g/L, NaNO3 1-3g/L。
Ferment liquid culture medium：Glucose 30-60g/L, analysis for soybean powder 15-35g/L, glycerine 70-100g/L, peptone 5-20g/
L, MgSO4·7H2O 0.5-2g/L, KH2PO41.5-5g/L, NaNO31-5g/L, ZnSO4·7H2O 1-4g/L。
Three kinds of pigment color values are measured and are calculated：The zymotic fluid of certain volume is taken, is centrifuged, is filtered, filtrate is dilute through deionized water
It releases to suitable multiple, measures its OD value under 390-420nm, 430-480nm, 490-520nm wavelength respectively with spectrophotometer,
OD values under three kinds of wavelength are multiplied by the color value that extension rate is extracellular yellow, orange, red trichromatism element respectively.Bacterial sediment after centrifugation
It is extracted 1-3 hours with 50-80 DEG C of the ethyl alcohol mixing of 50-90%, is then centrifuged for, filters, ethyl alcohol of the filtrate through 50-90% is diluted to
Suitable multiple, with its OD value is measured under the similary wavelength of spectrophotometer, the OD values under three kinds of wavelength are multiplied by extension rate i.e. respectively
For intracellular Huang, orange, red trichromatism element color value.Color value unit is represented with U/mL.Color value (U/mL)=OD values × extension rate.
The method provided in an embodiment of the present invention for promoting monascorubin production using composite extractant, including：
Step 1, first by the monascus purpureus of preservation in 30 DEG C of constant incubators activated spawn 3-4d, then will activate
Inoculation to seed liquor (liquid amount 50mL/250mL), seed liquor is cultivated in 28-32 DEG C, the shaking table of 160-200r/min
30-50h。
Step 2, the initial pH value for adjusting fermentation medium is 3.0-5.0.
Step 3, the Monascus liquid seeds in exponential phase are equipped with the inoculum concentration access of volume ratio 3%-8%
In the 250mL triangular flasks of 50mL fermentation mediums, while peanut oil is added in the fermentation medium, addition 70-180mL/L
Zymotic fluid.It is 150-250r/min in rotating speed, shaking flask culture 48-96h under conditions of 27-33 DEG C of temperature.
Step 4, fermentation medium adds nonionic surfactant into fermentation system after shaking flask culture 48-96h
Span80, additive amount are 2.8-3.4g/L zymotic fluids, continue shake flask fermentation 3-8 days.
Step 5, after fermentation, zymotic fluid is taken, 10000-20000r/min is centrifuged, and exo-cell pigment can be obtained in supernatant,
The ethyl alcohol of filter residue cell 50-90% volume fractions after filtering extracts 1-3h at 50-80 DEG C, can obtain para chrome.Pass through
Red, orange, yellow three kinds of pigment total outputs that ultraviolet specrophotometer is measured in zymotic fluid are respectively 190-200U/mL, 180-190U/
mL、180-190U/mL
Embodiment two：
It is identical with fermentation process step with the Spawn incubation of above-described embodiment one, except that fermentation medium is by following
It is prepared, minimal medium is rice meal 30-60g/L, glycerine 70-100g/L, peptone 5-20g/L, MgSO4·
7H2O 0.5-2g/L, KH2PO41.5-5g/L, NaNO31-5g/L, ZnSO4·7H2O 1-4g/L.Extraction phase is still nonionic table
Face activating agent Span80 and peanut oil, addition time are also identical with embodiment one.The fermentation condition identical with embodiment one issues
Ferment measures red, orange, yellow three pigments total color value after 5-10 days is respectively 180-190U/mL, 160-170U/mL, 180-190U/
mL。
Embodiment three：
With the step of above-described embodiment one and fermentative medium formula is identical, except that strain is different.Select this reality
Test room preservation of bacteria strain Monascus RP2.
Fermentation step and fermentation process and condition are identical with above-described embodiment one.After fermentation, pass through ultraviolet spectrometry light
Total color value that degree meter measures red, orange, yellow three pigments is respectively 150-160U/mL, 140-150U/mL, 140-150U/mL.
The composite extractant that the nonionic surfactant Span80 knowable to above-described embodiment is combined with peanut oil is to red yeast rice
It is mould to have good biocompatibility and pigment extraction ability.The present invention can efficiently produce monascorubin, be given birth in monascorubin
Production field has important practical value and economic value.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
Claims (6)
- A kind of 1. method for promoting monascorubin production using composite extractant, which is characterized in that described to utilize composite extractant The method of monascorubin production is promoted to include：By the Monascus liquid seeds in exponential phase in the inoculum concentration access fermentation medium of volume ratio 3%~8%； Peanut oil, addition 70mL/ are added in the fermentation medium simultaneouslyL zymotic fluids~180mL/L zymotic fluids；In 27 DEG C~33 DEG C of temperature Under the conditions of shaking flask culture；It is to containing addition nonionic surfactant Span80, additive amount in Monascus liquid seeds, peanut oil fermentation system 2.8g/L zymotic fluids~3.4g/L zymotic fluids, continue shake flask fermentation；The initial pH of the fermentation medium is 3.0-5.0.
- 2. promote the method for monascorubin production using composite extractant as described in claim 1, which is characterized in that the profit It is specifically included with the method that composite extractant promotes monascorubin to produce：Step 1, by monascus purpureus in constant incubator activated spawn, then by the inoculation activated to seed liquor, kind Sub- liquid is cultivated in shaking table；Step 2, the initial pH value for adjusting fermentation medium are 3.0-5.0；Monascus liquid seeds in exponential phase are equipped with by step 3 with the inoculum concentration access of volume ratio 3%~8% In the 250mL triangular flasks of 50mL fermentation mediums, while peanut oil is added in the fermentation medium, addition 70mL/L zymotic fluids ~180mL/L zymotic fluids；It is 150r/min~250r/min in rotating speed, shaking flask culture 48h under conditions of 27 DEG C~33 DEG C of temperature~ 96h；Step 4, fermentation medium add nonionic surfactant into fermentation system after shaking flask culture 48h~96h Span80, additive amount 2.8g/L zymotic fluids~3.4g/L zymotic fluids, continue shake flask fermentation 3 days~8 days；Step 5 after fermentation, takes zymotic fluid, centrifugation, and exo-cell pigment, the filter residue cell ethyl alcohol after filtering are obtained in supernatant Extraction, obtains para chrome.
- 3. promote the method for monascorubin production using composite extractant as claimed in claim 2, which is characterized in that step 1 It specifically includes：By monascus purpureus in 30 DEG C of constant incubators activated spawn 3d~4d, then the inoculation activated is arrived Seed liquor, seed liquor cultivate 30h~50h in 28 DEG C~32 DEG C, the shaking table of 160r/min~200r/min.
- 4. promote the method for monascorubin production using composite extractant as claimed in claim 2, which is characterized in that step 5 It specifically includes：After fermentation, zymotic fluid is taken, 10000r/min~20000r/min is centrifuged, and exo-cell pigment, mistake are obtained in supernatant The ethyl alcohol of 50%~90% volume fraction of filter residue cell after filter extracts 1h~3h at 50 DEG C~80 DEG C, obtains para chrome.
- 5. promote the method for monascorubin production using composite extractant as claimed in claim 2, which is characterized in that step 1 In, the strain is monascus purpureus NJ1 or monascus purpureus RP2.
- 6. a kind of composite extractant of method as described in claim 1 for monascorubin being promoted to produce using composite extractant, It is characterized in that, the composite extractant is made of nonionic surfactant Span80 and peanut oil；Non-ionic surface active Agent Span80 additive amounts are 2.8g/L zymotic fluids-3.4g/L zymotic fluids；Peanut oil additive amount is 70mL/L zymotic fluids-180mL/L zymotic fluids。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951801A (en) * | 2019-11-25 | 2020-04-03 | 北京工商大学 | Application of polyoxyethylene lauryl ether in production of monacolin K |
| CN112042858A (en) * | 2020-09-15 | 2020-12-08 | 江西理工大学 | Method for extracting biosurfactant of monascus pigment |
| CN114350526A (en) * | 2022-01-14 | 2022-04-15 | 北京工商大学 | Method for improving monascus pigment tone |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951801A (en) * | 2019-11-25 | 2020-04-03 | 北京工商大学 | Application of polyoxyethylene lauryl ether in production of monacolin K |
| CN110951801B (en) * | 2019-11-25 | 2021-04-13 | 北京工商大学 | Application of polyoxyethylene lauryl ether in production of monacolin K |
| CN112042858A (en) * | 2020-09-15 | 2020-12-08 | 江西理工大学 | Method for extracting biosurfactant of monascus pigment |
| CN112042858B (en) * | 2020-09-15 | 2022-11-29 | 江西理工大学 | Method for extracting biosurfactant of monascus pigment |
| CN114350526A (en) * | 2022-01-14 | 2022-04-15 | 北京工商大学 | Method for improving monascus pigment tone |
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