CN102443605A - Method for perstraction of fermented microbial intracellular product with non-ionic surfactant - Google Patents
Method for perstraction of fermented microbial intracellular product with non-ionic surfactant Download PDFInfo
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- CN102443605A CN102443605A CN2011103464131A CN201110346413A CN102443605A CN 102443605 A CN102443605 A CN 102443605A CN 2011103464131 A CN2011103464131 A CN 2011103464131A CN 201110346413 A CN201110346413 A CN 201110346413A CN 102443605 A CN102443605 A CN 102443605A
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Abstract
The invention discloses a method for perstraction of a fermented microbial intracellular product with a non-ionic surfactant. Specifically, during fermentation of a microbial intracellular product, a non-ionic surfactant micellar solution formed by addition of the non-ionic surfactant concentration or a cloud point system formed thereby is adopted as a microbial fermentation medium, by which the intracellular product can be penetrated to an extracellular environment so as to improve the product level of microbial fermentation. Simultaneously, perstraction and fermentation of the microbial intracellular product can be realized. The method of the invention is especially suitable for the fermentation process of an intracellular product, such as production of intracellular microbial enzymes, production of intracellular organic small molecule substances as well as production of intracellular oil compounds, etc. The method provided in the invention effectively eliminates intracellular product inhibition, enhances the concentration of a microbial fermentation product, and adjusts the composition of a microbial fermentation secondary metabolite, thus improving the volume yield of microbial fermentation as well as the efficiency of intracellular product fermentation, product release and other processes.
Description
Technical field
The present invention relates to a kind of method of extractive fermentation mikrobe intracellular product, specifically is a kind of method with nonionogenic tenside infiltration extractive fermentation mikrobe intracellular product.
Background technology
The microbial fermentation intracellular product accumulates the high yield substrate concentration usually and causes product to suppress in cell, main settling mode is through improving microbial cell density to improve production concentration.On the other hand, means such as intracellular product accumulation makes in downstream separation and the purge process must employing cytoclasis, solvent extraction have increased the complicacy and the running cost of process.Being employed in ferments in water-organic solvent biphasic system can be discharged into born of the same parents' external environment with intracellular product to change the cell membrane permeability, but the biocompatibility of organic solvent cell membrane and organic solvent are to the contradiction that exists between the extracting power of intracellular product.Therefore, utilize the infiltration extractive fermentation technology of organic solvent to be difficult to realize.
Nonionogenic tenside forms micellar solution in the aqueous solution.At a certain temperature, nonionogenic tenside micellar solution automatically is separated and forms tensio-active agent and concentrate phase and tensio-active agent dilute solution mutually.This biphasic system is called cloud point system.The application of cloud point system in extracting and separating that nonionic surfactant solution or nonionic surfactant solution form has a lot of reports.Through the prior art document is compared, the using cloud point system technology is extracting the microbial fermentation extracellular products to remove product to the inhibition of mikrobe existing relevant report [Wang Zhilong, Dai Zewen.Extract microbial fermentation in the cloud point system.Enzyme and microbial technique, 2010,46:407-418.Wang Z; Dai Z.Extractive microbial fermentation in cloud point system.Enzyme Microbial Technology; 2010; 46:407-418], but changing the cell membrane permeability in fermentation process, the cloud point system of application nonionic surfactant solution and formation thereof also do not report with the what is called infiltration extractive fermentation technology that discharges intracellular product while extraction product.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of extractive fermentation mikrobe intracellular product; Utilize the aqueous solution of nonionogenic tenside and the cloud point system of formation thereof, when the microbial fermentation intracellular product, intracellular product is penetrated into the extracellular fermented liquid and extracts product in the fermented liquid of extracellular simultaneously.
For addressing the above problem; The method of extractive fermentation mikrobe intracellular product of the present invention; Specifically: during the microbial fermentation intracellular product; The nonionogenic tenside micellar solution that forms with the nonionogenic tenside concentration of adding every liter of fermented liquid 0.2-128g or the cloud point system of its formation are as the fermentation media of mikrobe; The cloud point system that adopts nonionogenic tenside micellar solution or its formation is for intracellular product is penetrated into extracellular environment as the purpose of the fermentation media of microbial fermentation intracellular product, thereby improves the composition of the gentle adjusting microbial secondary of the product water meta-bolites of microbial fermentation.Realize concentrating and the separation and purification process of partial purification of title product through extraction product simultaneously with the simplification downstream.Promptly realize the infiltration extractive fermentation of mikrobe intracellular product.
Among the present invention, adopt the cloud point system of nonionogenic tenside micellar solution or its formation to realize that the key of the infiltration extractive fermentation of mikrobe intracellular product is that the perviousness that changes cell keeps the vitality of mikrobe simultaneously.
The ionic surfactant pack that the present invention relates to is drawn together high molecular polymer, the small molecules nonionogenic tenside of polymkeric substance nonionogenic tenside and application usually etc.
Infiltration extraction microbial fermentation technology of the present invention is particularly suitable for the fermentation of mikrobe intracellular product.Like fermentation of organic molecule such as grease, pigment in the fermentation of desmo enzyme, the cell etc.Particularly, with monascus ruber fermentative prodn parachrome as specific examples.
Preferably, the present invention utilizes the cloud point system of nonionogenic tenside micellar solution or its formation in mikrobe monascus ruber fermentative prodn monascorubin, intracellular product to be discharged into the extracellular, extracts the monascorubin in the cell fermentation liquid simultaneously.
The ionic surfactant pack that the present invention relates to is drawn together four types of T 46155 base polymer, methoxy polyoxyethylene base polymer, T 46155-polyoxypropylene kind block polymer, small molecules nonionogenic tensides etc.
Wherein, the T 46155 polymer is (obtaining PEG 4000 like commercially available commercial channel, PEG 10000 etc.); Methoxy polyoxyethylene base polymer MPEG 5000 (Sigma); Be total to polymer L64 (Zhejiang Real Madrid chemical company), F68 (Sigma)).Nonionogenic tenside is that (glucitols is (like span 20, Tween 80 (Shanghai reagent company); Fatty alcohol polyethenoxy ether class (Brij 30, (Fluka); Triton X series (like Triton X-45, Triton X-114, Triton X-100 (Fluka); Teritgol series (like Tergitol TMN-3 (Fulka)) etc.
Compared with prior art, the present invention has following beneficial effect: the present invention is through adding nonionogenic tenside in microbial fermentation solution, tensio-active agent has changed the cell membrane permeability.On the one hand, the title product (concentration is Cb) in the microorganism cells film can optionally be penetrated into extracellular environment (concentration is Cp).This has removed the restraining effect of product in the cell, with significantly improving title product effective volume during the fermentation, thereby significantly improves the output of title product, has regulated the metabolism of microbial secondary product simultaneously.For example adding nonionogenic tenside makes in the monascorubin yellow pigment form the branch rate obviously to increase.On the other hand; Title product adds in nonionogenic tenside micellar solution or its cloud point system and is dissolved in (concentration is Ce) in the surfactant micelle in the fermented liquid of extracellular; This extraction process has concentrated title product, has further removed the inhibition of product, has simplified the downstream separation process.This realization process key is that selected nonionogenic tenside can influence the cell membrane permeability and be unlikely to influence vital processes such as the growth of microorganism cells, synthetic secondary metabolite, promptly keeps the biocompatibility of mikrobe in surfactant micelle solution and formed cloud point system thereof.
Description of drawings
Fig. 1 is the schematic diagram of infiltration extractive fermentation;
Embodiment
Following instance will be described further the present invention.Present embodiment is that prerequisite is implemented with technical scheme of the present invention, provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.
It is disclosed existing bacterial classification that present embodiment relates to microbial strains Monascus anka, can obtain (deposit number CICC 5013) through Chinese industrial microbial strains preservation center, also can buy through businessman to obtain.This microorganism strains can obtain microbial secondary meta-bolites monascorubin through the solid state fermentation or the deep layer liquid state fermentation of routine.Its main moity of monascorubin is yellow pigment, citraurin and yellow pigment, is hydrophobic compound, mainly is distributed in the microorganism cells.Monascorubin has had the applicating history of more than one thousand years in China, is natural food color additive.Adopt modern fermentation technique to have important industrial value.
As shown in Figure 1, permeate the schematic diagram of extractive fermentation for the present invention.
Wherein, the seed slant culture is that PDA substratum (murphy juice 200g in the 1L water, glucose 20g, agar 15-20g) was cultivated 2 days under 30 degree conditions.
The seed activation substratum is (Semen Maydis powder 30g in the 1L water, NaNO
33g, KH
2PO
44g, FeSO
47H
2O0.01g), in 30 degree, 110 rev/mins shaking table, cultivated 2 days.
Fermention medium is (Semen Maydis powder 70g in the 1L water, KH
2PO
45g, CaCl
20.1g, FeSO
47H
2O0.01g), regulate pH to 4, in 30 degree, 110 rev/mins shaking table, cultivate 6-8 days.
The product monascorubin comprises main ingredients such as red pigment, citraurin and yellow pigment.Production concentration and extracellular products concentration are used spectrophotometry at 520nm, 470nm and 410nm place respectively in the corresponding cell.Extracellular products concentration is suitably diluted directly through centrifugal back and is measured.The absorbancy that supernatant is measured pigment is suitably diluted in 1 hour centrifugal back of alcohol immersion cell of fermentating liquid volumes such as the interior production concentration employing of cell.The pigment concentration of yellow pigment, citraurin, red pigment is expressed as the OD value under 410,470 and 510 nanometers respectively.
Embodiment 1
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at polymer P EG 4000 additions, citraurin, the optical density(OD) of red pigment is respectively 10,6 and 9.
Embodiment 2
At polymer P EG 10000 additions is 0.4g/100ml, condition under obtained the extracellular yellow pigment in 7 days by normal condition reaction, citraurin, the optical density(OD) of red pigment is respectively 10,7 and 8.
Embodiment 3
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at polymkeric substance MPEG 5000 additions, citraurin, the optical density(OD) of red pigment is respectively 5,4 and 5.
Embodiment 4
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at polymkeric substance nonionogenic tenside F68 addition, citraurin, the optical density(OD) of red pigment is respectively 5,4 and 6.
Embodiment 5
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at polymkeric substance nonionogenic tenside L64 addition, citraurin, the optical density(OD) of red pigment is respectively 6,5 and 5.5.
Embodiment 6
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside Tween 80 additions, citraurin, the optical density(OD) of red pigment is respectively 5,5 and 4.
Embodiment 7
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside Span 20 additions, citraurin, the optical density(OD) of red pigment is respectively 4,3 and 4.
Embodiment 8
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 16,8 and 15.
Embodiment 9
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside Triton X-114 addition, citraurin, the optical density(OD) of red pigment is respectively 3,2 and 3.
Embodiment 10
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside Triton X-45 addition, citraurin, the optical density(OD) of red pigment is respectively 4,3 and 4.
Embodiment 11
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside TMN-3 addition, citraurin, the optical density(OD) of red pigment is respectively 3,2 and 3.
Embodiment 12
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside Brij 30 additions, citraurin, the optical density(OD) of red pigment is respectively 0,0 and 0.
Embodiment 13
Be to obtain the extracellular yellow pigment in 6 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 12,6 and 11.
Embodiment 14
Be to obtain the extracellular yellow pigment in 8 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 17,9 and 16.
Embodiment 15
Be to obtain the extracellular yellow pigment in 9 days by the normal condition reaction under the condition of 0.4g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 18,9 and 17.
Embodiment 16
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.2g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 14,6 and 12.
Embodiment 17
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 0.8g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 27,16 and 18.
Embodiment 18
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 1.6g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 38,21 and 22.
Embodiment 19
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 3.2g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 49,23 and 24.
Embodiment 20
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 6.4g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 48,21 and 22.
Embodiment 21
Be to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 12.8g/100ml at nonionogenic tenside Triton X-100 addition, citraurin, the optical density(OD) of red pigment is respectively 37,17 and 18.
Embodiment 22
At nonionogenic tenside Triton X-100 and Triton X-45 is that 4: 6 and addition are to obtain the extracellular yellow pigment in 7 days by the normal condition reaction under the condition of 4g/100ml with compound proportion, citraurin, and the optical density(OD) of red pigment is respectively 53,25 and 26.This extracellular solution system issues the looks separation and forms cloud point system at leavening temperature, and wherein tensio-active agent concentrates the yellow pigment of phase, citraurin; The optical density(OD) of red pigment is respectively 108; 42 and 35, and yellow pigment in the tensio-active agent dilute phase, citraurin; The optical density(OD) of red pigment is respectively 8,3 and 5.
Embodiment 23
Under the condition of not adding nonionogenic tenside, obtained the extracellular yellow pigment in 7 days by the normal condition fermentation in the contradistinction system of the conventional aqueous solution, citraurin, the optical density(OD) of red pigment is respectively 8,6 and 10.Comparative example 19,20 and 21 etc. experimental result are added nonionogenic tenside the output of the outer monascorubin of born of the same parents are obviously increased, and obviously improved the branch rate of yellow pigment in the monascorubin.
It more than is the description of the preferred embodiment of the present invention; Should be understood that; Enforcement of the present invention is not limited to above-mentioned situation, and the present invention is particularly suitable for the fermenting process of product in the cell, like the production of oil compounds in the production of organic molecule material in the production of microbial enzyme in the cell, the cell and the cell etc.; Can remove intracellular product effectively and suppress, improve the concentration and the composition of regulating the microbial fermentation secondary metabolite of microbial fermentation product.Thereby the volume productivity and the efficient that has improved processes such as intracellular product fermentation, product release of microbial fermentation have been improved.
Although content of the present invention has been done detailed introduction through above-mentioned preferred embodiment, will be appreciated that above-mentioned description should not be considered to limitation of the present invention.After those skilled in the art have read foregoing, for multiple modification of the present invention with to substitute all will be conspicuous.Therefore, protection scope of the present invention should be limited appended claim.
Claims (7)
1. method with nonionogenic tenside infiltration extractive fermentation mikrobe intracellular product; It is characterized in that: during the microbial fermentation intracellular product; With the cloud point system that adds nonionogenic tenside micellar solution that nonionogenic tenside concentration forms or its formation fermentation media as mikrobe; Intracellular product is penetrated into extracellular environment, improves the product level of microbial fermentation, realize the infiltration extractive fermentation of mikrobe intracellular product simultaneously.
2. the method for employing nonionogenic tenside infiltration extractive fermentation mikrobe intracellular product according to claim 1; It is characterized in that: the infiltration extractive fermentation of described intracellular product; Comprise that the cloud point system that utilizes nonionogenic tenside micellar solution or its formation is discharged into the extracellular with the intracellular product of mikrobe monascus ruber fermentative prodn monascorubin, extracts the monascorubin in the microbial fermentation solution simultaneously.
3. the method with nonionogenic tenside infiltration extractive fermentation mikrobe intracellular product according to claim 1 and 2; It is characterized in that: said ionic surfactant pack is drawn together high molecular polymer, in polymkeric substance nonionogenic tenside and the small molecules nonionogenic tenside one or more.
4. the method with nonionogenic tenside infiltration extractive fermentation mikrobe intracellular product according to claim 3; It is characterized in that: said ionic surfactant pack is drawn together T 46155 polymer, methoxy polyoxyethylene base polymer, polymer L64, F68 altogether, and glucitols, fatty alcohol polyethenoxy ether class, Triton X series, Teritgol serial in one or more.
5. the method with nonionogenic tenside infiltration extractive fermentation mikrobe intracellular product according to claim 4, it is characterized in that: said nonionogenic tenside range of concentrations is at the 0.2-128g/L fermented liquid.
6. the method with nonionogenic tenside infiltration extractive fermentation mikrobe intracellular product according to claim 1 and 2; It is characterized in that: said nonionogenic tenside improves the production concentration of microbial fermentation, regulates the composition of microbial secondary meta-bolites simultaneously.
7. the method with nonionogenic tenside infiltration extractive fermentation mikrobe intracellular product according to claim 6, it is characterized in that: said nonionogenic tenside has improved the concentration of the outer monascorubins of born of the same parents and has improved the branch rate of yellow pigment in the monascorubin.
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Cited By (6)
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| CN103224958A (en) * | 2013-04-10 | 2013-07-31 | 上海交通大学 | Method for preparing monascus pigment through extraction fermentation and pH value regulation |
| CN104195178A (en) * | 2014-07-08 | 2014-12-10 | 上海交通大学 | High tone monascus red pigment preparation method |
| CN108251464A (en) * | 2018-01-23 | 2018-07-06 | 同济大学 | The method that grease is generated using microbial fermentation |
| CN109371053A (en) * | 2018-12-24 | 2019-02-22 | 江西科技师范大学 | A kind of High-productive Monascus Pigment Strain construction method |
| CN110205342A (en) * | 2019-06-28 | 2019-09-06 | 福州大学 | A kind of method of pigment production increase during immobilization Fermentation Condition of Monascus spp |
| CN112042858A (en) * | 2020-09-15 | 2020-12-08 | 江西理工大学 | Method for extracting biosurfactant of monascus pigment |
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Cited By (9)
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| CN103224958A (en) * | 2013-04-10 | 2013-07-31 | 上海交通大学 | Method for preparing monascus pigment through extraction fermentation and pH value regulation |
| CN103224958B (en) * | 2013-04-10 | 2016-02-10 | 上海交通大学 | Extractive fermentation and regulation and control pH prepare the method for monascorubin |
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| CN109371053A (en) * | 2018-12-24 | 2019-02-22 | 江西科技师范大学 | A kind of High-productive Monascus Pigment Strain construction method |
| CN110205342A (en) * | 2019-06-28 | 2019-09-06 | 福州大学 | A kind of method of pigment production increase during immobilization Fermentation Condition of Monascus spp |
| CN110205342B (en) * | 2019-06-28 | 2020-11-27 | 福州大学 | Method for increasing pigment yield in fermentation process of immobilized monascus |
| 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 |
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Effective date of registration: 20181210 Address after: 300467 Zhongbin Avenue 2633, Tianjin Eco-city, Binhai New Area, Tianjin Patentee after: Jacki (Tianjin) Biological Medicine Co., Ltd. Address before: 200240 800 Dongchuan Road, Shanghai, Minhang District, Shanghai. Patentee before: Shanghai Jiao Tong University |