CN102634470B - Cellulosimicrobium cellulans and method for producing trehalose through penetration fermentation of cellulosimicrobium cellulans - Google Patents
Cellulosimicrobium cellulans and method for producing trehalose through penetration fermentation of cellulosimicrobium cellulans Download PDFInfo
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- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 title claims abstract description 73
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 title claims abstract description 72
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
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- 239000008103 glucose Substances 0.000 claims abstract description 25
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Abstract
The invention discloses cellulosimicrobium cellulans and a method for producing trehalose through the penetration fermentation of the cellulosimicrobium cellulans. The new strain is named as the cellulosimicrobium cellulans S32, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) N0.5841. According to the method, glucose serves as a substrate; the penetration fermentation is performed on the cellulosimicrobium cellulans S32 after the cellulosimicrobium cellulans S32 is cultured; after being subjected to stress, cellulosimicrobium cellulans cells generate more trehalose synthetase systems and become permeability cells; and a great deal of intracellular trehalose synthetase systems are synthetized into extracellular trehalose. The conversion rate from the glucose to the trehalose reaches 60%, carbohydrate by-products are avoided, the product is easy to separate, the production processes are intensive, the equipment investment is less, and the production cost is lower. Due to the good non-specificity protective effect on large biological molecules, the prepared trehalose has a wide application prospect in the fields of medicaments, cosmetics, foods and the like.
Description
Technical field
The invention belongs to microbial fermentation and produce trehalose method and technology field, particularly utilize the method for the fine bacterium infiltration of fibrosis fiber fermentative production extracellular-trehalose take glucose as matrix.
Background technology
According to " Acta Biochimica et Biophysica Sinica " (Crowe et al, Biochim.Biophys.Acta, 1984,769:151-159) with " food chemistry " (Ohtani et al, Food Chem., 1994,2:91-95) introduce, trehalose (α-D-glucopyranosyl-α-D-glycopyranoside) is a kind of non-reduced glucoside that extensively is distributed in plant, insect, the fungus and bacterium, and this disaccharide can be used as a kind of file layout of the energy and the protective agent of the environmental stress such as dry or freezing.Owing in its molecular structure, not having reducing end, trehalose not only demonstrates the resistivity to extreme temperature, pH and Maillard reaction, but also have very high water holding activity, so trehalose has the function that helps stabilizing protein structure and microbial film integrity.Because these useful characteristics; according to " trehalose: the novel carbohydrate of 21 century " (Huang Ribo etc.; Beijing; Chemical Industry Press; the 1st edition 74-198 page or leaf in 2010) introduce; trehalose is at medicine; there are many application in the field such as makeup and food; such as can be used as antibody drug; vaccine; diagnostic reagent; active somatic cell or be organized in bio-activity protector when freezing and dry; can be used as the good auxiliary material of drug delivery system; can be used for the moisturizing of makeup; cytoprotective; prevent that grease from decomposing and the minimizing body odor; the functional components such as radioprotective; as age of starch; the inhibitor of protein denaturation and lipid oxidation, and the stablizer and the preservation agent that are used as vegetables or meat.
According to " glycobiology " (Elbein et al, Glycobiology, 2003,13:17-27) with " trehalose " (Huang Ribo etc., trehalose: the novel carbohydrate of 21 century, Beijing: Chemical Industry Press, the 1st edition 31-55 page or leaf in 2010) introduces in, present commercial trehalose production method mainly contains three kinds: (1) yeast cell extraction method is to take to collect yeast cell to extract with hot ethanol.But the method is consuming time longer, and efficient is low, extract solvent load large, and intracellular trehalose content is not high, causes thus cost higher, makes its food service industry that has the call at trehalose be difficult to promote the use of.(2) microbe fermentation method, the method are culturing micro-organisms on certain matrix, produce trehalose by microbial fermentation, extract purifying again from fermented liquid and obtain the trehalose of commercialization.Although effectively producing the microorganism of trehalose is extensively screened before, and the existing separated success of several microorganisms also is applied to trehalose fermentation commercial production system, but up to the present, because existing many production system transformation efficiencys are lower, and the carbohydrate by product is more in the fermented liquid, thereby makes the extraction of trehalose, refining relatively difficulty.(3) bacterial enzyme conversion method is after TreP relevant in some bacterial bodies is extracted, to utilize its transglycosylation to come trehalose synthesis in catalytic substrate.Present applicable enzyme transforming process has malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose lytic enzyme double-enzyme method, glycosyltransferase and amylase double-enzyme method and TreP method.Enzyme transforming process has the advantage that specificity is good, transformation efficiency is high, but used enzyme require is refining, because the expense of enzyme processed is higher, and the product separation process is complicated, so production cost is higher.The enzymatic conversion system that uses at present produces trehalose from Fructus Hordei Germinatus oligose, and the information of effectively producing trehalose from glucose (one of useful parent material of industrial production organic substance) lacks.
Summary of the invention
The invention provides the fine bacterium of a kind of fibrosis fiber (Cellulosimicrobium cellulans) S32, and utilize the fine bacterium of this fibrosis fiber to permeate the method for fermentative production trehalose, to overcome the above-mentioned deficiency of prior art, reach the technical purpose that production technique is intensive, transformation efficiency is high and product is easy to separate.
A kind of new fine bacteria strain of fibrosis fiber provided by the invention, obtain from the separation of Hefei district elm withered tree branch, the fine bacterium of called after fibrosis fiber (Cellulosimicrobium cellulans) S32, culture presevation number is CGMCC No.5841, preservation date is on March 2nd, 2012, preservation mechanism is China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
The method of utilizing the fine bacterium infiltration of fibrosis fiber fermentative production trehalose of the present invention, it is characterized in that: first with culture presevation number for the fine bacterium of fibrosis fiber (Cellulosimicrobium cellulans) the S32 bacterial strain of CGMCC No.5841 carries out activation culture, change over to by 5% inoculum size of culture volume and to be equipped with component and by quality grams contained in every liter of substratum to be: glucose 20, yeast powder 5, peptone 5, corn steep liquor 10, MgSO
41, KH
2PO
41 and CaCO
3In the fermentor tank of fermention medium 7(g/L), liquid amount is 70% of fermentor tank volume, and culture temperature is 30 ℃, air flow quantity 4L/min, rotating speed 300r/min; After cultivating 30h, in nutrient solution, be added into (the NH that final concentration is 5-10g/L
4)
2SO
4With the glucose of 60g/L, cultivate again 7h; Then fermented liquid is centrifugal or remove by filter strain cell, obtain containing the solution of trehalose; It is that 10000 ultra-filtration membrane removes macromolecular substance that relative molecular weight is held back in employing, with the anion-cation exchange resin desalination, separate trehalose and glucose; The trehalose component heating evaporation of collecting is concentrated, and spraying drying namely obtains the trehalose finished product again.
Behind above-mentioned production strain culturing 30h, preferably adding final concentration in the nutrient solution is the (NH of 8g/L
4)
2SO
4
The inventive method is because take glucose as substrate, utilize the fine bacteria strain of fibrosis fiber to cultivate after, the fine bacterium of fibrosis fiber is permeated fermentation take glucose as matrix, thereby can synthesize in a large number extracellular-trehalose.Adopt the inventive method to produce trehalose, production technique is intensive, facility investment is few and be easy to separation without carbohydrate by product, product, its outstanding feature be the trehalose transformation efficiency up to 60%, production cost is reduced, thereby lays a good foundation for the widespread use of trehalose.Adopt the prepared trehalose of the inventive method because it has broad application prospects in fields such as medicine, makeup and food to the good non-specific provide protection of biomacromolecule.
Owing to having taked in the inventive method in fermention medium, to add an amount of (NH
4)
2SO
4Improve the osmotic pressure of culture environment, coerce and cultivate the fine bacterium of fibrosis fiber so that stress produce more TreP system in the strain cell, simultaneously cytolemma has lost original selection perviousness, the trehalose that produces in the cell can free diffusing in the fermented liquid of extracellular.After measured, under the same fermentation condition, do not add (NH
4)
2SO
4The fine fermented liquid of fibrosis fiber in do not have trehalose to generate, and add an amount of (NH
4)
2SO
4The fine fermented liquid of fibrosis fiber in the trehalose fermentation unit can reach 34g/L.
Embodiment
Below in conjunction with specific embodiment the present invention is further elaborated.
Be used for raw materials of glucose, yeast powder, peptone, corn steep liquor, the MgSO of the composition of preparation substratum in the embodiment of the invention
4, KH
2PO
4And CaCO
3And (NH
4)
2SO
4Be the commercially available prod.
The fine bacteria strain of fibrosis fiber that the present invention uses obtains autonomous separation the from the Hefei district elm withered tree branch, the fine bacterium of called after fibrosis fiber (Cellulosimicrobium cellulans) S32, culture presevation number is CGMCC No.5841, preservation date is on March 2nd, 2012, preservation mechanism is China Committee for Culture Collection of Microorganisms common micro-organisms center, address: Institute of Microorganism, Academia Sinica.
Embodiment 1:
The present embodiment utilizes the fine bacterium of fibrosis fiber to permeate the method for fermentative production trehalose, comprises that the fine bacterium of fibrosis fiber is cultivated, the infiltration fermentation separates with the extraction of trehalose with the synthetic of trehalose.Concrete operation step is as follows:
(1) the fine bacterium of fibrosis fiber is cultivated: get culture presevation number and carry out activation culture for the fine bacterium of fibrosis fiber (Cellulosimicrobium cellulans) the S32 bacterial strain of CGMCC No.5841,5% inoculum size by culture volume changes in the 5L fermentor tank that is equipped with fermention medium, liquid amount 3.5L, 30 ℃ of culture temperature, air flow quantity 4L/min, rotating speed 300r/min, incubation time 30h.
Above-mentioned fermention medium component by quality grams contained in every liter of substratum is: glucose 20, yeast powder 5, peptone 5, corn steep liquor 10, MgSO
41, KH
2PO
41 and CaCO
37(g/L).
(2) infiltration fermentation: in the nutrient solution of cultivating behind the 30h, add 28g (NH
4)
2SO
4With 210g glucose, cultivate again 7h.Add ammonium salt (NH
4)
2SO
4Can improve the osmotic pressure of production strain culturing environment, induce its cellular stress to produce more TreP system, and become Permeabilized cells; Simultaneously glucose enters into and is converted into trehalose in the cell, trehalose can free diffusing in the fermented liquid of extracellular.
(3) isolation and purification of trehalose: fermented liquid is centrifugal or remove by filter strain cell, obtain the solution that content of trehalose is 34g/L.It is that 10000 ultra-filtration membrane removes macromolecular substance that relative molecular weight is held back in employing, with the anion-cation exchange resin desalination, separate trehalose and glucose.The trehalose component heating evaporation of collecting is concentrated, and spraying drying obtains trehalose powder 102g again.
Embodiment 2:
(1) the fine bacterium of fibrosis fiber is cultivated: get the fine bacterium preservation of bacteria strain of fibrosis fiber activation culture, 5% inoculum size by culture volume changes in the 5L fermentor tank that is equipped with fermention medium, liquid amount 3.5L, 30 ℃ of culture temperature, air flow quantity 4L/min, rotating speed 300r/min, incubation time 30h.
Identical among described fermention medium and the embodiment 1, component by quality grams contained in every liter of substratum is: glucose 20, yeast powder 5, peptone 5, corn steep liquor 10, MgSO
41, KH
2PO
41 and CaCO
37(g/L).
(2) infiltration fermentation: in the nutrient solution of cultivating behind the 30h, add 17.5g (NH
4)
2SO
4With 210g glucose, cultivate again 7h.
(3) isolation and purification of trehalose: fermented liquid is centrifugal or remove by filter strain cell, obtain the solution that content of trehalose is 32g/L.It is that 10000 ultra-filtration membrane removes macromolecular substance that relative molecular weight is held back in employing, with the anion-cation exchange resin desalination, separate trehalose and glucose.The trehalose component heating evaporation of collecting is concentrated concentrated with the trehalose component heating evaporation of collecting, and spraying drying obtains trehalose powder 96g again.
Embodiment 3:
(1) the fine bacterium of fibrosis fiber is cultivated: get the fine bacterium preservation of bacteria strain of fibrosis fiber activation culture, 5% inoculum size by culture volume changes in the 5L fermentor tank that is equipped with fermention medium, liquid amount 3.5L, 30 ℃ of culture temperature, air flow quantity 4L/min, rotating speed 300r/min, incubation time 30h.Identical among described fermention medium and the embodiment 1, component by quality grams contained in every liter of substratum is: glucose 20, yeast powder 5, peptone 5, corn steep liquor 10, MgSO
41, KH
2PO
41 and CaCO
37(g/L).
(2) infiltration fermentation: in the nutrient solution of cultivating behind the 30h, add 35g (NH
4)
2SO
4With 210g glucose, cultivate again 7h.
(3) isolation and purification of trehalose: the centrifugal or filtration sterilization strain cell with fermented liquid obtains the solution that content of trehalose is 31g/L.It is that 10000 ultra-filtration membrane removes macromolecular substance that relative molecular weight is held back in employing, with the anion-cation exchange resin desalination, separate trehalose and glucose.The trehalose component heating evaporation of collecting is concentrated, and spraying drying obtains trehalose powder 93g again.
The inventive method is because take glucose as substrate, utilize the fine bacteria strain of fibrosis fiber to cultivate after, the fine bacterium of fibrosis fiber is permeated fermentation take glucose as matrix, thereby can synthesize in a large number extracellular-trehalose.Adopt the inventive method to produce trehalose, production technique is intensive, facility investment is few and be easy to separation without carbohydrate by product, product, its outstanding feature be the trehalose transformation efficiency up to 60%, production cost is reduced, thereby lays a good foundation for the widespread use of trehalose.Adopt the prepared trehalose of the inventive method because it has broad application prospects in fields such as medicine, makeup and food to the good non-specific provide protection of biomacromolecule.
Owing to having taked in the inventive method in fermention medium, to add an amount of (NH
4)
2SO
4Improve the osmotic pressure of culture environment, coerce and cultivate the fine bacterium of fibrosis fiber so that stress produce more TreP system in the strain cell, simultaneously cytolemma has lost original selection perviousness, the trehalose that produces in the cell can free diffusing in the fermented liquid of extracellular.After measured, under the same fermentation condition, do not add (NH
4)
2SO
4The fine fermented liquid of fibrosis fiber in do not have trehalose to generate, and add an amount of (NH
4)
2SO
4The fine fermented liquid of fibrosis fiber in the trehalose fermentation unit can reach 34g/L.
Claims (3)
1. the fine bacteria strain of fibrosis fiber obtains from the separation of Hefei district elm withered tree branch, the fine bacterium of called after fibrosis fiber (
Cellulosimicrobium cellulans) S32, culture presevation number is CGMCC No.5841.
2. method of utilizing the fine bacterium of fibrosis fiber infiltration fermentative production trehalose is characterized in that: first with culture presevation number for the fine bacterium of fibrosis fiber of CGMCC No.5841 (
Cellulosimicrobium cellulans) the S32 bacterial strain carries out activation culture, change over to by 5% inoculum size of culture volume to be equipped with component and by quality grams contained in every liter of substratum to be: glucose 20, yeast powder 5, peptone 5, corn steep liquor 10, MgSO
41, KH
2PO
41 and CaCO
3In the fermentor tank of 7 fermention medium, liquid amount is 70% of fermentor tank volume, and culture temperature is 30 ℃, air flow quantity 4L/min, rotating speed 300r/min; After cultivating 30h, in nutrient solution, be added into (the NH that final concentration is 5-10g/L
4)
2SO
4With the glucose of 60g/L, cultivate again 7h; Then fermented liquid is centrifugal or remove by filter strain cell, obtain containing the solution of trehalose; It is that 10000 ultra-filtration membrane removes macromolecular substance that relative molecular weight is held back in employing, with the anion-cation exchange resin desalination, separate trehalose and glucose; The trehalose component heating evaporation of collecting is concentrated, and spraying drying namely obtains the trehalose finished product again.
3. utilize as claimed in claim 2 the method for the fine bacterium infiltration of fibrosis fiber fermentative production trehalose, be characterised in that behind described production strain culturing 30h, in nutrient solution, add (NH
4)
2SO
4To final concentration be 8g/L.
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| CN104232498B (en) * | 2013-06-19 | 2016-08-10 | 中国科学院大连化学物理研究所 | A kind of fine bacteria strain of fibrosis fiber and application thereof |
| CN103468610B (en) * | 2013-08-30 | 2015-03-04 | 河北出入境检验检疫局检验检疫技术中心 | Fiber fiber microbe and active protein, and application thereof |
| CN104830910B (en) * | 2015-05-25 | 2017-11-07 | 江苏师范大学 | The method for preparing microbial flocculant using the fine bacterium of fibrosis fiber |
| CN108841898A (en) * | 2018-07-16 | 2018-11-20 | 安徽民祯生物工程有限公司 | A kind of method of permeability yeast cells production trehalose |
| CN115247142B (en) * | 2022-08-16 | 2023-05-12 | 安徽农业大学 | Fiber-based fiber micro-bacteria and application thereof in straw field composting |
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| JP2005210977A (en) * | 2004-01-30 | 2005-08-11 | Hitachi Kiden Kogyo Ltd | Cell-wall digesting enzyme-productive bacterium |
| CN101381708A (en) * | 2007-09-04 | 2009-03-11 | 中国科学院大连化学物理研究所 | Fibrized cellulomonas cartae, hydrolase and application thereof in taxane conversion aspect |
| CN101691554A (en) * | 2009-06-30 | 2010-04-07 | 广西科学院 | Cellulosimicrobium cellulans capable of producing acrylic acid |
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Patent Citations (3)
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| JP2005210977A (en) * | 2004-01-30 | 2005-08-11 | Hitachi Kiden Kogyo Ltd | Cell-wall digesting enzyme-productive bacterium |
| CN101381708A (en) * | 2007-09-04 | 2009-03-11 | 中国科学院大连化学物理研究所 | Fibrized cellulomonas cartae, hydrolase and application thereof in taxane conversion aspect |
| CN101691554A (en) * | 2009-06-30 | 2010-04-07 | 广西科学院 | Cellulosimicrobium cellulans capable of producing acrylic acid |
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| Jian-min Song和Dong-zhi Wei.Production and characterization of cellulases and xylanases of cellulosimicrobiumcellulans grown in pretreated and extracted bagasse and minimal nutrient medium M9.《Biomass and Bioenergy》.2010,第34卷(第12期),1930-1934. |
| Production and characterization of cellulases and xylanases of cellulosimicrobiumcellulans grown in pretreated and extracted bagasse and minimal nutrient medium M9;Jian-min Song和Dong-zhi Wei;《Biomass and Bioenergy》;20101231;第34卷(第12期);1930-1934 * |
| 一株纤维化纤维微细菌的生物学特性及其对几种苯环类化合物的利用研究;陈燕红等;《微生物学通报》;20080720;第35卷(第7期);35(7)：102l一1027 * |
| 陈燕红等.一株纤维化纤维微细菌的生物学特性及其对几种苯环类化合物的利用研究.《微生物学通报》.2008,第35卷(第7期),35(7)：102l一1027. |
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