CN103224960A - Method for producing erythritol by continuously fermenting and extracting Torula.sp strain - Google Patents
Method for producing erythritol by continuously fermenting and extracting Torula.sp strain Download PDFInfo
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Abstract
The invention relates to a method for producing erythritol by continuously fermenting and extracting a Torula.sp strain, and particularly relates to a Torula.sp B84512 strain belonging to the technical field of biology. According to the method for producing erythritol through continuous fermentation and extraction by taking the Torula.sp B84512 as an original strain, the fermentation period of erythritol is prolonged by filtering and recovering a filtrate through a filter membrane and adding glucose, the substrate conversion rate and the product yield are increased, the extraction process is simplified, the product quality is stable, and the production cost is low; and the process can be further popularized to realize industrial large-scale production.
Description
Technical field
The present invention relates to a kind of continuously fermenting and extract the method for producing erythritol, be specifically related to strain torula (Torula.sp) B84512 bacterial strain, belong to biological technical field with the torula bacterial strain.
Background technology
Along with growth in the living standard, " three height " crowd's quantity is exponential growth, and low sugar, less salt, food low in calories become people's first-selection now.Erythritol since its have that heat is low, mouthfeel is good, agent of low hygroscopicity, to diabetics's safety, very stable and can not cause advantage such as gastrointestinal upset to hot acid, become most popular functional food additives of 21 century.
The production method of erythritol mainly contains chemical synthesis and microbe fermentation method.Chemical synthesis mainly is starch or the Mierocrystalline cellulose of crossing through periodate oxidation with acid-alkali treatment, and hydrogenating reduction obtains under the High Temperature High Pressure, but by product is more in the product, and yield is low, and product is difficult to be separated, and is difficult to realize industrialization.And, generate glucose through enzyme liberating with wheat or corn and other starches raw material, by osmophilic yeast or other bacterial strains, condensing crystal separates dry and gets behind the high density bottom fermentation.Compare with chemical synthesis, this method production process gentle control, low in the pollution of the environment, product safety easily have more the production advantage.
Summary of the invention
The object of the present invention is to provide a kind of torula (Torula.sp) B84512 to continuously ferment to extract the method for erythritol, with the fermentation efficiency that improves erythritol and inversion rate of glucose, simplification extraction process.
According to technical scheme provided by the invention, a kind of continuously fermenting with the torula bacterial strain extracted the method for producing erythritol, and step is:
(1) preparation of cell liquid culture: torula (Torula.sp) B84512 bacterial strain one ring on the picking YPD solid medium, be seeded in the 500 mL Erlenmeyer flasks that the 50mL seed culture medium is housed, under 25-35 ℃, place on the shaking table and cultivate 30-48h to logarithmic phase, promptly make the cell liquid culture of torula (Torula.sp) B84512 bacterial strain with the rotating speed of 150-200 r/min;
The YPD solid medium is as follows: yeast powder 10 g/L; Peptone 20 g/L; Glucose 20 g/L; 7.0,121 ℃ of high pressure steam sterilization 20min of pH; Add 20 g/L agar powders on the basis of solid YPD substratum liquid medium within;
Seed culture medium: glucose 200 g/L, yeast powder 10 g/L, urea 1 g/L, CuSO
45H
2O 0.01 g/L, MnSO
4H
2O 0. 01 g/L, the PH nature, all the other are pure water, 115 ℃ of high pressure steam sterilization 15min;
(2) prepare fermention medium, medium component is as follows by weight percentage: glucose 20%-30%, yeast powder 0.5%-1%, CuSO
45H
2O 0.0005%-0.001%, MnSO
4H
2O 0.0005%-0.001%, all the other are pure water, the pH nature, 110-120 ℃, 0.1-0.2MPa steam sterilizing 10-20min, stand-by;
(3) fermentation unit connects: the feed supplement bottle links to each other with the 5L fermentor tank, and the 5L fermentor tank connects microfiltration membrane, nanofiltration membrane successively, and nanofiltration membrane is connected with the erythritol storage tank;
(4) fermentation culture conditions: with the cell liquid culture of torula (Torula.sp) the B84512 bacterial strain of step (1) preparation with weight ratio, be that the inoculum size that the weight of cell liquid culture accounts for fermention medium 4%-8% is seeded in the 5L fermentor tank that sterilized 2-3L fermention medium is housed, in temperature is 28-30 ℃, rotating speed is 300-600r/min, and air flow is that the condition bottom fermentation of 0.5-1vvm is cultivated 60-84h;
When 5L fermentor tank mesoerythrit concentration reached 60g/L, by the micro-filtrate membrane filtration fermentation system, filtrate continued to filter through nanofiltration membrane, and the macromole liquid of being held back by microfiltration membrane is back to fermentation system with the flow velocity of 0.5-1mL/min and utilizes; Behind the nanofiltration membrane selective filter, promptly obtain the fermented liquid of erythritol, be stored in the erythritol storage tank, the liquid of being held back by nanofiltration membrane is back to fermentation system with the flow velocity of 0.5-1mL/min; Velocity flow with 0.4-0.6mL/min adds the glucose that mass concentration is 15%-25% simultaneously, and for fermentation system provides competent substrate, and it is constant to keep the fermentation cumulative volume, and continuous fermentation culture can be continuously fermented and be extracted the fermented liquid that obtains erythritol.
Described glucose substrate conversion efficiency reaches more than 45%, and the broth extraction yield of erythritol reaches 97%.
The pore size filter of described millipore filtration is 0.1-1 μ m; The pore size filter of hole filter membrane received is 0.1-100nm.
Erythritol assay: adopt the HPLC method to detect erythritol.Chromatographic column is the Sugar-D chromatographic column; Moving phase is acetonitrile: water=75:25; Flow velocity is 1ml/min, and detector is a differential refraction detector.The erythritol standard substance come from sigma.
The biological material specimens preservation: a strain torula (
Torula.sp) the B84512 bacterial strain, deposit number is CGMCC7582, preservation date is on May 12nd, 2013, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address.
The present invention has following advantage: of the present invention with torula (
Torula.sp) B84512 is that starting strain continuously ferments and extract to produce the method for erythritol, prolonged the fermentation period of erythritol, improved the transformation efficiency and the product yield of substrate, simplified extraction process, constant product quality, production cost is low, and this technology can further extend to large-scale industrialization production.
Description of drawings
Fig. 1 fermentation system connection diagram of the present invention.
Description of reference numerals: 1. feed supplement bottle: in adorn 20% glucose solution; 2. 5L fermentor tank; 3. microfiltration membrane: be used to filter yeast, make it be back to fermentor tank; 4. nanofiltration membrane: be used for the selective filter erythritol; 5. erythritol storage tank.
Arrow is a flow direction.
Embodiment
The 5L fermentor tank of embodiment 1 torula (Torula.sp) the B84512 system of continuously fermenting prepares erythritol
1, culture medium prescription
(1) seed culture medium: glucose 200 g/L, yeast powder 10 g/L, urea 1 g/L, CuSO4 5,H20 0.01 g/L, MnSO4 H20 0. 01 g/L, PH nature, 115 ℃ of high pressure steam sterilization 15min.
(2) fermention medium: glucose 300 g/L, yeast powder 10 g/L, urea 1 g/L, CuSO4 5,H20 0.01 g/L, MnSO4 H20 0. 01 g/L, PH nature, 115 ℃ of high pressure steam sterilization 15min.
2, cultural method
(1) seed culture method
Torula (Torula.sp) B84512 bacterial strain one ring on the picking YPD solid medium, be seeded in the 500 mL Erlenmeyer flasks that 50 mL seed culture mediums are housed, under 30 ℃, place on the shaking table and cultivate 30-48 h to logarithmic phase, promptly make the seed liquid culture of torula (Torula.sp) B84512 bacterial strain with the rotating speed of 150-200 r/min.
(2) the 5L fermentor tank extracting method that continuously ferments
With the seed liquid culture of above-mentioned torula (Torula.sp) B84512 bacterial strain with 4-8%(v/v) inoculum size be seeded in the 5L fermentor tank that sterilized 3L fermention medium is housed, under 30 ℃ of conditions, rotating speed is 300-600r/min, air flow is 1vvm, be set at 0.5vvm behind the 50h, about 70h secondary fermentation liquid mesoerythrit output reaches 60g/L, and glucose concn is reduced to about 50g/L, open feed supplement this moment, adds 20% glucose with the speed of 0.5 ml/min; Open micro-filtration, nanofiltration device simultaneously, with the speed purifying erythritol mother liquor of 0.5mL/min.Ferment to 240h, bacterial strain is active to descend, and erythritol drops to below the 0.5g/L/h than synthesis rate, stops fermentation, gets the filtering fermentation liquor degerming, and the filtered solution that obtains through nanofiltration mixes with the erythritol mother liquor.Consumption of glucose 1920g in this fermenting process as calculated, the fermentation substrate transformation efficiency reaches more than 45%, obtains the about 7L of erythritol mother liquor, and concentration is 120g/L, amounts to 840g, and product yield reaches 97%.
Embodiment 2
A kind of continuously fermenting with the torula bacterial strain extracted the method for producing erythritol, and step is:
(1) preparation of cell liquid culture: torula (Torula.sp) B84512 bacterial strain one ring on the picking YPD solid medium, be seeded in the 500 mL Erlenmeyer flasks that the 50mL seed culture medium is housed, under 35 ℃, place on the shaking table and cultivate 36h to logarithmic phase, promptly make the cell liquid culture of torula (Torula.sp) B84512 bacterial strain with the rotating speed of 200 r/min;
The YPD solid medium is as follows: yeast powder 10 g/L; Peptone 20 g/L; Glucose 20 g/L; 7.0,121 ℃ of high pressure steam sterilization 20min of pH; Add 20 g/L agar powders on the basis of solid YPD substratum liquid medium within;
Seed culture medium: glucose 200 g/L, yeast powder 10 g/L, urea 1 g/L, CuSO
45H
2O 0.01 g/L, MnSO
4H
2O 0. 01 g/L, the PH nature, all the other are pure water, 115 ℃ of high pressure steam sterilization 15min;
(2) prepare fermention medium, medium component is as follows by weight percentage: glucose 30%, yeast powder 1%, CuSO
45H
2O 0.001%, MnSO
4H
2O 0.001%, and all the other are pure water, the pH nature, and 120 ℃, 0.2MPa steam sterilizing 20min, stand-by;
(3) fermentation unit connects: as shown in Figure 1, feed supplement bottle 1 links to each other with 5L fermentor tank 2, and 5L fermentor tank 2 connects microfiltration membrane 3, nanofiltration membrane 4 successively, and nanofiltration membrane 4 is connected with erythritol storage tank 5;
(4) fermentation culture conditions: with the cell liquid culture of torula (Torula.sp) the B84512 bacterial strain of step 1 preparation with weight ratio, be that the inoculum size that the weight of cell liquid culture accounts for fermention medium 8% is seeded in the 5L fermentor tank 2 that sterilized 2L fermention medium is housed, in temperature is 30 ℃, rotating speed is 300r/min, and air flow is that the condition bottom fermentation of 1vvm is cultivated 60-84h;
When 5L fermentor tank 2 mesoerythrit concentration reach 60g/L, filter fermentation systems by microfiltration membrane 3, filtrate continues to filter through nanofiltration membrane 4, and the macromole liquid of being held back by microfiltration membrane 3 is back to fermentation system with the flow velocity of 1mL/min and utilizes; Behind nanofiltration membrane 4 selective filters, promptly obtain the fermented liquid of erythritol, be stored in the erythritol storage tank 5, the liquid of being held back by nanofiltration membrane 4 is back to fermentation system with the flow velocity of 0.5mL/min; Simultaneously adding mass concentration with the velocity flow of 0.6mL/min is 15% glucose, and for fermentation system provides competent substrate, and it is constant to keep the fermentation cumulative volume, and constantly fermentation culture can be continuously fermented and extracted the fermented liquid that obtains erythritol.
Described glucose substrate conversion efficiency reaches more than 45%, and the broth extraction yield of erythritol reaches 97%.
The pore size filter of described millipore filtration is 0.1-1 μ m; The pore size filter of hole filter membrane received is 0.1-100nm.
Embodiment 3
A kind of continuously fermenting with the torula bacterial strain extracted the method for producing erythritol, and step is:
(1) preparation of cell liquid culture: torula (Torula.sp) B84512 bacterial strain one ring on the picking YPD solid medium, be seeded in the 500 mL Erlenmeyer flasks that the 50mL seed culture medium is housed, under 30 ℃, place on the shaking table and cultivate 42h to logarithmic phase, promptly make the cell liquid culture of torula (Torula.sp) B84512 bacterial strain with the rotating speed of 180 r/min;
The YPD solid medium is as follows: yeast powder 10 g/L; Peptone 20 g/L; Glucose 20 g/L; 7.0,121 ℃ of high pressure steam sterilization 20min of pH; Add 20 g/L agar powders on the basis of solid YPD substratum liquid medium within;
Seed culture medium: glucose 200 g/L, yeast powder 10 g/L, urea 1 g/L, CuSO
45H
2O 0.01 g/L, MnSO
4H
2O 0. 01 g/L, the PH nature, all the other are pure water, 115 ℃ of high pressure steam sterilization 15min;
(2) prepare fermention medium, medium component is as follows by weight percentage: glucose 25%, yeast powder 0.8%, CuSO
45H
2O 0.0005%, MnSO
4H
2O 0.001%, and all the other are pure water, the pH nature, and 115 ℃, 0.1-0.2MPa steam sterilizing 15min, stand-by;
(3) fermentation unit connects: as shown in Figure 1, feed supplement bottle 1 links to each other with 5L fermentor tank 2, and 5L fermentor tank 2 connects microfiltration membrane 3, nanofiltration membrane 4 successively, and nanofiltration membrane 4 is connected with erythritol storage tank 5;
(4) fermentation culture conditions: with the cell liquid culture of torula (Torula.sp) the B84512 bacterial strain of step (1) preparation with weight ratio, be that the inoculum size that the weight of cell liquid culture accounts for fermention medium 6% is seeded in the 5L fermentor tank 2 that sterilized 2-3L fermention medium is housed, in temperature is 28-30 ℃, rotating speed is 500r/min, and air flow is that the condition bottom fermentation of 1vvm is cultivated 84h;
When 5L fermentor tank 2 mesoerythrit concentration reach 60g/L, filter fermentation systems by microfiltration membrane 3, filtrate continues to filter through nanofiltration membrane 4, and the macromole liquid of being held back by microfiltration membrane 3 is back to fermentation system with the flow velocity of 0.5mL/min and utilizes; Behind nanofiltration membrane 4 selective filters, promptly obtain the fermented liquid of erythritol, be stored in the erythritol storage tank 5, the liquid of being held back by nanofiltration membrane 4 is back to fermentation system with the flow velocity of 0.8mL/min; Simultaneously adding mass concentration with the velocity flow of 0.5mL/min is 20% glucose, and for fermentation system provides competent substrate, and it is constant to keep the fermentation cumulative volume, and constantly fermentation culture can be continuously fermented and extracted the fermented liquid that obtains erythritol.
Described glucose substrate conversion efficiency reaches more than 45%, and the broth extraction yield of erythritol reaches 97%.
The pore size filter of described millipore filtration is 0.1-1 μ m; The pore size filter of hole filter membrane received is 0.1-100nm.
Claims (3)
1. one kind continuously ferments with the torula bacterial strain and extract to produce the method for erythritol, it is characterized in that:
(1) preparation of cell liquid culture: torula (Torula.sp) B84512 bacterial strain one ring on the picking YPD solid medium, be seeded in the 500 mL Erlenmeyer flasks that the 50mL seed culture medium is housed, under 25-35 ℃, place on the shaking table and cultivate 30-48h to logarithmic phase, promptly make the cell liquid culture of torula (Torula.sp) B84512 bacterial strain with the rotating speed of 150-200 r/min;
The YPD solid medium is as follows: yeast powder 10 g/L; Peptone 20 g/L; Glucose 20 g/L; 7.0,121 ℃ of high pressure steam sterilization 20min of pH; Add 20 g/L agar powders on the basis of solid YPD substratum liquid medium within;
Seed culture medium: glucose 200 g/L, yeast powder 10 g/L, urea 1 g/L, CuSO
45H
2O 0.01 g/L, MnSO
4H
2O 0. 01 g/L, the PH nature, all the other are pure water, 115 ℃ of high pressure steam sterilization 15min;
(2) prepare fermention medium, medium component is as follows by weight percentage: glucose 20%-30%, yeast powder 0.5%-1%, CuSO
45H
2O 0.0005%-0.001%, MnSO
4H
2O 0.0005%-0.001%, all the other are pure water, the pH nature, 110-120 ℃, 0.1-0.2MPa steam sterilizing 10-20min, stand-by;
(3) fermentation unit connects: feed supplement bottle (1) links to each other with 5L fermentor tank (2), and 5L fermentor tank (2) connects microfiltration membrane (3), nanofiltration membrane (4) successively, and nanofiltration membrane (4) is connected with erythritol storage tank (5);
(4) fermentation culture conditions: with the cell liquid culture of torula (Torula.sp) the B84512 bacterial strain of step (1) preparation with weight ratio, be that the inoculum size that the weight of cell liquid culture accounts for fermention medium 4%-8% is seeded in the 5L fermentor tank (2) that sterilized 2-3L fermention medium is housed, in temperature is 28-30 ℃, rotating speed is 300-600r/min, and air flow is that the condition bottom fermentation of 0.5-1vvm is cultivated 60-84h;
When 5L fermentor tank (2) mesoerythrit concentration reaches 60g/L, filter fermentation system by microfiltration membrane (3), filtrate continues to filter through nanofiltration membrane (4), and the macromole liquid of being held back by microfiltration membrane (3) is back to fermentation system with the flow velocity of 0.5-1mL/min and utilizes; Behind nanofiltration membrane (4) selective filter, promptly obtain the fermented liquid of erythritol, be stored in the erythritol storage tank (5), the liquid of being held back by nanofiltration membrane (4) is back to fermentation system with the flow velocity of 0.5-1mL/min; Velocity flow with 0.4-0.6mL/min adds the glucose that mass concentration is 15%-25% simultaneously, and for fermentation system provides competent substrate, and it is constant to keep the fermentation cumulative volume, and continuous fermentation culture can be continuously fermented and be extracted the fermented liquid that obtains erythritol.
2. continuously ferment with the torula bacterial strain according to claim 1 and extract the method for producing erythritol, it is characterized in that: described glucose substrate conversion efficiency reaches more than 45%, and the broth extraction yield of erythritol reaches 97%.
3. continuously ferment with the torula bacterial strain according to claim 1 and extract the method for producing erythritol, it is characterized in that: the pore size filter of described millipore filtration is 0.1-1 μ m; The pore size filter of hole filter membrane received is 0.1-100nm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106497988A (en) * | 2016-09-27 | 2017-03-15 | 天津科技大学 | A kind of method that Lactobacillus buchneri microbe conversion separation coupling prepares Mannitol |
| CN107446813A (en) * | 2017-08-22 | 2017-12-08 | 江西省科学院微生物研究所 | A kind of 2 PE of continuous conversion production 2 PE of device and its continuous conversion production method |
| CN114075580A (en) * | 2020-11-06 | 2022-02-22 | 山东福洋生物科技股份有限公司 | Method for improving erythritol product concentration and production conversion rate |
| CN115044489A (en) * | 2022-06-13 | 2022-09-13 | 南京同凯兆业生物技术有限责任公司 | Method for culturing saccharomyces cerevisiae by using continuous fermentation and separation integrated technology |
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| CN102964213A (en) * | 2012-11-23 | 2013-03-13 | 赛普特环保技术(厦门)有限公司 | Method for recovering erythritol from erythritol crystallization mother liquor |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106497988A (en) * | 2016-09-27 | 2017-03-15 | 天津科技大学 | A kind of method that Lactobacillus buchneri microbe conversion separation coupling prepares Mannitol |
| CN106497988B (en) * | 2016-09-27 | 2019-12-13 | 天津科技大学 | method for preparing mannitol by lactobacillus buchneri fermentation-transformation-separation coupling |
| CN107446813A (en) * | 2017-08-22 | 2017-12-08 | 江西省科学院微生物研究所 | A kind of 2 PE of continuous conversion production 2 PE of device and its continuous conversion production method |
| CN107446813B (en) * | 2017-08-22 | 2023-10-31 | 江西省科学院微生物研究所 | Device for producing 2-PE through continuous conversion and method for producing 2-PE through continuous conversion |
| CN114075580A (en) * | 2020-11-06 | 2022-02-22 | 山东福洋生物科技股份有限公司 | Method for improving erythritol product concentration and production conversion rate |
| CN114075580B (en) * | 2020-11-06 | 2023-03-17 | 山东福洋生物科技股份有限公司 | Method for improving erythritol product concentration and production conversion rate |
| CN115044489A (en) * | 2022-06-13 | 2022-09-13 | 南京同凯兆业生物技术有限责任公司 | Method for culturing saccharomyces cerevisiae by using continuous fermentation and separation integrated technology |
| CN115044489B (en) * | 2022-06-13 | 2024-02-20 | 南京同凯兆业生物技术有限责任公司 | Method for culturing saccharomyces cerevisiae by utilizing continuous fermentation and separation integrated technology |
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