CN103667367A - Method for producing mannitol by taking brown sugar as carbon source through fermentation of leukonid - Google Patents
Method for producing mannitol by taking brown sugar as carbon source through fermentation of leukonid Download PDFInfo
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- CN103667367A CN103667367A CN201310537851.5A CN201310537851A CN103667367A CN 103667367 A CN103667367 A CN 103667367A CN 201310537851 A CN201310537851 A CN 201310537851A CN 103667367 A CN103667367 A CN 103667367A
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Abstract
The invention discloses a method for producing mannitol by taking brown sugar as a carbon source through fermentation of leukonid, and relates to a microbial fermentation process. The method comprises the following steps: dissolving 19-21g of brown sugar, 1.9-2.1g of yeast extract powder, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate and 2g of ammonium citrate in 1000ml of water to prepare a culture medium; filling 50ml of culture medium into a 250ml triangular flask, and sterilizing; adding an activated leukonid strain into the culture medium according to a bacterium inoculation amount of 1%, and culturing for 16-18 hours while shaking at 120 rpm at 30 DEG C; and centrifuging a culture solution at 10000 rpm for 10 minutes, removing precipitates (thalli), adding an equal amount of ethanol into the supernatant, centrifuging at 12000 rpm for 15 minutes, removing precipitates (glucan), and crystallizing the supernatant at 4 DEG C, thus obtaining the mannitol. Thus, the cost of the culture medium is effectively lowered.
Description
Technical field
The present invention relates to fermentative production N.F,USP MANNITOL technical field, specifically relate to take brown sugar and as carbon source, prepare substratum and apply this substratum and ferment and produce the method for N.F,USP MANNITOL by leukonid.
Background technology
N.F,USP MANNITOL is widely used in the industries such as medicine, food, chemical industry and electronics.Whole world N.F,USP MANNITOL consumption accounts for 11% of polyvalent alcohol total amount, is about every year 150000 tons.The U.S. is the maximum country of consumption of N.F,USP MANNITOL.Other is as all larger for the production of the consumption of chewing gum, buccal tablet, chew tablet in European countries.The current N.F,USP MANNITOL consumption of China is to be mainly directly used in to produce formula mannitol injection liquid, medicine supplementary additive and sugar-free type foodstuff additive etc.Because food service industry especially chewing gum increases fast to N.F,USP MANNITOL demand, N.F,USP MANNITOL market capacity expands rapidly.N.F,USP MANNITOL is in pharmaceutical industries large usage quantity, and current China only formula mannitol injection liquid will consume 4500 tons, N.F,USP MANNITOL every year, and within 2003, pharmaceutical industries consumption N.F,USP MANNITOL is 5600 tons.China is fat and diabetes are comparatively serious in recent years, and has more and more serious trend, so huge to sugar-free the nutritious sweeting agent N.F,USP MANNITOL of tool potential of demand, and China's food in 2004 and additive agent field thereof are to approximately 3000 tons of the demands of N.F,USP MANNITOL.
The method of producing at present N.F,USP MANNITOL mainly contains four kinds:
(1) marine alga extraction method: extract 1 ton of N.F,USP MANNITOL and approximately need 13～15 tons of dry sea-tangles, production process produces a large amount of waste water, and energy consumption is high, seriously polluted, and yield is low;
(2) shortening method: the shortening method that sucrose or glucose is raw material of take has raw materials enjoy stable sources, and product term is unrestricted, and cost is low, but its productive rate is lower, and has sorbyl alcohol association;
(3) enzyme transforming process: enzyme process hydrogenation need add expensive coenzyme in system, uneconomical;
(4) microbe fermentation method: it is more that occurring in nature can synthesize the microbe species of N.F,USP MANNITOL, has some bacterial strains to have the ability of producing N.F,USP MANNITOL in bacterium, yeast and mould.In the process of lactic acid bacteria tranformation N.F,USP MANNITOL, N.F,USP MANNITOL is primary product, simultaneously lactic acid producing, acetic acid, ethanol and carbonic acid gas, and do not produce the by products such as other polyvalent alcohol, thereby be easy to purifies and separates and refining, and mild condition, transformation efficiency are higher.Milk-acid bacteria is a kind of comparatively safe bacterial classification simultaneously, and the research of producing N.F,USP MANNITOL with milk-acid bacteria has obvious advantage.At present, microbe fermentation method is in the laboratory study stage.
Summary of the invention
The object of the invention is for the technological deficiency existing in prior art, and provide a kind of take that brown sugar is carbon source prepare substratum and leukonid is applied the method that N.F,USP MANNITOL is produced in this substratum fermentation; This substratum is according to the composition of brown sugar, to improve MRS substratum to make, and has reduced the cost of substratum, has shortened fermentation time, thereby has overcome the bottleneck problem of Production by Microorganism Fermentation N.F,USP MANNITOL.
The present invention solves this technical problem adopted technical scheme: the method that the brown sugar of take produces N.F,USP MANNITOL as the fermentation of carbon source leukonid, is characterized in that concrete steps are as follows:
(1) take brown sugar prepares substratum as carbon source:
(1.1) prepare raw material
Accurately taking 19～21 grams, brown sugar, yeast, to soak 1.9～2.1 grams, powder, 5 grams of sodium acetates, 2 grams of dipotassium hydrogen phosphates, 2 grams of ammonium citrates standby;
(1.2) allocate and dissolve
The beaker of placing dress 300ml water on magnetic stirring apparatus, the raw material that step (1.1) is taken joins in the water of beaker, after fully dissolving, adds 700ml water, makes substratum;
(1.3) packing and sterilizing
After the substratum 50ml that adds step (1.2) to prepare in 250ml triangular flask, seal, be placed in 121 ℃ of sterilizings of high-pressure sterilizing pot 20 minutes;
(2) N.F,USP MANNITOL is produced in leukonid fermentation
(2.1) activated spawn
Leukonid is connect to bacterium amount with 1% and join in the 10ml MRS substratum in 50ml triangular flask, shaking culture 12～16 hours; The Classification And Nomenclature of described leukonid is Leuconostoc mesenteroides, Latin name is Leuconostoc mesenteroides, preservation people and on December 31st, 2009 transfer to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) to carry out preservation, and depositary institution provides survival proof on September 18th, 2013 according to applying for of preservation people.
(2.2) connect bacterium
Bacterial classification step (2.1) having been activated at Bechtop connects bacterium amount with 1% and joins in the 50ml substratum in the 250ml triangular flask after step (1.3) sterilizing;
(2.3) cultivate
The triangular flask that step (2.2) is connect after bacterium is placed in shaking table, and the velocity fluctuation with 120 revs/min at 30 ℃ is cultivated 16～18 hours, obtains nutrient solution;
(2.4 separation and purification
The nutrient solution that step (2.3) is made through 10000 revs/min centrifugal 10 minutes, remove bacterial sediment and obtain supernatant A; The ethanol that adds equivalent in supernatant A, through 12000 revs/min centrifugal 15 minutes, remove dextran and precipitate to obtain supernatant liquor B, supernatant liquor B is made to N.F,USP MANNITOL 4 ℃ of crystallizations.
In above-mentioned steps (2), leukonid fermentation is produced in the technique of N.F,USP MANNITOL, and reagent used is provided by supplier, and composition is all known; It is known that involved equipment and process working method is those skilled in the art of the present technique.Need to illustrate, the Classification And Nomenclature of the leukonid that the present invention is used is Leuconostoc mesenteroides, and Latin name is Leuconostoc mesenteroides; Preservation date is on December 31st, 2009, and depositary institution's full name is China Committee for Culture Collection of Microorganisms's common micro-organisms center; Deposit number: CGMCC1.10327.
The invention has the beneficial effects as follows:
(1) compare with marine alga extraction method, shortening method, enzyme transforming process, special feature of the present invention is: by lactic-acid-bacterium, produce in specific manner this principal product of N.F,USP MANNITOL, so production process is eco-friendly, meets industrial development direction;
(2) compare with the technology of existing Production by Microorganism Fermentation N.F,USP MANNITOL, of the present invention take method that brown sugar is that the fermentation of carbon source leukonid produces N.F,USP MANNITOL with take sucrose or fructose, glucose and compare as the method that carbon source through fermentation lactic-acid-bacterium produces N.F,USP MANNITOL, effectively reduced the cost of substratum.This is that price is lower because brown sugar is the product of slightly carrying from sugarcane or beet; Sucrose is highly purified industrial chemicals, and it is high that the price of sucrose or fructose, glucose is wanted relatively; Than take the technique that sucrose is raw material leukonid fermentative production N.F,USP MANNITOL in prior art, raw material of the present invention has been removed consumption that peptone, yeast soak powder and has been reduced much in forming, so preparation cost just reduces a lot.
Biomaterial preservation explanation
Classification And Nomenclature: Leuconostoc mesenteroides
Latin name: Leuconostoc mesenteroides;
Preservation date: on December 31st, 2009
Depositary institution's full name: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Depositary institution is called for short: CGMCC
Deposit number: 1.10327
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1
The method that the brown sugar of take produces N.F,USP MANNITOL as the fermentation of carbon source leukonid, its step is as follows.
The first step, prepares raw material
Accurately take 19 grams, brown sugar, yeast soaks 1.9 grams, powder, 5 grams of sodium acetates, 2 grams of dipotassium hydrogen phosphates, 2 grams of ammonium citrates;
Second step, allocates and dissolves
The beaker of placing dress 300ml water on magnetic stirring apparatus, joins according to the raw material of preparing in the first step in the water of beaker, after fully dissolving, adds 700ml water.
The 3rd step, packing and sterilizing
After adding 50ml substratum in 250ml triangular flask, seal, be placed in 121 ℃ of sterilizings of high-pressure sterilizing pot 20 minutes.
The 4th step, activated spawn
In the bacterial classification of subzero 80 ℃ of Refrigerator stores, connect in the 10ml MRS substratum that bacterium amount joins 50ml triangular flask shaking culture 14 hours with 1%.
The 5th step, connects bacterium
At Bechtop, the bacterial classification having activated is connect in the 50ml substratum that bacterium amount joins 250ml triangular flask with 1%.
The 6th step, cultivates
The triangular flask that connects bacterium is placed in shaking table, in 30 ℃ 120 revs/min vibrations, supports 18 hours.
The 7th step, separation and purification
Nutrient solution through 10000 revs/min centrifugal 10 minutes, remove precipitation (thalline), toward the ethanol that adds equivalent in supernatant liquor, through 12000 revs/min centrifugal 15 minutes, remove and precipitate (dextran), supernatant liquor is obtained to N.F,USP MANNITOL 4 ℃ of crystallizations.
Embodiment 2
The method that the brown sugar of take produces N.F,USP MANNITOL as the fermentation of carbon source leukonid, its step is as follows.
The first step, prepares raw material
Accurately take 21 grams, brown sugar, yeast soaks 2.1 grams, powder, 5 grams of sodium acetates, 2 grams of dipotassium hydrogen phosphates, 2 grams of ammonium citrates.
Second step, allocates and dissolves
The beaker of placing dress 300ml water on magnetic stirring apparatus, joins according to the raw material of preparing in the first step in the water of beaker, after fully dissolving, adds 700ml water.
The 3rd step, packing and sterilizing
After adding 50ml substratum in 250ml triangular flask, seal, be placed in 121 ℃ of sterilizings of high-pressure sterilizing pot 20 minutes.
The 4th step, activated spawn
In the bacterial classification of subzero 80 ℃ of Refrigerator stores, connect in the 10ml MRS substratum that bacterium amount joins 50ml triangular flask shaking culture 13 hours with 1%.
The 5th step, connects bacterium
At Bechtop, the bacterial classification having activated is connect in the 50ml substratum that bacterium amount joins 250ml triangular flask with 1%.
The 6th step, cultivates
The triangular flask that connects bacterium is placed in shaking table, in 30 ℃ 120 revs/min vibrations, supports 18 hours.
The 7th step, separation and purification
Nutrient solution through 10000 revs/min centrifugal 10 minutes, remove precipitation (thalline), toward the ethanol that adds equivalent in supernatant liquor, through 12000 revs/min centrifugal 15 minutes, remove and precipitate (dextran), supernatant liquor is obtained to N.F,USP MANNITOL 4 ℃ of crystallizations.
Embodiment 3
The method that the brown sugar of take produces N.F,USP MANNITOL as the fermentation of carbon source leukonid, its step is as follows.
The first step, prepares raw material
Accurately take 20 grams, brown sugar, yeast soaks 2 grams, powder, 5 grams of sodium acetates, 2 grams of dipotassium hydrogen phosphates, 2 grams of ammonium citrates.
Second step, allocates and dissolves
The beaker of placing dress 300ml water on magnetic stirring apparatus, joins according to the raw material of preparing in the first step in the water of beaker, after fully dissolving, adds 700ml water.
The 3rd step, packing and sterilizing
After adding 50ml substratum in 250ml triangular flask, seal, be placed in 121 ℃ of sterilizings of high-pressure sterilizing pot 16 minutes.
The 4th step, activated spawn
In the bacterial classification of subzero 80 ℃ of Refrigerator stores, connect in the 10ml MRS substratum that bacterium amount joins 50ml triangular flask shaking culture 14 hours with 1%.
The 5th step, connects bacterium
At Bechtop, the bacterial classification having activated is connect in the 50ml substratum that bacterium amount joins 250ml triangular flask with 1%.
The 6th step, cultivates
The triangular flask that connects bacterium is placed in shaking table, in 30 ℃ 120 revs/min vibrations, supports 18 hours.
The 7th step, separation and purification
Nutrient solution through 10000 revs/min centrifugal 10 minutes, remove precipitation (thalline), toward the ethanol that adds equivalent in supernatant liquor, through 12000 revs/min centrifugal 15 minutes, remove and precipitate (dextran), supernatant liquor is obtained to N.F,USP MANNITOL 4 ℃ of crystallizations.
Embodiment 4
The method that the brown sugar of take produces N.F,USP MANNITOL as the fermentation of carbon source leukonid, its step is as follows.
The first step, prepares raw material
Accurately take 21 grams, brown sugar, yeast soaks 1.9 grams, powder, 5 grams of sodium acetates, 2 grams of dipotassium hydrogen phosphates, 2 grams of ammonium citrates, be dissolved in 1000ml water.
Second step, allocates and dissolves
The beaker of placing dress 300ml water on magnetic stirring apparatus, joins according to the raw material of preparing in the first step in the water of beaker, after fully dissolving, adds 700ml water.
The 3rd step, packing and sterilizing
After adding 50ml substratum in 250ml triangular flask, seal, be placed in 121 ℃ of sterilizings of high-pressure sterilizing pot 20 minutes.
The 4th step, activated spawn
In the bacterial classification of subzero 80 ℃ of Refrigerator stores, connect in the 10ml MRS substratum that bacterium amount joins 50ml triangular flask shaking culture 15 hours with 1%.
The 5th step, connects bacterium
At Bechtop, the bacterial classification having activated is connect in the 50ml substratum that bacterium amount joins 250ml triangular flask with 1%.
The 6th step, cultivates
The triangular flask that connects bacterium is placed in shaking table, in 30 ℃ 120 revs/min vibrations, supports 17 hours.
The 7th step, separation and purification
Nutrient solution through 10000 revs/min centrifugal 10 minutes, remove precipitation (thalline), toward the ethanol that adds equivalent in supernatant liquor, through 12000 revs/min centrifugal 15 minutes, remove and precipitate (dextran), supernatant liquor is obtained to N.F,USP MANNITOL 4 ℃ of crystallizations.
Magnesium sulfate and manganous sulfate are essential during the fermentation compositions, but in this patent, have used brown sugar just need not add magnesium sulfate and manganous sulfate, and this is because all contain magnesium ion and mn ion in brown sugar.Through measuring, the obtained mannitol content that ferments in embodiment 1-4 is as shown in table 1.
The mannitol content that table 1 embodiment 1-4 obtains
| Project | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 |
| Mannitol content | 7.18g/L | 7.21g/L | 7.30g/L | 7.25g/L |
In above-described embodiment 1～4, described brown sugar, yeast soak powder, sodium acetate, dipotassium hydrogen phosphate and ammonium citrate to be provided by supplier, and composition is all known; Bacterial classification used is that our company preserves; It is known that involved equipment and process working method is those skilled in the art of the present technique.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (1)
1. the method that the brown sugar of take produces N.F,USP MANNITOL as the fermentation of carbon source leukonid, is characterized in that concrete steps are as follows:
(1) take brown sugar prepares substratum as carbon source:
(1.1) prepare raw material
Accurately taking 19～21 grams, brown sugar, yeast, to soak 1.9～2.1 grams, powder, 5 grams of sodium acetates, 2 grams of dipotassium hydrogen phosphates, 2 grams of ammonium citrates standby;
(1.2) allocate and dissolve
The beaker of placing dress 300ml water on magnetic stirring apparatus, the raw material that step (1.1) is taken joins in the water of beaker, after fully dissolving, adds 700ml water, makes substratum;
(1.3) packing and sterilizing
After the substratum 50ml that adds step (1.2) to prepare in 250ml triangular flask, seal, be placed in 121 ℃ of sterilizings of high-pressure sterilizing pot 20 minutes;
(2) N.F,USP MANNITOL is produced in leukonid fermentation
(2.1) activated spawn
Leukonid is connect to bacterium amount with 1% and join in the 10ml MRS substratum in 50ml triangular flask, shaking culture 12～16 hours;
(2.2) connect bacterium
Bacterial classification step (2.1) having been activated at Bechtop connects bacterium amount with 1% and joins in the 50ml substratum in the 250ml triangular flask after step (1.3) sterilizing;
(2.3) cultivate
The triangular flask that step (2.2) is connect after bacterium is placed in shaking table, and the velocity fluctuation with 120 revs/min at 30 ℃ is cultivated 16～18 hours, obtains nutrient solution;
(2.4) separation and purification
The nutrient solution that step (2.3) is made through 10000 revs/min centrifugal 10 minutes, remove bacterial sediment and obtain supernatant A; The ethanol that adds equivalent in supernatant A, through 12000 revs/min centrifugal 15 minutes, remove dextran and precipitate to obtain supernatant liquor B, supernatant liquor B is made to N.F,USP MANNITOL 4 ℃ of crystallizations.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017831A (en) * | 2014-06-10 | 2014-09-03 | 河北工业大学 | Method for producing mannitol by taking sugarcane juice as raw material through leuconostoc mesenteroides fermentation |
| CN107881140A (en) * | 2017-11-22 | 2018-04-06 | 河北工业大学 | The Leuconostoc mesenteroides mutant strain of one plant height production mannitol and its application process |
| CN108285878A (en) * | 2017-11-24 | 2018-07-17 | 延边大学 | The method that the bright string strain bacterium HM1 bacterial strains of lemon of high yield mannitol and fermentation prepare mannitol |
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| CN101736058A (en) * | 2008-11-26 | 2010-06-16 | 中国科学院大连化学物理研究所 | Method for producing mannitol by taking jerusalem artichoke as raw materials through biotransformation |
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2013
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101736058A (en) * | 2008-11-26 | 2010-06-16 | 中国科学院大连化学物理研究所 | Method for producing mannitol by taking jerusalem artichoke as raw materials through biotransformation |
Non-Patent Citations (3)
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| 张晓慧等: "乙醇沉淀法测定葡聚糖浓度方法探讨", 《广州化工》, vol. 39, no. 20, 23 October 2011 (2011-10-23), pages 88 - 91 * |
| 金红星等: "以蔗糖为原料明串珠菌发酵生产甘露醇", 《食品与发酵工业》, vol. 37, no. 2, 28 February 2011 (2011-02-28) * |
| 金红星等: "明串珠菌发酵产甘露醇的研究进展", 《中国酿造》, vol. 31, no. 10, 15 October 2012 (2012-10-15), pages 4 - 6 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017831A (en) * | 2014-06-10 | 2014-09-03 | 河北工业大学 | Method for producing mannitol by taking sugarcane juice as raw material through leuconostoc mesenteroides fermentation |
| CN107881140A (en) * | 2017-11-22 | 2018-04-06 | 河北工业大学 | The Leuconostoc mesenteroides mutant strain of one plant height production mannitol and its application process |
| CN107881140B (en) * | 2017-11-22 | 2020-03-27 | 河北工业大学 | Leuconostoc mesenteroides mutant strain capable of producing mannitol in high yield and application method thereof |
| CN108285878A (en) * | 2017-11-24 | 2018-07-17 | 延边大学 | The method that the bright string strain bacterium HM1 bacterial strains of lemon of high yield mannitol and fermentation prepare mannitol |
| CN108285878B (en) * | 2017-11-24 | 2022-10-28 | 延边大学 | Citrinia citrulline HM1 strain with high mannitol yield and method for preparing mannitol by fermentation |
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Application publication date: 20140326 |