CN104017831B - The method producing mannitol for the fermentation of raw material Leuconostoc mesenteroides with Caulis Sacchari sinensis juice - Google Patents
The method producing mannitol for the fermentation of raw material Leuconostoc mesenteroides with Caulis Sacchari sinensis juice Download PDFInfo
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- CN104017831B CN104017831B CN201410254455.6A CN201410254455A CN104017831B CN 104017831 B CN104017831 B CN 104017831B CN 201410254455 A CN201410254455 A CN 201410254455A CN 104017831 B CN104017831 B CN 104017831B
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- sacchari sinensis
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Abstract
The method that the present invention produces mannitol with Caulis Sacchari sinensis juice for the fermentation of raw material Leuconostoc mesenteroides, relate to the preparation method of mannitol, by the Caulis Sacchari sinensis juice that squeezes by centrifugation with filtration, add brown sugar and obtain Caulis Sacchari sinensis juice brown sugar filtrate, the more aseptic Caulis Sacchari sinensis juice brown sugar filtrate prepared through cold sterilization is stand-by as culture medium；With 1%, the Leuconostoc mesenteroides strain activated is connect bacterium amount join in aseptic Caulis Sacchari sinensis juice brown sugar filtrate culture medium, then prepare fermentation liquid and isolated and purified prepared mannitol through cultivating.The inventive method reduces the cost of culture medium, thus overcomes the bottleneck problem of Production by Microorganism Fermentation mannitol.
Description
Technical field
Technical scheme relates to the preparation method of mannitol, the method specifically producing mannitol with Caulis Sacchari sinensis juice for the fermentation of raw material Leuconostoc mesenteroides.
Background technology
Mannitol is widely used in the industries such as medicine, food, chemical industry and electronics.Whole world mannitol consumption figure accounts for the 11% of polyhydric alcohol total amount, annual about 150,000 tons.The U.S. is the maximum country of consumption of mannitol.Other is such as European countries, the biggest for producing the consumption of chewing gum, buccal tablet and chew tablet.China's current mannitol consumption is mainly directly used in production formula mannitol injection liquid, medicine auxiliary additive and Sugarless type food additive etc..Owing to mannitol demand is quickly increased by food service industry especially chewing gum, mannitol market capacity expands rapidly.Mannitol is in pharmaceuticals industry large usage quantity, and current China only formula mannitol injection liquid will consume 4500 tons of mannitol, pharmaceuticals industry 5600 tons of mannitol of consumption in 2003 every year.China is fat and diabetes are more serious in recent years, and has increasingly severe trend, therefore to sugar-free and to have nutritious sweeting agent mannitol potential of demand huge, China's food in 2004 and the additive agent field demand about 3000 tons to mannitol thereof.
Prior art produces the method for mannitol mainly four kinds:
(1) Thallus Laminariae (Thallus Eckloniae) extraction method: extracting 1 ton of mannitol and about need the dry Thallus Laminariae (Thallus Eckloniae) of 13-15 ton, production process produces a large amount of waste water, and energy consumption is high, and seriously polluted, yield is low；
(2) catalytic hydrogenation method: nickel (Ni) the catalysis high-temperature and high-pressure hydrogenation as raw material has raw material sources stably with sucrose or glucose, and product term is unrestricted, low cost, but its productivity is relatively low, and the sorbitol of association is not readily separated；
(3) enzyme transforming process: enzyme process hydrogenation needs to add expensive coenzyme (NADH) in system, uneconomical；
(4) microbe fermentation method: the microbe species that can synthesize mannitol in nature is more, has some bacterial strains to have the ability producing mannitol in antibacterial, yeast and mycete.Wherein, with lactic acid bacteria microbe conversion mannitol, with mannitol as primary product, lactic acid producing, acetic acid, ethanol and carbon dioxide simultaneously, and do not produce the by-products such as other polyhydric alcohol, thus gained mannitol product is easily isolated purification and refines, and process conditions are gentle, and conversion ratio is higher.Lactic acid bacteria is the GRAS bacterium of U.S. FDA accreditation simultaneously, is a kind of comparatively safe strain, thus produces the research and development of mannitol with lactic acid bacteria and have obvious advantage.Lactic acid bacteria is divided into again homofermentative lactic bacteria and heterofermentative lactic bacteria, use homofermentative lactic bacteria through intermediate compounds such as G6P, fructose-1, 6-diphosphate, 1-phosphomamlose alcohol, glucose can be converted into mannitol, and yield is less than heterofermentative lactic bacteria；Use the leukonid in heterofermentative lactic bacteria then the fructose moiety in fructose or sucrose can be converted into mannitol, such as, disclosed method and strain leukonid mutant strain and the construction method and methods for using them thereof of CN 201410065372.2 disclosure producing mannitol with brown sugar for the fermentation of carbon source leukonid of CN201310537851.5, these methods not only yield is higher than homofermentative lactic bacteria, and because the chromogene group size of leukonid only has about 2Mb, therefore fermentation period is short.But CN201310537851.5 and CN
201410065372.2 yet suffer from the defect that culture medium cost is higher, and these defects become the bottleneck problem of Production by Microorganism Fermentation mannitol.
Summary of the invention
The technical problem to be solved is: provide the method producing mannitol for the fermentation of raw material Leuconostoc mesenteroides with Caulis Sacchari sinensis juice, it is a kind of preparation culture medium with Caulis Sacchari sinensis juice as raw material and method that Leuconostoc mesenteroides applies the fermentation of this culture medium to produce mannitol, reduce the cost of culture medium, thus overcome the bottleneck problem of Production by Microorganism Fermentation mannitol.
The present invention solves this technical problem and be the technical scheme is that the method producing mannitol for the fermentation of raw material Leuconostoc mesenteroides with Caulis Sacchari sinensis juice, and step is as follows:
The first step, prepares culture medium with Caulis Sacchari sinensis juice-brown sugar for raw material:
(1.1) apparatus is prepared:
The filter A kraft paper accompanying 0.2 μm filter membrane is packaged, then puts into high-pressure sterilizing pot together with the 100mL triangular flask of sealing, in 121 DEG C of sterilizings 20 minutes, get out the filter A accompanying 0.2 μm filter membrane and the 100mL triangular flask of sterilizing of sterilizing；
(1.2) Caulis Sacchari sinensis juice is squeezed:
Weighing 3350 grams of Caulis Sacchari sinensis, peeling rear cutout into strips, is put into and is squeezed Caulis Sacchari sinensis juice in fruit juice mixer；
(1.3) the centrifugal and filtration of Caulis Sacchari sinensis juice:
Take the prepared centrifuge that 1050mL juice from sugar cane rotating speed is 13000 revs/min of previous step (1.2) 30 minutes, obtain sugar cane juice supernatant A 1010mL；Again sugar cane juice supernatant A is filtered with the filter B accompanying 0.2 μm filter membrane and obtain Caulis Sacchari sinensis juice liquor B 1005mL；
(1.4) interpolation brown sugar acquisition Caulis Sacchari sinensis juice-brown sugar filtrate:
In above-mentioned Caulis Sacchari sinensis juice liquor B 1005mL, add 2.5 grams of brown sugar and stirring makes it be completely dissolved, thus obtain Caulis Sacchari sinensis juice-brown sugar liquor C；
(1.5) cold sterilization:
The filter A accompanying 0.2 μm filter membrane of Caulis Sacchari sinensis juice-brown sugar liquor C above-mentioned steps (1.1) ready sterilizing above-mentioned steps (1.4) prepared near the alcohol burner of superclean bench filters, prepare aseptic Caulis Sacchari sinensis juice-brown sugar filtrate D 1000mL, the 100mL triangular flask of above-mentioned steps (1.1) ready sterilizing adds the aseptic Caulis Sacchari sinensis juice-brown sugar filtrate D just prepared
Sealing after 40mL, the culture medium as next step is stand-by；
Second step, ferments with Leuconostoc mesenteroides and produces mannitol:
(2.1) activation Leuconostoc mesenteroides strain:
Leuconostoc mesenteroides is connect in the 10mL MRS culture medium that bacterium amount joins in the triangular flask having been placed on 50mL with 1%, shaken cultivation 12～16 hours；
(2.2) Leuconostoc mesenteroides is connect:
Leuconostoc mesenteroides strain above-mentioned steps (2.1) activated near the alcohol burner of superclean bench connects bacterium amount with 1% and joins above-mentioned steps (1.5) in the Caulis Sacchari sinensis juice being placed in 100mL triangular flask-brown sugar filtrate D 40mL that cold sterilization prepares；
(2.3) prepared fermentation liquid is cultivated:
Triangular flask after above-mentioned steps (2.2) is connect Leuconostoc mesenteroides is placed in shaking table, and at 30 DEG C, the velocity fluctuation with 120 revs/min is cultivated 24 hours, prepares fermentation liquid 40mL；
(2.4) isolated and purified:
Above-mentioned steps (2.3) is cultivated the prepared centrifuge that fermentation liquid 40mL rotating speed is 10000 revs/min 10 minutes, removes bacterial sediment and obtain fermented supernatant fluid A 37mL；In fermented supernatant fluid A 37mL, add the ethanol of equivalent volumes, with the centrifuge that rotating speed is 12000 revs/min 15 minutes, remove dextran sedimentation and obtain fermented supernatant fluid B 74mL, by fermented supernatant fluid B
74mL i.e. prepares 1.16 grams of mannitol 4 DEG C of crystallizations.
The above-mentioned method producing mannitol for the fermentation of raw material Leuconostoc mesenteroides with Caulis Sacchari sinensis juice, the Classification And Nomenclature of Leuconostoc mesenteroides used is Leuconostoc mesenteroides, Latin name is Leuconostoc mesenteroides, this microorganism (strain) is received by preservation center in November, 2009, and register on the books, preservation date is December in 2009 31, and depositary institution's full name is China Committee for Culture Collection of Microorganisms's common micro-organisms center；Deposit number: CGMCC1.10327.China Committee for Culture Collection of Microorganisms's common micro-organisms center sent viability statement on 09 12nd, 2013.
The invention has the beneficial effects as follows: compared with prior art, the marked improvement of the inventive method is as follows:
(1) method that the present invention is belonging to produce mannitol with lactic acid bacteria, compared with existing Sargassum extraction method, catalytic hydrogenation method and enzyme transforming process, not only energy consumption is low and yield is high, does not more importantly pollute in production process, it is eco-friendly, meets industrial development direction.
(2) compared with prior art CN201310537851.5, CN 201410065372.2 and other prior art, the cost of the culture medium of the inventive method is greatly lowered.Following table lists the Cost comparisons of the inventive method and the culture medium of prior art, it is seen that the economic benefit of the inventive method is the most prominent.
| Invention | The present invention | CN201310537851.5 | CN 01410065372.2 | Other prior art |
| Culture medium | Caulis Sacchari sinensis juice-brown sugar | Brown sugar is the MRS of carbon source | Sucrose is the MRS of carbon source | Fructose_glucose is the MRS of carbon source |
| Composition (unit/liter) | 1.28 | 2.69 | 2.73 | 3.13 |
Detailed description of the invention
Embodiment
The first step, prepares culture medium with Caulis Sacchari sinensis juice-brown sugar for raw material:
(1.1) apparatus is prepared:
The filter A kraft paper accompanying 0.2 μm filter membrane is packaged, then puts into high-pressure sterilizing pot together with the 100mL triangular flask of sealing, in 121 DEG C of sterilizings 20 minutes, get out the filter A accompanying 0.2 μm filter membrane and the 100mL triangular flask of sterilizing of sterilizing；
(1.2) Caulis Sacchari sinensis juice is squeezed:
Weighing 3350 grams of Caulis Sacchari sinensis, peeling rear cutout into strips, is put into and is squeezed Caulis Sacchari sinensis juice in fruit juice mixer；
(1.3) the centrifugal and filtration of Caulis Sacchari sinensis juice:
Take the prepared centrifuge that 1050mL juice from sugar cane rotating speed is 13000 revs/min of previous step (1.2) 30 minutes, obtain sugar cane juice supernatant A 1010mL；Again sugar cane juice supernatant A is filtered with the filter B accompanying 0.2 μm filter membrane and obtain Caulis Sacchari sinensis juice liquor B 1005mL；
(1.4) interpolation brown sugar acquisition Caulis Sacchari sinensis juice-brown sugar filtrate:
In above-mentioned Caulis Sacchari sinensis juice liquor B 1005mL, add 2.5 grams of brown sugar and stirring makes it be completely dissolved, thus obtain Caulis Sacchari sinensis juice-brown sugar liquor C；
(1.5) cold sterilization:
The filter A accompanying 0.2 μm filter membrane of Caulis Sacchari sinensis juice-brown sugar liquor C above-mentioned steps (1.1) ready sterilizing above-mentioned steps (1.4) prepared near the alcohol burner of superclean bench filters, prepare aseptic Caulis Sacchari sinensis juice-brown sugar filtrate D 1000mL, the 100mL triangular flask of above-mentioned steps (1.1) ready sterilizing adds the aseptic Caulis Sacchari sinensis juice-brown sugar filtrate D just prepared
Sealing after 40mL, the culture medium as next step is stand-by；
Second step, ferments with Leuconostoc mesenteroides and produces mannitol:
(2.1) activation Leuconostoc mesenteroides strain:
Leuconostoc mesenteroides is connect in the 10mL MRS culture medium that bacterium amount joins in the triangular flask having been placed on 50mL with 1%, shaken cultivation 12～16 hours；
(2.2) Leuconostoc mesenteroides is connect:
Leuconostoc mesenteroides strain above-mentioned steps (2.1) activated near the alcohol burner of superclean bench connects bacterium amount with 1% and joins above-mentioned steps (1.5) in the Caulis Sacchari sinensis juice being placed in 100mL triangular flask-brown sugar filtrate D 40mL that cold sterilization prepares；
(2.3) prepared fermentation liquid is cultivated:
Triangular flask after above-mentioned steps (2.2) is connect Leuconostoc mesenteroides is placed in shaking table, and at 30 DEG C, the velocity fluctuation with 120 revs/min is cultivated 24 hours, prepares fermentation liquid 40mL；
(2.4) isolated and purified:
Above-mentioned steps (2.3) is cultivated the prepared centrifuge that fermentation liquid 40mL rotating speed is 10000 revs/min 10 minutes, removes bacterial sediment and obtain fermented supernatant fluid A 37mL；In fermented supernatant fluid A 37mL, add the ethanol of equivalent volumes, with the centrifuge that rotating speed is 12000 revs/min 15 minutes, remove dextran sedimentation and obtain fermented supernatant fluid B 74mL, by fermented supernatant fluid B
74mL i.e. prepares 1.16 grams of mannitol 4 DEG C of crystallizations.
The Classification And Nomenclature of Leuconostoc mesenteroides used in above-described embodiment is Leuconostoc mesenteroides, Latin name is Leuconostoc mesenteroides, this microorganism (strain) is received by preservation center in November, 2009, and register on the books, preservation date is December in 2009 31, and depositary institution's full name is China Committee for Culture Collection of Microorganisms's common micro-organisms center；Deposit number: CGMCC1.10327.China Committee for Culture Collection of Microorganisms's common micro-organisms center sent viability statement on 09 12nd, 2013；Other raw material is all by commercially available.
Claims (1)
1. the method producing mannitol with Caulis Sacchari sinensis juice for the fermentation of raw material Leuconostoc mesenteroides, it is characterised in that step is as follows:
The first step, prepares culture medium with Caulis Sacchari sinensis juice-brown sugar for raw material:
(1.1) apparatus is prepared:
The filter A kraft paper accompanying 0.2 μm filter membrane is packaged, then puts into together with the 100mL triangular flask of sealing
In high-pressure sterilizing pot, in 121 DEG C of sterilizings 20 minutes, get out the filter A accompanying 0.2 μm filter membrane of sterilizing and sterilizing
100mL triangular flask；
(1.2) Caulis Sacchari sinensis juice is squeezed:
Weighing 3350 grams of Caulis Sacchari sinensis, peeling rear cutout into strips, is put into and is squeezed Caulis Sacchari sinensis juice in fruit juice mixer；
(1.3) the centrifugal and filtration of Caulis Sacchari sinensis juice:
Take the prepared centrifuge that 1050mL juice from sugar cane rotating speed is 13000 revs/min of previous step (1.2) 30 minutes,
Obtain sugar cane juice supernatant A 1010mL；Again sugar cane juice supernatant A is filtered with the filter B accompanying 0.2 μm filter membrane and obtain
Obtain Caulis Sacchari sinensis juice liquor B 1005mL；
(1.4) interpolation brown sugar acquisition Caulis Sacchari sinensis juice-brown sugar filtrate:
In above-mentioned Caulis Sacchari sinensis juice liquor B 1005mL, add 2.5 grams of brown sugar and stirring makes it be completely dissolved, thus obtain Caulis Sacchari sinensis
Juice-brown sugar liquor C；
(1.5) cold sterilization:
Caulis Sacchari sinensis juice-brown sugar liquor C the above-mentioned steps near the alcohol burner of superclean bench, above-mentioned steps (1.4) prepared
(1.1) the filter A accompanying 0.2 μm filter membrane of ready sterilizing filters, and prepares aseptic Caulis Sacchari sinensis juice-brown sugar filter
Liquid D 1000mL, adds just prepared aseptic in the 100mL triangular flask of above-mentioned steps (1.1) ready sterilizing
Caulis Sacchari sinensis juice-brown sugar filtrate D 40mL after seal, the culture medium as next step is stand-by；
Second step, ferments with Leuconostoc mesenteroides and produces mannitol:
(2.1) activation Leuconostoc mesenteroides strain:
Leuconostoc mesenteroides is connect in the 10mL MRS culture medium that bacterium amount joins in the triangular flask having been placed on 50mL with 1%,
Shaken cultivation 12～16 hours；
(2.2) Leuconostoc mesenteroides is connect:
Near the alcohol burner of superclean bench, the Leuconostoc mesenteroides strain that above-mentioned steps (2.1) has activated is connect bacterium with 1%
Amount joins the Caulis Sacchari sinensis juice being placed in 100mL triangular flask-brown sugar filtrate D 40mL that above-mentioned steps (1.5) prepares through cold sterilization
In；
(2.3) prepared fermentation liquid is cultivated:
Triangular flask after above-mentioned steps (2.2) is connect Leuconostoc mesenteroides is placed in shaking table, with 120 revs/min at 30 DEG C
Velocity fluctuation cultivate 24 hours, prepare fermentation liquid 40mL；
(2.4) isolated and purified:
Above-mentioned steps (2.3) is cultivated the centrifuge 10 that fermentation liquid 40mL rotating speed is 10000 revs/min prepared
Minute, remove bacterial sediment and obtain fermented supernatant fluid A 37mL；The ethanol of equivalent volumes is added in fermented supernatant fluid A 37mL,
With the centrifuge that rotating speed is 12000 revs/min 15 minutes, remove dextran sedimentation and obtain fermented supernatant fluid B 74mL, will
Fermented supernatant fluid B 74mL i.e. prepares 1.16 grams of mannitol 4 DEG C of crystallizations；
The Classification And Nomenclature of above-mentioned Leuconostoc mesenteroides used is Leuconostoc mesenteroides, and Latin name is Leuconostoc
Mesenteroides, deposit number: CGMCC1.10327.
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| CN201410254455.6A CN104017831B (en) | 2014-06-10 | 2014-06-10 | The method producing mannitol for the fermentation of raw material Leuconostoc mesenteroides with Caulis Sacchari sinensis juice |
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