CN1288249C - Method for preparing xylitol by microbial mixed fermentation - Google Patents
Method for preparing xylitol by microbial mixed fermentation Download PDFInfo
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- CN1288249C CN1288249C CN 200410031949 CN200410031949A CN1288249C CN 1288249 C CN1288249 C CN 1288249C CN 200410031949 CN200410031949 CN 200410031949 CN 200410031949 A CN200410031949 A CN 200410031949A CN 1288249 C CN1288249 C CN 1288249C
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- xylitol
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Abstract
The present invention belongs to a method for preparing xylitol by microbial mixed fermentation, which comprises the following specific steps: glucose is used as raw material; firstly, a saccharomyces cerevisiae FX-6201CGMCC0283.1 strain and an acetobacter suboxydans MF-21CGMCC0283.2 strain are respectively cultivated; subsequently, the two strains are mixed and fermented, ethanol is used for extracting fermentation liquor, solid substances are extracted to obtain crystallized xylitol, and then, the crystallized xylitol is filtered and dried to obtain xylitol crystals.
Description
Technical field
The invention belongs to microbial fermentation, concretely, relate to the method that an Accharomyces cerevisiae FX-6201CGMCC0283.1 and a strain weak oxide acetobacter MF-21CGMCC 0283.2 mixed fermentation prepare Xylitol.
Background technology
Xylitol is five carbon polyols, natural being present in the fruits and vegetables such as strawberry, Pueraria lobota lettuce.Because its sugariness is high and have metabolism not rely on characteristics such as Regular Insulin, anti-dental caries, is diabetes.Odontopathy patient's ideal sucrose surrogate.The preparation of Xylitol can have three approach: directly extract from plant (1); (2) chemical method or biological process wood sugar shortening; (3) utilize method of microorganism.
Because Xylitol naturally occurring content in plant is too low, directly extract very uneconomical.At present industrial main method preparation (Wisnik with the wood sugar shortening, J.et al.:Ind.Eng.Chem.Prod.Res.Dev., 3:232-236,1974), but this method complex process, and cost is very high, the biological process hydrogenation is also obtained many progress (Leathers in recent years, T.D.:FEMSYeast Research, 3:133-140,2003; Jiangning etc.: ZL01110778.2,2004), but with wood sugar is that raw material is after all than glucose price height.Utilizing microorganism direct fermentation glucose to prepare Xylitol is ideal method very economically, but also finds can direct fermentation glucose to obtain the microorganism of Xylitol so far at nature.Hiroshi, people such as O. once fermented step by step with three kinds of microorganisms and had prepared Xylitol (Appl.Microbiol., 18:1031-1035 from glucose, 1969), proved the possibility of microbial fermentation, but three bacterium substep fermenting process is very complicated, does not have practical value from the glucose making xylitol.In recent years, Harkki, the method for human recombinant DNAs such as A.M. has made up engineering bacteria, can obtain Xylitol by direct fermentation glucose, and apply for international monopoly (WO 94/10325,1994), but transformation efficiency has only 3-4%, and distance applications is still very remote.
Summary of the invention
The object of the present invention is to provide a kind of method of practicable double-bacterium ferment to prepare Xylitol, specifically, the invention provides the method that a kind of usefulness one Accharomyces cerevisiae FX-6201CGMCC 0283.1 and a strain weak oxide acetobacter MF-21CGMCC 0283.2 mixed fermentation prepare Xylitol.
We utilize glucose fermentation by microorganism, optimized seed selection one saccharomycete (Saccharomyces sp.) FX-6021 and a strain bacterium (Acetobacter suboxydans) MF-21, they on December 6th, 1996 at the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms, China, Beijing, the Zhong Guan-cun; Postcode: 100080) carried out preservation, preserving number is respectively CGMCC 0283.1 and CGMCC 0283.2.Carry out mixed fermentation after two bacterial strains are cultivated respectively, can directly obtain Xylitol from glucose.With yeast yeast saccharomyces cerevisiae (Saccharomyces sp.) FX-6021 CGMCC 0283.1 in containing the nutritional medium of high concentration glucose, cultivated 3-5 days in 24-32 ℃, the composition of yeast culture base is: glucose 10-70%, peptone 1%, yeast extract paste 1%, extractum carnis 0.5%, NaCl 0.1%, CaCO
30.05%, pH4-8.Then with containing calcium carbonate culture-medium, be lime carbonate 0.1-5%, glucose 2%, yeast extract paste 1%, peptone 0.5%, NaCl 0.1%, pH 5-9, in mix with 24-37 ℃ of bacterium weak oxide acetobacter (Acetobacter suboxydans) MF-21 CGMCC 0283.2 that cultivated 6-36 hour, continue at 24-34 ℃ the fermentation 16-120 hour, after glucose in the detection substratum all is converted into Xylitol, with the centrifugal thalline of removing of fermented liquid, supernatant liquor activated carbon decolorizing, evaporated under reduced pressure, with the crystallization of hot ethanol extracting postcooling, promptly obtain xylitol crystal." the composition % " of substratum of the present invention is meant the per-cent of the weight of its composition to volume of water.
Thereby a kind of method for preparing Xylitol provided by the present invention is characterized in that, is raw material with glucose, cultivates earlier yeast saccharomyces cerevisiae FX-6201CGMCC 0283.1 and weak oxide acetobacter MF-21CGMCC 0283.2 respectively; After above-mentioned nutrient solution mixing, continuation mixed fermentation, fermentation reaches terminal point, with ethanol extractive fermentation liquid, extracts solid matter and gets the Xylitol crystallization, filtration, drying, the method for acquisition xylitol crystal.
The main process of method of the present invention is:
(1) yeast FX-6201CGMCC 0283.1 is cultivated in containing the nutritional medium of glucose;
(2) bacterium MF-21CGMCC 0283.2 is cultivated in substratum calciferous;
(3) with fermenting after two kinds of cultures mixing, obtain Xylitol.
In the method for the invention, used glucose prepare for the starch acid hydrolysis or enzyme process prepares.The content of glucose is in the 10%-70% scope in the used substratum, preferred 20%-50%.The used medium pH that contains glucose is 4-8, and preferred pH is 5.The culture temperature of yeast FX-6201CGMCC 0283.1 in containing dextrose culture-medium is 24-32 ℃, preferred 30 ℃.The incubation time of yeast FX-6201CGMCC 0283.1 in containing dextrose culture-medium is 3-5 days, preferred 4 days.It is 0.1%-5% that used substratum calciferous contains lime carbonate, preferred 1-2.5%.Used medium pH calciferous is 5-9, and preferred pH is 7.The culture temperature of bacterium MF-21CGMCC 0283.2 in substratum calciferous is 24-37 ℃, preferred 30 ℃.Bacterium MF-21CGMCC 0283.2 incubation time in substratum calciferous is 6-36 hour, preferred 10-24 hour.The temperature of mixed fermentation is 24-37 ℃, preferred 30 ℃.The time of mixed fermentation is 16-120 hour, preferred 40-80 hour.
Different is for the present invention and prior art:
1. preparation method of the present invention is a main raw material with glucose, and glucose is cheap, so that the cost for preparing Xylitol with the present invention is the chemical method of raw material than with the wood sugar is low.
2. the present invention mixes one-step fermentation with two bacterium, and whole technology is fermented simply step by step than three bacterium, and available common fermentation equipment.
3. the bacterial classification of institute of the present invention seed selection, it utilizes the transformation efficiency of glucose product Xylitol more much higher than the recombinant bacterial strain of having reported.
Therefore, the present invention has characteristics such as raw material is cheap, with low cost, technology simple, Device-General, transformation efficiency height, is suitable for industrial application.
Embodiment
For the ease of understanding and implement the present invention, below give further instruction by several specific embodiments.
Embodiment 1:
(1) S. cervisiae FX 6021 CGMCC 0283.1 access is equipped with in the 250ml triangular flask of 10ml yeast culture base A, in 30 ℃, 220rpm rotary shaker cultivation 96hr.The composition of yeast culture base A is: glucose (starch acid hydrolysis preparation) 30%, and peptone 1%, yeast extract paste 1%, extractum carnis 0.5%, NaCl 0.1%, CaCl
20.05%, pH 5.0.
(2) weak oxide acetobacter MF-21 CGMCC 0283.2 access is equipped with in the 250ml triangular flask of 10ml bacteria culture medium, in 30 ℃, 250rpm rotary shaker cultivation 24hr.The composition of bacteria culture medium is: glucose 2%, and yeast extract paste 1%, peptone 0.5%, NaCl 0.1%, CaCO
32%, pH 6.8.
(3) with the fermented liquid in (1), (2) after aseptic condition mixes down, continue on shaking table to cultivate 50hr with 30 ℃, 220rpm.During fermentation ends in the fermented liquid Xylitol concentration be 25g/L.
(4) fermented liquid centrifugal (Beckman GS-15R, 8000rpm, 10min, 20 ℃) is got supernatant liquor 19ml, add the 0.38g gac, transfer pH=5.0, boil, suction filtration gets colorless clear liquid behind the cool to room temperature.Clear liquid in 50 ℃ of evaporated under reduced pressure, with anhydrous hot ethanol extracting solid matter, is got the Xylitol crystallization after the cooling, filter,, get xylitol crystal 0.41g the crystal drying.
Embodiment 2:
(1) yeast FX 6021 accesses is equipped with in the 250ml triangular flask of 10ml yeast culture base (as embodiment 1 (1)), in 30 ℃, 220rpm rotary shaker cultivation 96hr.
(2) bacterium MF-21 access is equipped with in the 250ml triangular flask of 10ml bacteria culture medium (as embodiment 1 (2)), in 30 ℃, 250rpm rotary shaker cultivation 24hr.
(3) getting in (2) fermented liquid 3ml changes under aseptic condition the 20ml bacteria culture medium is housed
In the 250ml triangular flask of (as embodiment 1 (2)), in 30 ℃, 250rpm rotary shaker cultivation 8hr.
(4) get in (3) fermented liquid 10ml and fermented liquid (1) after mixing under the aseptic condition, continuation is cultivated 60hr with 30 ℃, 220rpm on shaking table.During fermentation ends in the fermented liquid Xylitol concentration be 31g/L.
(5) with the centrifugal supernatant liquor 19ml that gets of fermented liquid, add the 0.38g gac, transfer pH=5.0, boil, suction filtration gets colorless clear liquid behind the cool to room temperature.Clear liquid in 50 ℃ of evaporated under reduced pressure, with anhydrous hot ethanol extracting solid matter, is got the Xylitol crystallization after the cooling, filter,, get xylitol crystal 0.47g the crystal drying.
Embodiment 3
(1) yeast FX 6021 CGMCC 0283.1 access is equipped with in the 250ml triangular flask of 20ml yeast culture base B, in 30 ℃, 220rpm rotary shaker cultivation 100hr.The composition of yeast culture base B is: glucose (amylorrhexis preparation) 30%, and peptone 1%, yeast extract paste 1%, extractum carnis 0.5%, NaCl 0.1%, CaCl
20.05%, pH 5.0.
(2) bacterium MF-21 CGMCC 0283.2 access is equipped with in the 250ml triangular flask of 20ml bacteria culture medium (as embodiment 1 (2)), in 30 ℃, 250rpm rotary shaker cultivation 20hr.
(3) with the fermented liquid in (1), (2) after aseptic condition mixes down, continue on shaking table to cultivate 55hr with 30 ℃, 220rpm.During fermentation ends in the fermented liquid Xylitol concentration be 26g/L.
(4) with the centrifugal supernatant liquor 38.5ml that gets of fermented liquid, add the 0.77g gac, transfer pH=5.0, boil, suction filtration gets colorless clear liquid behind the cool to room temperature.Clear liquid in 50 ℃ of evaporated under reduced pressure, with anhydrous hot ethanol extracting solid matter, is got the Xylitol crystallization after the cooling, filter,, get xylitol crystal 0.86g the crystal drying.
Embodiment 4:
(1) yeast FX 6021 CGMCC 0283.1 access is equipped with in the 250ml triangular flask of 10ml yeast culture base B (as embodiment 3 (1)), in 30 ℃, 220rpm rotary shaker cultivation 96hr.
(2) bacterium MF-21 CGMCC 0283.2 access is equipped with in the 250ml triangular flask of 10ml bacteria culture medium (as embodiment 1 (2)), in 30 ℃, 250rpm rotary shaker cultivation 24hr.
(3) get (2) middle fermented liquid 2ml and under aseptic condition, change in the 250ml triangular flask that 20ml substratum (as embodiment 1 (2)) is housed, in 30 ℃, 250rpm rotary shaker cultivation 10hr.
(4) get in (3) fermented liquid 10ml and fermented liquid (1) after mixing under the aseptic condition, continuation is cultivated 46hr with 30 ℃, 220rpm on shaking table.During fermentation ends in the fermented liquid Xylitol concentration be 25g/L.
(5) with the centrifugal supernatant liquor 19ml that gets of fermented liquid, add the 0.38g gac, transfer pH=5.0, boil, suction filtration gets colorless clear liquid behind the cool to room temperature.Clear liquid in 50 ℃ of evaporated under reduced pressure, with anhydrous hot ethanol extracting solid matter, is got the Xylitol crystallization after the cooling, filter,, get xylitol crystal 0.40gg the crystal drying.
Claims (22)
1. method with preparing xylitol by micro-organism mixture fermentation, it is characterized in that, with glucose is raw material, cultivates yeast saccharomyces cerevisiae FX-6201CGMCC 0283.1 earlier respectively and cultivate weak oxide acetobacter MF-21CGMCC0283.2 in containing the nutritional medium of glucose in substratum calciferous; Above-mentioned two bacteria culture fluids are mixed the back continue fermentation; Fermenting reaches terminal point, with ethanol extractive fermentation liquid, extracts solid matter and gets the Xylitol crystallization, again by filtering drying, the method for acquisition xylitol crystal.
2. preparation Xylitol method as claimed in claim 1 is characterized in that, and is that used glucose prepares for the starch acid hydrolysis or enzyme process prepares.
3. the method for claim 1, in its used substratum the content of glucose be with its weight and volume of water per-cent in the 10%-70% scope.
4. method as claimed in claim 3, in its used substratum the content of glucose be with its weight and volume of water per-cent in the scope of 20%-50%.
5. the method for claim 1, the pH scope of the substratum that contains glucose that it is used is 4-8.
6. method as claimed in claim 5, the medium pH that contains glucose that it is used is 5.
7. the method for claim 1, the culture temperature scope of its yeast FX-6201CGMCC 0283.1 in containing dextrose culture-medium is 24-32 ℃.
8. method as claimed in claim 7, the culture temperature of its yeast FX-6201CGMCC 0283.1 in containing dextrose culture-medium is 30 ℃.
9. the method for claim 1, the incubation time of its yeast FX-6201CGMCC 0283.1 in containing dextrose culture-medium is 3-5 days.
10. method as claimed in claim 9, the incubation time of its yeast FX-6201CGMCC 0283.1 in containing dextrose culture-medium is 4 days.
11. the method for claim 1, the substratum calciferous that its bacterium MF-21CGMCC 0283.2 is used, the weight of its lime carbonate and volume of water per-cent are 0.1%-5%.
12. method as claimed in claim 11, its weight calciferous and volume of water per-cent are 1%-2.5%.
13. the method for claim 1, its used medium pH calciferous is 5-9.
14. method as claimed in claim 13, its used medium pH calciferous is 7.
15. the method for claim 1, the culture temperature of its bacterium MF-21CGMCC 0283.2 in substratum calciferous is 24-37 ℃.
16. method as claimed in claim 15, the culture temperature of its bacterium MF-21CGMCC 0283.2 in substratum calciferous is 30 ℃.
17. the method for claim 1, its bacterium MF-21CGMCC 0283.2 incubation time in substratum calciferous is 6-36 hour.
18. method as claimed in claim 17, the incubation time of its bacterium MF-21CGMCC 0283.2 in substratum calciferous is 10-24 hour.
19. the method for claim 1, the temperature of its mixed fermentation are 24-37 ℃.
20. method as claimed in claim 19, the temperature of its mixed fermentation are 30 ℃.
21. the method for claim 1, the time of its mixed fermentation is 16-120 hour.
22. method as claimed in claim 21, the time of its mixed fermentation is 40-80 hour.
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| CN 200410031949 CN1288249C (en) | 2004-03-31 | 2004-03-31 | Method for preparing xylitol by microbial mixed fermentation |
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| CN 200410031949 CN1288249C (en) | 2004-03-31 | 2004-03-31 | Method for preparing xylitol by microbial mixed fermentation |
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Cited By (1)
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| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2010009984A (en) * | 2008-03-13 | 2010-09-30 | Dsm Ip Assets Bv | Selection of organisms capable of fermenting mixed substrates. |
| CN101709309B (en) * | 2009-12-22 | 2012-05-09 | 安徽丰原发酵技术工程研究有限公司 | Method for combined fermentation of ethanol and xylitol |
| CN104762333B (en) * | 2015-03-09 | 2018-05-22 | 浙江工业大学 | The method that xylitol is prepared using winter bamboo shoot shell |
| CN112442556A (en) * | 2020-12-07 | 2021-03-05 | 浙江华康药业股份有限公司 | Method for reducing sugar in xylitol crystal |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
| US11840500B2 (en) | 2016-02-19 | 2023-12-12 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
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