CN105463041A - Preparation method of glucosamine - Google Patents
Preparation method of glucosamine Download PDFInfo
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- CN105463041A CN105463041A CN201510969047.3A CN201510969047A CN105463041A CN 105463041 A CN105463041 A CN 105463041A CN 201510969047 A CN201510969047 A CN 201510969047A CN 105463041 A CN105463041 A CN 105463041A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
Abstract
The invention relates to fermentation engineering, in particular to a preparation method of glucosamine, comprising the following steps: (1) inoculating a glucosamine producing strain into a seed medium according to an inoculum size of 0.4-0.6 percent and culturing to a logarithmic phase; (2) inoculating a product obtained in step (1) into a fermentation medium according to an inoculum size of 5 to 10%, controlling dissolved oxygen to be 20-40 percent and pH to be 7 plus/minus 0.2, fermenting under the condition of the temperature being 37 plus/minus 0.5 DEG C, starting to slowly and continuously add a glucose solution with the concentration of 59-61 percent when the concentration of glucose is reduced to 0.01-0.2g/L, adding trace element mother liquor into the glucose solution, and maintaining the concentration of residual sugar in fermentation liquor to be 0.05-0.8g/L to the end of fermentation, wherein the maximal speed of slowly and continuously adding the glucose solution is 0.5-1.6 percent. By adopting the preparation method provided by the invention, the content and percent conversion of the glucosamine are remarkably increased.
Description
Technical field
The present invention relates to fermentation engineering, particularly a kind of preparation method of glucosamine.
Background technology
Glucosamine is the compound that a hydroxyl of glucose is replaced by an amino, and molecular formula is C
6h
13o
5n, is commonly called as aminosugar, is called for short ammonia sugar.Extensively be present in occurring in nature, glucosamine usually with N-acetyl derivative (as chitin) or with N-sulfuric ester and N-acetyl-3-O-lactic acid ether (teichoic acid) form be present in microorganism, animal-origin polysaccharide and in conjunction with in polysaccharide.
Glucosamine is the important nutrient forming chondrocyte, is the natural tissues composition of healthy articular cartilage.With advancing age, in human body, the shortage of glucosamine can be more and more serious, and then cause continuous degeneration and the wearing and tearing of joint cartilage.The U.S., Europe and Japanese a large amount of medical researches show: glucosamine can help repair and safeguard cartilage, and can stimulate the growth of chondrocyte.
At present, glucosamine mainly adopts chitin in acidolysis shrimp shell or crab shell to produce, and the waste liquid environmental pollution of this method is comparatively serious, and the product utilizing this kind of method to obtain easily causes allergic reaction, and is not suitable for seafood allergic human population and takes.Relative to chitin hydrolysis method, Production by Microorganism Fermentation is not by resource limit, and environmental pollution is little, and product, without fishy smell, does not exist irritated effect.
Find through retrieval, publication number is that the application for a patent for invention of CN102031232 discloses a kind of method and the special strain therefore of producing aminosugar.Its deposit number of special strain therefore producing aminosugar is CGMCCNo.4218.Produce the method for aminosugar, comprise the following steps: will special strain therefore 28 ~ 37 DEG C of fermentation culture in product enzymic fermentation substratum of aminosugar be produced, and obtain the combination product containing aminosugar; By the described combination product containing aminosugar of non-specific saccharase hydrolysis, filter, concentrated, drying obtains aminosugar powder.
In addition, now conventional bacterial strain also comprises intestinal bacteria and subtilis, and wherein due to intestinal bacteria, to have clear, the easy cultivation of hereditary property, fermentation period short and can the characteristic such as high expression of goal gene in advance, is widely applied.But can produce acetic acid during Escherichia coli Growth, especially in recombinant bacterium high-density culture, acetic acid and L-glutamic acid produce and accumulate the growth that not only can affect thalline, also can suppress the synthesis of product.Therefore, the generation reducing the acetic acid in fermenting process is not only conducive to prior colibacillary high-density culture, and very important with the industrialization realizing glucosamine for improving aminoglucose candy output.
Summary of the invention
The object of this invention is to provide a kind of preparation method of glucosamine.
For achieving the above object, the technical scheme that concrete employing is following:
A preparation method for glucosamine, comprises the following steps:
(1) glucosamine produced bacterium access seed culture medium and be cultured to logarithmic phase;
(2) product step (1) obtained accesses fermention medium by inoculum size 5 ~ 10%, pass into oxygen, control dissolved oxygen is 20 ~ 40%, pH is 7 ± 0.2, be ferment under the condition of 37 ± 0.5 DEG C in temperature, when the concentration of glucose reduces to 0.01 ~ 0.2g/L, start stream and add the glucose solution that concentration is 59 ~ 61% (m/m), and in glucose solution, add micro-mother liquor, the max-flow acceleration of glucose solution is 0.5 ~ 1.6%, and maintaining remaining sugar concentration in fermented liquid is that 0.05 ~ 0.8g/L is to fermentation termination.
In the inventive solutions, glucosamine production bacterium selects the one in intestinal bacteria, subtilis, is preferably Recombinant organism.Described intestinal bacteria are optional commercially available all products, and the present invention does not make particular determination to this.
In the inventive solutions, the operation steps that step (1) is concrete is: glucosamine is produced bacterium and access seed culture medium by inoculum size 0.4 ~ 0.6%, 37 ± 0.5 DEG C, pH is 7 ± 0.2, be cultured to logarithmic phase under the condition of dissolved oxygen 20 ~ 30%, step (1) generally needs cultivation 12 ~ 14h just can reach logarithmic phase.
Glucosamine preferably, is produced bacterium and is accessed seed culture medium by inoculum size 0.5% by step (1), 37 DEG C, pH is 7, be cultured to logarithmic phase under the condition of dissolved oxygen 20 ~ 30%.
When being produced by glucosamine after bacterium is cultured to logarithmic phase in step (1), the OD value (660nm) of seeding tank is generally 2.0 ~ 2.5.
In step (2), reach fermentation termination when the content of glucosamine does not increase, generally need fermentation 35 ~ 42h just can reach fermentation termination.
In the inventive solutions, concrete, described seed culture medium comprises glucose 1.9 ~ 2.1g/L, KH
2pO
40.5 ~ 0.7g/L, K
2hPO
43H
2o3.1 ~ 3.2g/L, Trisodium Citrate 2H
2o0.09 ~ 0.11g/L, ammonium sulfate 0.9 ~ 1.1g/L, MgSO
47H
2o0.05 ~ 0.07g/L, CaCl
22H
2o0.002 ~ 0.004g/L, micro-mother liquor 0.009 ~ 0.011g/L.
Preferably, described seed culture medium comprises glucose 2g/L, KH
2pO
40.6g/L, K
2hPO
43H
2o3.145g/L, Trisodium Citrate 2H
2o0.1g/L, ammonium sulfate 1g/L, MgSO
47H
2o0.06g/L, CaCl
22H
2o0.003g/L, micro-mother liquor 0.01g/L.
Concrete, described fermention medium comprises glucose 0.4 ~ 0.6g/L, KH
2pO
40.6 ~ 0.7g/L, Citric acid monohydrate Food grade 0.3 ~ 0.4g/L, MgSO
47H
2o0.2 ~ 0.3g/L, CaCl
22H
2o0.002 ~ 0.003g/L, defoamer 0.02 ~ 0.03g/L, micro-mother liquor 0.009 ~ 0.011g/L.
Preferably, described fermention medium comprises glucose 0.5g/L, KH
2pO
40.667g/L, Citric acid monohydrate Food grade 0.355g/L, MgSO
47H
2o0.25g/L, CaCl
22H
2o0.0025g/L, defoamer 0.025g/L, micro-mother liquor 0.01g/L.
In the fermentation medium described defoamer be chosen as ordinary skill in the art means, such as can select polyether antifoam agent, silicone antifoam agent etc.
Microorganism is to Fe
2+, Mn
2+, Pb
2+, Co
2+, Ni
2+, Zn
2+little etc. micro-demand, but trace element cuts much ice to microbial growth, in the inventive solutions, all containing micro-mother liquor in the glucose solution that seed culture medium, fermention medium and step (2) stream add, concrete, described micro-mother liquor comprises: FeSO
47H
2o4 ~ 6g/L, H
3bO
30.09 ~ 0.11g/L, CoCl
26H
2o0.09 ~ 0.11g/L, MnSO
4h
2o0.3 ~ .04g/L, ZnSO
47H
2o3.7 ~ 3.9g/L, NaMoO
42H
2o0.09 ~ 0.11g/L, CoSO
40.09 ~ 0.11g/L.
Preferably, micro-mother liquor comprises FeSO
47H
2o5g/L, H
3bO
30.1g/L, CoCl
26H
2o0.1g/L, MnSO
4h
2o0.33g/L, ZnSO
47H
2o3.8g/L, NaMoO
42H
2o0.1g/L, CoSO
40.1g/L.
In step (2), inoculum size is excessive or too small, all can affect fermentation, and inoculum size is crossed conference and caused dissolved oxygen not enough, affects Product formation, and too much can move into metabolic waste; Inoculum size is too small then can the Extending culture time, reduces the productivity of fermentor tank.In the inventive solutions, the inoculum size of step (2) is preferably 10%.
Glucosamine produces bacterium needs a large amount of oxygen to participate in metabolism in process of production, and oxyty is too high or too low, all can affect thalli growth and product generation.Oxygen has potential toxic side effect to aerobic microbiological, and oxygen concn is too high can produce Crabtree effect; Too low oxyty then causes a large amount of generations of acetic acid, thalli growth and specific growth rate is suppressed, even can makes thalline autolyze, have a strong impact on Product formation.Therefore, maintain the oxyty of certain level, be not only conducive to thalli growth, also help and improve aminoglucose sugar level.In the inventive solutions, preferably, controlling dissolved oxygen in step (2) is 25 ~ 30%, the max-flow acceleration of glucose solution is 1.3 ~ 1.6%, more preferably, the max-flow acceleration of glucose solution is 1.6%, now can be conducive to thalli growth, reduce the generation of acetic acid, improve glucosamine transformation efficiency.
In the inventive solutions, in step (2), stream adds the mode of glucose is ordinary skill in the art means, and the present invention does not make particular determination to this.
Preferably, maintaining remaining sugar concentration in fermented liquid in step (2) is that 0.05 ~ 0.2g/L is to fermentation termination.
Invention further provides the preferred forms of the preparation method of glucosamine, specifically comprise the following steps:
(1) glucosamine is produced bacterium and accesses seed culture medium by inoculum size 0.5%, 37 DEG C, pH is 7 ± 0.2, be cultured to logarithmic phase under the condition of dissolved oxygen 20 ~ 30%;
(2) product that step (1) obtains is accessed fermention medium by inoculum size 10%, pass into oxygen, control dissolved oxygen is 25 ~ 30%, pH is 7 ± 0.2, be ferment under the condition of 37 ± 0.5 DEG C in temperature, when the concentration of glucose reduces to 0.01 ~ 0.2g/L, start stream and add the glucose solution that concentration is 60%, and in glucose solution, add micro-mother liquor, the max-flow acceleration of glucose solution is 1.6%, and maintaining remaining sugar concentration in fermented liquid is that 0.05 ~ 0.2g/L is to fermentation termination.
The invention provides a kind of preparation method of glucosamine, and add by control dissolved oxygen level, fermentor tank inoculum size, glucose limitation stream, add the measures such as trace element, the content of glucosamine and transformation efficiency are significantly improved.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
The preparation method of a kind of glucosamine of the present embodiment, comprises the following steps:
(1) intestinal bacteria are accessed seed culture medium, inoculum size is 0.5%, at 37 DEG C, pH be 7.0 and dissolved oxygen 20 ~ 30% condition under in 5L automatically control in fermentor tank cultivate 12 ~ 14h to logarithmic phase, OD value (660nm) is 2.0 ~ 2.5;
(2) in product access fermention medium step (1) obtained, inoculum size is 10%, initial air flow is 0.8L/min, mixing speed 300 ~ 800rpm, dissolved oxygen level is 25 ~ 30%, be 7 ± 0.2 by Feeding ammonia water control pH, culture temperature is 37 ± 0.5 DEG C, (in fermenting process, prevent foaming from escaping liquid to steep enemy's froth breaking, external bubble is utilized to oppose bottle froth breaking) when the concentration of glucose reduces to 0.01 ~ 0.2g/L, start stream add concentration be 60% glucose solution carry out benefit sugar, and in glucose solution, add micro-mother liquor, the max-flow acceleration of glucose solution is 0.5 ~ 1.6% (namely mend sugared speed and be less than 12g/L per hour, ) to maintain remaining sugar concentration in fermented liquid be 0.05 ~ 0.2g/L, fermentation stops to 40h.
Wherein, seed culture medium comprises glucose 2g/L, KH
2pO
40.6g/L, K
2hPO
43H
2o3.145g/L, Trisodium Citrate 2H
2o0.1g/L, ammonium sulfate 1g/L, MgSO
47H
2o0.06g/L, CaCl
22H
2o0.003g/L, micro-mother liquor 0.01g/L.
Fermention medium comprises glucose 0.5g/L, KH
2pO
40.667g/L, Citric acid monohydrate Food grade 0.355g/L, MgSO
47H2O0.25g/L, CaCl
22H
2o0.0025g/L, defoamer 0.025g/L.
Trace element mother liquor comprises: FeSO
47H
2o5g/L, H
3bO
30.1g/L, CoCl
26H
2o0.1g/L, MnSO
4h
2o0.33g/L, ZnSO
47H
2o3.8g/L, NaMoO
42H
2o0.1g/L, CoSO
40.1g/L.
Product during fermentation termination is as follows: glucosamine is 90.5g/L, acetic acid content 1.8g/L, content of glutamic acid 4.8g/L, glucosamine and sugared transformation efficiency 36.9%.
Embodiment 2
The difference of the present embodiment and embodiment 1 is only, remaining sugar concentration in step (2), to mend sugared speed different with fermentation time, in the present embodiment, under the condition controlling residual sugar 0.2 ~ 0.8g/L, maintain the sugared speed 1.6% of maximum benefit, fermentation accumulation glucosamine, fermentation culture 35h.
Product during fermentation termination is as follows: glucosamine is 62g/L, acetic acid content 8.9g/L, content of glutamic acid 16.1g/L, glucosamine and sugared transformation efficiency 21.2%.
Embodiment 3
The difference of the present embodiment and embodiment 1 is only, the inoculum size in step (2), fermentation time are different.In the present embodiment, the inoculum size in step (2) is 5%, and fermented incubation time is 42h.
Product during fermentation termination is as follows: glucosamine is 85g/L, acetic acid content 1.9g/L, content of glutamic acid 5.1g/L, glucosamine and sugared transformation efficiency 29%.
Embodiment 4
The difference of the present embodiment and embodiment 1 is only, the control condition of the dissolved oxygen in step (2), to mend sugared speed different with fermentation time.In the present embodiment, step (2), under the condition controlling dissolved oxygen 20 ~ 25%, maintains and mends sugared speed 1.3%, fermentation period 37h.
Product during fermentation termination is as follows: glucosamine is 78g/L, acetic acid content 3.7g/L, content of glutamic acid 6.6g/L, glucosamine and sugared transformation efficiency 26.6%.
Embodiment 5
The difference of the present embodiment and embodiment 1 is only, the control condition of the dissolved oxygen in step (2) is different with fermentation time.In the present embodiment, step (2) control dissolved oxygen 30 ~ 40% condition under, fermentation culture 36.5h.
Product during fermentation termination is as follows: glucosamine is 85.1g/L, acetic acid content 3.7g/L, content of glutamic acid 5.6g/L, glucosamine and sugared transformation efficiency 29%.
Embodiment 6
The difference of the present embodiment and embodiment 1 is only, step does not add trace element when stream adds glucose in (2), and fermentation time is also different simultaneously.In the present embodiment, step (2) does not add trace element when stream adds glucose, fermentation culture 41h.
Product during fermentation termination is as follows: glucosamine is 89.7g/L, acetic acid content 5.1g/L, content of glutamic acid 5.7g/L, glucosamine and sugared transformation efficiency 29.5%.
Product as can be seen from the fermentation termination of embodiment 1 ~ embodiment 6:
Embodiment 1 is compared with embodiment 2, and glucosamine output increased 46.7%, acetic acid content reduces by 79.8%, and content of glutamic acid reduces by 70.2%, and glucosamine and sugared transformation efficiency improve 74.1%.
Embodiment 1 is compared with embodiment 3, and glucosamine output increased 6.5%, acetic acid content reduces by 5%, and content of glutamic acid reduces by 5.9%, and glucosamine and sugared transformation efficiency improve 27.2%.
Embodiment 1 is compared with embodiment 4, and glucosamine output increased 16%, acetic acid content reduces by 51.3%, and content of glutamic acid reduces by 27.3%, and glucosamine and sugared transformation efficiency improve 38.7%.
Embodiment 1 is compared with embodiment 5, and glucosamine output increased 6.3%, acetic acid content reduces by 51.3%, and content of glutamic acid reduces by 2%, and glucosamine and sugared transformation efficiency improve 27.2%.
Embodiment 1 is compared with embodiment 6, and aminoglucose candy output is substantially identical, and acetic acid content reduces by 64.7%, and content of glutamic acid reduces by 15.8%, and glucosamine and sugared transformation efficiency improve 25.1%.
As can be seen here, embodiment 1 has best implementation result.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a preparation method for glucosamine, is characterized in that, comprises the following steps:
(1) glucosamine produced bacterium access seed culture medium and be cultured to logarithmic phase;
(2) product step (1) obtained accesses fermention medium by inoculum size 5 ~ 10%, pass into oxygen, control dissolved oxygen is 20 ~ 40%, pH is 7 ± 0.2, be ferment under the condition of 37 ± 0.5 DEG C in temperature, when the concentration of glucose reduces to 0.01 ~ 0.2g/L, start stream and add the glucose solution that concentration is 59 ~ 61%, and in glucose solution, add micro-mother liquor, the max-flow acceleration of glucose solution is 0.5 ~ 1.6%, and maintaining remaining sugar concentration in fermented liquid is that 0.05 ~ 0.8g/L is to fermentation termination.
2. preparation method according to claim 1, is characterized in that, described seed culture medium comprises glucose 1.9 ~ 2.1g/L, KH
2pO
40.5 ~ 0.7g/L, K
2hPO
43H
2o3.1 ~ 3.2g/L, Trisodium Citrate 2H
2o0.09 ~ 0.11g/L, ammonium sulfate 0.9 ~ 1.1g/L, MgSO
47H
2o0.05 ~ 0.07g/L, CaCl
22H
2o0.002 ~ 0.004g/L, micro-mother liquor 0.009 ~ 0.011g/L.
3. preparation method according to claim 2, is characterized in that, described seed culture medium comprises glucose 2g/L, KH
2pO
40.6g/L, K
2hPO
43H
2o3.145g/L, Trisodium Citrate 2H
2o0.1g/L, ammonium sulfate 1g/L, MgSO
47H
2o0.06g/L, CaCl
22H
2o0.003g/L, micro-mother liquor 0.01g/L.
4. preparation method according to claim 1, is characterized in that, described fermention medium comprises glucose 0.4 ~ 0.6g/L, KH
2pO
40.6 ~ 0.7g/L, Citric acid monohydrate Food grade 0.3 ~ 0.4g/L, MgSO
47H
2o0.2 ~ 0.3g/L, CaCl
22H
2o0.002 ~ 0.003g/L, defoamer 0.02 ~ 0.03g/L, micro-mother liquor 0.009 ~ 0.011g/L.
5. preparation method according to claim 4, is characterized in that, described fermention medium comprises glucose 0.5g/L, KH
2pO
40.667g/L, Citric acid monohydrate Food grade 0.355g/L, MgSO
47H
2o0.25g/L, CaCl
22H
2o0.0025g/L, defoamer 0.025g/L, micro-mother liquor 0.01g/L.
6. preparation method according to claim 1, is characterized in that, described micro-mother liquor comprises: FeSO
47H
2o4 ~ 6g/L, H
3bO
30.09 ~ 0.11g/L, CoCl
26H
2o0.09 ~ 0.11g/L, MnSO
4h
2o0.3 ~ .04g/L, ZnSO
47H
2o3.7 ~ 3.9g/L, NaMoO
42H
2o0.09 ~ 0.11g/L, CoSO
40.09 ~ 0.11g/L.
7. preparation method according to claim 6, is characterized in that, described micro-mother liquor comprises FeSO
47H
2o5g/L, H
3bO
30.1g/L, CoCl
26H
2o0.1g/L, MnSO
4h
2o0.33g/L, ZnSO
47H
2o3.8g/L, NaMoO
42H
2o0.1g/L, CoSO
40.1g/L.
8. preparation method according to claim 1, is characterized in that, controlling dissolved oxygen in step (2) is 25 ~ 30%, and the flow acceleration of glucose solution is 0.3 ~ 1.6%.
9. preparation method according to claim 1, is characterized in that, maintaining remaining sugar concentration in fermented liquid in step (2) is that 0.05 ~ 0.2g/L is to fermentation termination.
10. the preparation method according to any one of claim 1 ~ 9, is characterized in that, comprises the following steps:
(1) glucosamine is produced bacterium and accesses seed culture medium by inoculum size 0.5%, 37 DEG C, pH is 6 ~ 8, be cultured to logarithmic phase under the condition of dissolved oxygen 20 ~ 30%;
(2) product that step (1) obtains is accessed fermention medium by inoculum size 10%, pass into oxygen, control dissolved oxygen is 25 ~ 30%, pH is 7 ± 0.2, be ferment under the condition of 37 ± 0.5 DEG C in temperature, when the concentration of glucose reduces to 0.01 ~ 0.2g/L, start stream and add the glucose solution that concentration is 60%, and in glucose solution, add micro-mother liquor, the max-flow acceleration of glucose solution is 1.6%, and maintaining remaining sugar concentration in fermented liquid is that 0.05 ~ 0.2g/L is to fermentation termination.
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| CN110577977A (en) * | 2019-10-09 | 2019-12-17 | 山东润德生物科技有限公司 | Method for producing glucosamine by fermenting escherichia coli |
| CN110760622A (en) * | 2019-12-17 | 2020-02-07 | 大自然生物集团有限公司 | Material supplementing method for producing glucosamine through fermentation of bacillus subtilis |
| CN110760622B (en) * | 2019-12-17 | 2023-06-27 | 大自然生物集团有限公司 | Feeding method for producing glucosamine by fermenting bacillus subtilis |
| CN111494309A (en) * | 2020-03-29 | 2020-08-07 | 耶赛明(南通)保健有限公司 | Glucosamine liquid preparation and preparation method thereof |
| CN111494309B (en) * | 2020-03-29 | 2021-07-23 | 技源健康科技(江苏)有限公司 | Glucosamine liquid preparation and preparation method thereof |
| CN114592020A (en) * | 2020-12-07 | 2022-06-07 | 上海医药工业研究院 | Method for producing N-acetylglucosamine by fermentation |
| CN114592020B (en) * | 2020-12-07 | 2023-07-14 | 上海医药工业研究院 | Method for producing N-acetylglucosamine by fermentation |
| CN112592944A (en) * | 2020-12-17 | 2021-04-02 | 内蒙古金达威药业有限公司 | Production method of glucosamine |
| CN112592944B (en) * | 2020-12-17 | 2023-09-29 | 内蒙古金达威药业有限公司 | Production method of glucosamine |
| CN113142215A (en) * | 2021-04-09 | 2021-07-23 | 浙江大学 | Application of glucosamine serving as pre-harvest sprouting inhibitor in inhibition of pre-harvest sprouting of hybrid rice |
| CN113142215B (en) * | 2021-04-09 | 2022-05-06 | 浙江大学 | Application of glucosamine serving as pre-harvest sprouting inhibitor in inhibition of pre-harvest sprouting of hybrid rice |
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