1. A process for refining glucosamine complex salt prepared by a microbial fermentation method is characterized by comprising the following steps:

(1) inoculating a fermentation microbial inoculum to a culture medium for fermentation to obtain fermentation liquor containing glucosamine, removing thalli in the obtained fermentation liquor to obtain clear liquid, adding a hydrochloric acid solution into the clear liquid for hydrolysis, and adding a neutralizing agent to neutralize redundant hydrochloric acid after the hydrolysis is finished to obtain hydrolysate containing glucosamine hydrochloride;

(2) adding a decolorizing agent into the hydrolysate, stirring, standing, and separating to obtain decolorized solution for later use;

(3) carrying out ultrafiltration treatment on the decolorized solution, and then concentrating the obtained filtrate to obtain a concentrated solution for later use;

(4) and adding sodium chloride into the concentrated solution, and then preparing the concentrated solution into freeze-dried powder to obtain the refined glucosamine compound salt.

2. The process for refining glucosamine complex salt prepared by microbial fermentation according to claim 1, wherein in the step (1), the concentration of the hydrochloric acid solution is preferably controlled to be 30-35%, and the addition amount of the hydrochloric acid solution is more than 25% of the volume of the hydrolysate.

3. The process for refining glucosamine complex salt prepared by microbial fermentation according to claim 1, wherein in the step (1), the hydrolysis temperature is 60-80 ℃ and the hydrolysis time is 3-5 hours.

4. The process for refining glucosamine complex salt prepared by microbial fermentation according to claim 1, wherein in step (1), the neutralizing agent comprises at least one of sodium hydroxide, sodium carbonate and sodium bicarbonate.

5. The process for refining glucosamine complex salt prepared by microbial fermentation according to claim 1, wherein in the step (2), the decolorizing agent is activated carbon; preferably, the mass volume ratio (m/v) of the activated carbon to the hydrolysate is 0.3-0.8 g: 10 ml.

6. The process for refining the glucosamine complex salt prepared by the microbial fermentation method according to claim 1, wherein the ultrafiltration treatment in the step (3) is completed in an ultrafiltration device, and the pore size of the ultrafiltration membrane is 35-50 nm.

7. The process for refining glucosamine complex salt prepared by microbial fermentation according to claim 1, wherein in the step (4), the preparation method of the lyophilized powder comprises the following steps: freezing the concentrated solution into solid, vacuumizing to sublimate water in the solid, and grinding the solid into micro powder to obtain the product.

8. The process for refining glucosamine complex salt prepared by microbial fermentation according to claim 7, wherein the concentrated solution is rapidly frozen to-8-15 ℃ to form a solid, and then the solid is vacuumized to 0.1-0.25 mbra to sublimate water in the solid.

9. The process for refining glucosamine complex salt prepared by any one of claims 1 to 8, wherein the amount of sodium chloride added in step (4) is 0.1 to 0.15% by mass of the concentrated solution.

Background

The research shows that: glucosamine sulfate as a latest generation product of the glucosamine osteoarthritis treatment drug can be directly absorbed by human bodies, and the glucosamine sulfate is a high-specificity drug which is internationally recognized for treating osteoarthritis and has the structure improvement effect on primary joint degenerative change and secondary osteoarthritis sports articular cartilage injury by using nutrient substances for supplementing proteoglycan synthetic matrixes. The early supplement of glucosamine sulfate can well prevent and treat articular cartilage diseases.

However, glucosamine sulfate is very hygroscopic and undergoes non-enzymatic browning reactions or oxidation reactions. Therefore, it is very difficult to mass-produce and store high-purity glucosamine sulfate. Moreover, the instability of glucosamine sulfate greatly limits the large-scale production and application of glucosamine sulfate, and the stability of glucosamine sulfate containing sodium chloride (namely, double salt) is obviously improved relatively. Compared with the method for extracting glucosamine from biomasses such as shrimp shells by using concentrated hydrochloric acid hydrolysis, the method for preparing the glucosamine by using a microbial fermentation method is more environment-friendly. The method is generally characterized in that the glucosamine is synthesized by microbial fermentation in a culture medium, and then a metabolite is extracted from a fermentation liquid to obtain a glucosamine crude product. Currently, some methods used for refining a glucosamine crude product include dissolving the crude product, heating for concentration, adding ethanol to the concentrated solution, and cooling for recrystallization. However, the method has the following defects: (1) face the problem of separation and recovery of ethanol. (2) The product quality needs to be improved. (3) The recrystallization yield is low, and the problem of recycling of mother liquor is faced.

Disclosure of Invention

The invention aims to provide a process for refining glucosamine complex salt prepared by a microbial fermentation method, which can not only obtain the refined glucosamine complex salt, but also avoid the problems of low recrystallization yield and cyclic utilization of mother liquor. In order to realize the purpose, the invention discloses the following technical scheme:

a process for refining glucosamine complex salt prepared by a microbial fermentation method comprises the following steps:

(1) inoculating the fermentation inoculum to a culture medium for fermentation to obtain fermentation liquor containing glucosamine, removing thalli in the obtained fermentation liquor to obtain clear liquid, adding a hydrochloric acid solution into the clear liquid for hydrolysis, and adding a neutralizing agent to neutralize redundant hydrochloric acid after the hydrolysis is finished to obtain hydrolysis liquid containing glucosamine hydrochloride.

(2) Adding a decolorizing agent into the hydrolysate, stirring, standing, and separating to obtain decolorized solution for later use.

(3) And carrying out ultrafiltration treatment on the decolorized solution, and then concentrating the obtained filtrate to obtain a concentrated solution for later use.

(4) And adding sodium chloride into the concentrated solution, and then preparing the concentrated solution into freeze-dried powder to obtain the refined glucosamine compound salt.

Further, in the step (1), the concentration of the hydrochloric acid solution is preferably controlled to be 30-35%, and the addition amount of the hydrochloric acid solution is more than 25% of the volume of the hydrolysate, such as 25-30%.

Further, in the step (1), the hydrolysis temperature is 60-80 ℃, and the hydrolysis time is 3-5 hours. Compared with the hydrolysis by adopting concentrated hydrochloric acid, the hydrolysis by adopting sulfuric acid has lower reaction temperature and milder reaction, and the concentrated hydrochloric acid is easier to volatilize when being hydrolyzed under heating condition, and the problem can be avoided by adopting sulfuric acid.

Further, in the step (1), the neutralizing agent includes at least one of sodium hydroxide, sodium carbonate and sodium bicarbonate. The excessive hydrochloric acid in the hydrolysate is removed by the alkaline neutralizers to obtain the glucosamine sodium sulfate, and simultaneously, the generated carbon dioxide overflows from the hydrolysate, thereby avoiding introducing new impurities into the product.

Further, in the step (2), the decolorizing agent is activated carbon, and optionally, the mass-to-volume ratio (m/v) of the activated carbon to the hydrolysate is 0.3-0.8 g: 10 ml. The activated carbon can effectively remove the pigment and partial impurities in the hydrolysate.

Further, in the step (3), the ultrafiltration treatment is completed in ultrafiltration equipment, wherein the aperture of the ultrafiltration membrane is 35-50 nm. Macromolecular impurities in the decolored solution can be removed through ultrafiltration treatment, and the product purity is improved.

Further, in the step (4), the preparation method of the freeze-dried powder comprises the following steps: freezing the concentrated solution into solid, vacuumizing to sublimate water in the solid, and grinding the solid into micro powder to obtain the product.

Further, the concentrated solution is rapidly frozen to the temperature of-8-15 ℃ to form a solid, then the solid is vacuumized to 0.1-0.25 mbra, so that the water in the solid is sublimated, and the process is a physical change process, so that the glucosamine sulfate in the solid cannot be influenced.

Further, in the step (4), the addition amount of the sodium chloride is 0.1-0.15% of the mass of the concentrated solution. The stability of the glucosamine sulfate can be improved by adding a small amount of sodium chloride, and the product deterioration caused by non-enzymatic browning reaction or oxidation reaction due to moisture absorption of the glucosamine sulfate is avoided.

Compared with the prior art, the invention has the following beneficial effects: in the conventional process for preparing a glucosamine product by using hydrolyzed chitin, when a crude glucosamine product is refined in a subsequent process, a method of dissolving the crude glucosamine product, heating and concentrating the dissolved crude glucosamine product, adding ethanol into a concentrated solution, and cooling the concentrated solution for recrystallization is generally adopted. However, this method has major disadvantages in that the recovery treatment of the mother liquor after the recrystallization, the recovery treatment of ethanol, and the low yield of the recrystallization cause a problem of cost increase. In the invention, firstly, the impurities in the product are effectively removed through the activated carbon and ultrafiltration treatment, and the purity of the product is improved. The concentrated solution after impurity removal is directly prepared into freeze-dried powder, so that mother solution generation is avoided, namely, the problem of recycling residual mother solution after concentration and crystallization in the traditional process is solved, because in the traditional process, partial product in the concentrated solution is gradually crystallized and separated out after the temperature of the concentrated solution is reduced, then the crystallized products are separated out by methods such as filtration, centrifugation and the like, but in the residual liquid (namely, the mother solution), more than 10% of the product can not be separated out and remains in the mother solution, and the partial mother solution is often used for recrystallization again after being applied, but the method can not fundamentally solve the problem because the crystallization rate of the product in the mother solution is not improved essentially, and only mechanical recrystallization is performed on the mother solution for many times. The invention solves the problem that the traditional process can generate mother liquor by adopting the technical idea of preparing the purified concentrated solution into the freeze-dried powder, and also solves the problem of low product yield caused by the fact that the mother liquor carries a part of products which can not be recovered. Meanwhile, no chemical reagent is needed in the preparation process of the freeze-dried powder, so that the problems that ethanol needs to be recycled and the process is complicated and the cost is increased due to the fact that ethanol is used as a precipitator in the traditional process are solved.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. The invention will now be further illustrated by specific examples.

In the following examples, the seed culture medium was uniformly prepared using the existing recipe: 11.5g/L of peptone, 20g/L of yeast extract powder, 10g/L of cysteine, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 5.5g/L of glycerol, and the pH is adjusted to 6.5 by using ammonia water. During the culture, the culture temperature is controlled at 37 ℃, the shaking table speed is 230rpm, and the culture time is 12 h.

The fermentation medium uniformly adopts the existing formula: 25g/L of glucose, 20g/L of yeast extract powder, 3g/L of sodium nitrate, 1.05g/L of dipotassium phosphate, 0.1g/L of sodium chloride, 0.5g/L of magnesium sulfate, 0.03g/L of ferrous sulfate, 3.2g/L of lactose and 5.5g/L of glycerol. During the culture, the culture temperature is 37 ℃, the shaking table speed is 230rpm, the culture time is 40h, and the feed supplement speed of glucose is adjusted to be 3.8L/h.

The fermentation inoculum adopts the following components in a uniform volume ratio of 3: 0.6: 1.5 of Escherichia coli, saccharomyces cerevisiae and staphylococcus.

Example 1

A process for refining glucosamine complex salt prepared by a microbial fermentation method comprises the following steps:

(1) inoculating the mixed fermentation microbial inoculum into a seed culture medium for activated culture, inoculating the mixed fermentation microbial inoculum into the fermentation culture medium according to 13% of the inoculation amount for fermentation culture, and obtaining fermentation liquor containing glucosamine after completion.

(2) Centrifuging the fermentation liquor to remove solid substances such as thalli, collecting supernatant, and adding a hydrochloric acid solution with the mass concentration of 30% into the supernatant, wherein: the addition of the hydrochloric acid solution is 30% of the volume of the hydrolysate, the hydrolysate is hydrolyzed for 4 hours under the heating condition of 65 ℃, sodium hydroxide is added to neutralize the redundant hydrochloric acid after the hydrolysis is finished, and then the hydrolysate containing glucosamine sulfate is filtered and separated.

(3) According to the mass volume ratio of the active carbon to the hydrolysate of 0.35 g: and (3) adding active carbon into the hydrolysate obtained in the step (2) according to the proportion of 10ml, decoloring for 25 minutes at the temperature of 40 ℃, and filtering the active carbon to obtain a decolored solution.

(4) And (3) putting the decolorized solution into a membrane separation system, and purifying the decolorized solution by adopting an ultrafiltration membrane, wherein the mass transfer driving force is the water pressure difference (15 kg). In the ultrafiltration process, the decolorized solution flows across the surface of the membrane under the push of pressure, water and glucosamine sulfate molecules pass through the ultrafiltration membrane to become filtrate, and macromolecular impurities are intercepted by the ultrafiltration membrane.

(5) And (3) concentrating the filtrate obtained in the step (4) at 65 ℃ to 50% of the original volume to obtain a concentrated solution, and then adding 0.1% of sodium chloride by mass into the concentrated solution. And finally, placing the concentrated solution in a freeze dryer, quickly freezing to the temperature of minus 10 ℃ to form a solid, then vacuumizing to 0.2mbra to sublimate water in the solid, and grinding the solid into micro powder after the sublimation is finished to obtain the refined glucosamine composite salt powder.

Example 2

A process for refining glucosamine complex salt prepared by a microbial fermentation method comprises the following steps:

(1) inoculating the mixed fermentation microbial inoculum into a seed culture medium for activated culture, inoculating the mixed fermentation microbial inoculum into the fermentation culture medium according to 13% of the inoculation amount for fermentation culture, and obtaining fermentation liquor containing glucosamine after completion.

(2) Centrifuging the fermentation liquor to remove solid substances such as thalli and the like, and collecting supernatant, wherein: adding hydrochloric acid solution 30% of the hydrolysate volume, hydrolyzing the hydrolysate at 80 deg.C for 5 hr, adding sodium carbonate to neutralize excessive hydrochloric acid, and filtering to separate out hydrolysate containing glucosamine sulfate.

(3) According to the mass volume ratio of the active carbon to the hydrolysate of 0.7 g: and (3) adding active carbon into the hydrolysate obtained in the step (2) according to the proportion of 10ml, decoloring for 20 minutes at the temperature of 40 ℃, and filtering the active carbon to obtain a decolored solution.

(4) And (3) putting the decolorized solution into a membrane separation system, and purifying the decolorized solution by adopting an ultrafiltration membrane, wherein the mass transfer driving force is the water pressure difference (15 kg). In the ultrafiltration process, the decolorized solution flows across the surface of the membrane under the push of pressure, water and glucosamine sulfate molecules pass through the ultrafiltration membrane to become filtrate, and macromolecular impurities are intercepted by the ultrafiltration membrane.

(5) And (3) concentrating the filtrate obtained in the step (4) at 65 ℃ to 50% of the original volume to obtain a concentrated solution, and then adding 0.15% of sodium chloride by mass into the concentrated solution to improve the stability of the product. And finally, placing the concentrated solution in a freeze dryer, quickly freezing to the temperature of minus 8 ℃ to form a solid, then vacuumizing to 0.2mbra to sublimate water in the solid, and grinding the solid into micro powder after the sublimation is finished to obtain the refined glucosamine composite salt powder.

Example 3

A process for refining glucosamine complex salt prepared by a microbial fermentation method comprises the following steps:

(1) inoculating the mixed fermentation microbial inoculum into a seed culture medium for activated culture, inoculating the mixed fermentation microbial inoculum into the fermentation culture medium according to 13% of the inoculation amount for fermentation culture, and obtaining fermentation liquor containing glucosamine after completion.

(2) Centrifuging the fermentation liquor to remove solid substances such as thalli and the like, and collecting supernatant, wherein: adding hydrochloric acid solution with the volume of 25% of the hydrolysate, hydrolyzing the hydrolysate at 60 deg.C for 3 hr, adding sodium bicarbonate to neutralize excessive hydrochloric acid, and filtering to separate the hydrolysate containing glucosamine sulfate.

(3) According to the mass volume ratio of the active carbon to the hydrolysate of 0.8 g: and (3) adding active carbon into the hydrolysate obtained in the step (2) according to the proportion of 10ml, decoloring for 20 minutes at the temperature of 40 ℃, and filtering the active carbon to obtain a decolored solution.

(4) And (3) putting the decolorized solution into a membrane separation system, and purifying the decolorized solution by adopting an ultrafiltration membrane, wherein the mass transfer driving force is the water pressure difference (15 kg). In the ultrafiltration process, the decolorized solution flows across the surface of the membrane under the push of pressure, water and glucosamine sulfate molecules pass through the ultrafiltration membrane to become filtrate, and macromolecular impurities are intercepted by the ultrafiltration membrane.

(5) And (3) concentrating the filtrate obtained in the step (4) at 65 ℃ to 50% of the original volume to obtain a concentrated solution, and then adding 0.15% of sodium chloride by mass into the concentrated solution to improve the stability of the product. And finally, placing the concentrated solution in a freeze dryer, quickly freezing to the temperature of minus 12 ℃ to form a solid, then vacuumizing to 0.25mbra to sublimate water in the solid, and grinding the solid into micro powder after the sublimation is finished to obtain the refined glucosamine composite salt powder.

Example 4

A process for refining glucosamine complex salt prepared by a microbial fermentation method comprises the following steps:

(1) inoculating the mixed fermentation microbial inoculum into a seed culture medium for activated culture, inoculating the mixed fermentation microbial inoculum into the fermentation culture medium according to 13% of the inoculation amount for fermentation culture, and obtaining fermentation liquor containing glucosamine after completion.

(2) Centrifuging the fermentation liquor to remove solid substances such as thalli and the like, and collecting supernatant, wherein: adding hydrochloric acid solution with the volume of 25% of the hydrolysate, hydrolyzing the hydrolysate at 70 deg.C for 4 hr, adding sodium hydroxide to neutralize excessive hydrochloric acid, and filtering to separate the hydrolysate containing glucosamine sulfate.

(3) According to the mass volume ratio of the active carbon to the hydrolysate of 0.3 g: and (3) adding active carbon into the hydrolysate obtained in the step (2) according to the proportion of 10ml, decoloring for 20 minutes at the temperature of 40 ℃, and filtering the active carbon to obtain a decolored solution.

(4) And (3) putting the decolorized solution into a membrane separation system, and purifying the decolorized solution by adopting an ultrafiltration membrane, wherein the mass transfer driving force is the water pressure difference (15 kg). In the ultrafiltration process, the decolorized solution flows across the surface of the membrane under the push of pressure, water and glucosamine sulfate molecules pass through the ultrafiltration membrane to become filtrate, and macromolecular impurities are intercepted by the ultrafiltration membrane.

(5) And (3) concentrating the filtrate obtained in the step (4) at 65 ℃ to 50% of the original volume to obtain a concentrated solution, and then adding 0.12% of sodium chloride by mass into the concentrated solution to improve the stability of the product. And finally, placing the concentrated solution in a freeze dryer, quickly freezing to the temperature of minus 15 ℃ to form a solid, then vacuumizing to 0.1mbra to sublimate water in the solid, and grinding the solid into micro powder after the sublimation is finished to obtain the refined glucosamine composite salt powder.

Example 5

A process for refining glucosamine complex salt prepared by a microbial fermentation method comprises the following steps:

(1) inoculating the mixed fermentation microbial inoculum into a seed culture medium for activated culture, inoculating the mixed fermentation microbial inoculum into the fermentation culture medium according to 13% of the inoculation amount for fermentation culture, and obtaining fermentation liquor containing glucosamine after completion.

(2) Centrifuging the fermentation liquor to remove solid substances such as thalli and the like, and collecting supernatant, wherein: the addition of hydrochloric acid solution is 25% of the volume of the hydrolysate, then the hydrolysate is hydrolyzed for 4 hours under the heating condition of 65 ℃, sodium hydroxide is added to neutralize the redundant hydrochloric acid after the hydrolysis is finished, and then the hydrolysate containing glucosamine sulfate is filtered and separated for later use.

(3) According to the mass volume ratio of the active carbon to the hydrolysate of 0.35 g: and (3) adding active carbon into the hydrolysate obtained in the step (2) according to the proportion of 10ml, decoloring for 25 minutes at the temperature of 40 ℃, and filtering the active carbon to obtain a decolored solution.

(4) And (3) putting the decolorized solution into a membrane separation system, and purifying the decolorized solution by adopting an ultrafiltration membrane, wherein the mass transfer driving force is the water pressure difference (15 kg). In the ultrafiltration process, the decolorized solution flows across the surface of the membrane under the push of pressure, water and glucosamine sulfate molecules pass through the ultrafiltration membrane to become filtrate, and macromolecular impurities are intercepted by the ultrafiltration membrane.

(5) And (3) concentrating the filtrate obtained in the step (4) at 65 ℃ to 50% of the original volume to obtain a concentrated solution, cooling the concentrated solution to 5 ℃ for crystallization, filtering and separating out crystals after the crystals are separated out, and drying the crystals to obtain the glucosamine complex salt product.

Example 6

A process for refining glucosamine complex salt prepared by a microbial fermentation method comprises the following steps:

(1) inoculating the mixed fermentation microbial inoculum into a seed culture medium for activated culture, inoculating the mixed fermentation microbial inoculum into the fermentation culture medium according to 13% of the inoculation amount for fermentation culture, and obtaining fermentation liquor containing glucosamine after completion.

(2) Centrifuging the fermentation liquor to remove solid substances such as thalli and the like, and collecting supernatant, wherein: adding hydrochloric acid solution with the volume of 25% of the hydrolysate, hydrolyzing the hydrolysate at 65 deg.C for 4 hr, adding sodium carbonate to neutralize excessive hydrochloric acid, and filtering to separate the hydrolysate containing glucosamine sulfate.

(3) According to the mass volume ratio of the active carbon to the hydrolysate of 0.7 g: and (3) adding active carbon into the hydrolysate obtained in the step (2) according to the proportion of 10ml, decoloring for 20 minutes at the temperature of 40 ℃, and filtering the active carbon to obtain a decolored solution.

(4) And (3) putting the decolorized solution into a membrane separation system, and purifying the decolorized solution by adopting an ultrafiltration membrane, wherein the mass transfer driving force is the water pressure difference (15 kg). In the ultrafiltration process, the decolorized solution flows across the surface of the membrane under the push of pressure, water and glucosamine sulfate molecules pass through the ultrafiltration membrane to become filtrate, and macromolecular impurities are intercepted by the ultrafiltration membrane.

(5) And (3) concentrating the filtrate obtained in the step (4) at 65 ℃ to 50% of the original volume to obtain a concentrated solution, adding 20% of absolute ethyl alcohol by volume into the concentrated solution, cooling to 5 ℃ for crystallization, filtering to separate out crystals after the crystals are separated out, and drying the crystals to obtain the glucosamine complex salt product.

The purity and yield of the glucosamine complex salt prepared in examples 1 to 6 were calculated, and the results are shown in table 1.

TABLE 1

	Example 1	Example 2	Example 3	Example 4	Example 5	Example 6
							Purity/%)	99.89	99.95	99.92	99.96	99.88	99.95
Yield/%	95.8	94.6	96.4	95.5	79.7	86.2

The detection results in table 1 show that the yield of examples 1 to 4 is significantly higher than that of examples 5 and 6, because the purified concentrated solution is made into lyophilized powder, the problem that the traditional process generates mother solution is avoided, and the problem that the yield of the product is low due to the fact that the mother solution carries a part of the product which cannot be recovered is also avoided. Meanwhile, because no chemical reagent is needed in the preparation process of the freeze-dried powder, the problems that ethanol needs to be recycled and the process is complicated and the cost is increased due to the fact that ethanol is used as a precipitator in the traditional process are solved.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.