CN110028533A - A kind of method and application of the refining amino glucosamine salt hydrochlorate from microbial fermentation solution - Google Patents
A kind of method and application of the refining amino glucosamine salt hydrochlorate from microbial fermentation solution Download PDFInfo
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Abstract
The method and application of the invention discloses a kind of from microbial fermentation solution refining amino glucosamine salt hydrochlorate, the refining methd include: to removal thallus after microbial fermentation solution in dilute hydrochloric acid is added, acidolysis is carried out in boiling water bath, active carbon is added to be adsorbed, it is subsequently added into alkali, pH value is adjusted to neutrality, hyperfiltration treatment is carried out using polypropylene filter, finally treatment fluid is successively concentrated in vacuo, low temperature crystallization, washing and vacuum drying, the amino acid hydrochloride salt refined.The present invention uses the process for refining of " acidolysis+activated carbon adsorption+ultrafiltration+concentration+crystallization+washing+drying ", microbial fermentation solution specifically containing N- acetyl D-amino glucose and D- Glucosamine is isolated and purified, the rate of recovery and the high aminoglucose hydrochloride of product purity are obtained；Solve the problems, such as that Glucosamine recovery rate is lower in microbial fermentation solution in the prior art and the period is long.
Description
Technical field
The present invention relates to the separation and extraction technology field of microbial fermentation product, more particularly to it is a kind of from microbial fermentation solution
The method and application of middle refining amino glucosamine salt hydrochlorate.
Background technique
Aminoglucose hydrochloride, also known as D-Glucosamine Hydrochloride are a kind of natural mucopolysaccharides, are a kind of no gas
Taste but slightly bitter white crystalline powder, it is soluble easily in water, do not dissolve in ethyl alcohol.Aminoglucose hydrochloride chemical industry, medicine,
The fields such as food and health care all have extensive use.Ammonia sugar can specifically act on articular cartilage, restore cartilage cell just
Normal metabolic function can stimulate cartilage cell to generate the proteoglycan with normal multimeric structure, inhibit the enzyme of damaged cartilage,
Delay the pathologic process of Osteoarthritis and the progress of disease, improves joint motion, relieve pain, therefore clinically a large amount of
Treatment for osteoarthritis.In addition, ammonia sugar also has important physiological action in vivo, such as there is the removing toxic substances of liver kidney, play anti-
Scorching, protect liver effect；And as antibacterial-anti-inflammatory drug, treat gastric ulcer etc..
The production method of aminoglucose hydrochloride can be divided into acid-hydrolysis method, enzymatic isolation method and microbe fermentation method, first two
Method obtains aminoglucose hydrochloride through acidolysis or enzymatic hydrolysis and produces using the chitin and chitosan extracted in shrimp and crab shells as raw material
Product.Acid hydrolyzation brings serious problem of environmental pollution due to the use of a large amount of concentrated acids, enzymatic isolation method due to degrading enzyme specificity and
The limitation of degradation efficiency has that the production cycle is long and low efficiency, production cost are higher.Production by Microorganism Fermentation amino
Glucose hydrochloride product salt, not by resource constraint, environmental pollution is small, and product is not present allergy effect, is in recent years without fishlike smell
Carry out the hot spot of domestic and foreign scholars' research and concern.
But microbe fermentation method is prepared in the production technology of glucosamine product, in addition to fermentation strain vigor and
Outside the control of technological condition for fermentation, it is also fermentation that separation how is extracted from fermentation liquid and obtains the glucosamine product of high-purity
A problem in production.In the production of existing real attenuation, using Recombinant organism as fermentation strain fermenting and producing
Fermentation liquid in, the product form containing two kinds of ammonia sugar of N-acetylglucosamine and Glucosamine isolates and purifies difficulty.This
Outside, ammonia sugar is mixed with impurity a large amount of in fermentation liquid, and such as ferment the raw material not run out of, the by-product in production process
Deng all limiting the extraction of target product, this also becomes the technical problem of restriction micro-organisms fermenting and producing ammonia sugar technique.
Existing isolation technics is based on storng-acid cation exchange resin, and there are purification cycles for separation purifying technique
It is long, and in fermentation liquid aminoglucose hydrochloride the rate of recovery less than 70%, cause the significant wastage of ammonia sugar product.
Therefore, how also to become urgently to be resolved in ammonia sugar zymotechnique from high efficiency extraction Glucosamine in fermentation liquid to ask
Topic.
Summary of the invention
In view of this, providing a kind of technique conjunction the technical problem to be solved by the present invention is in view of the deficiencies of the prior art
It manages, the aminoglucose hydrochloride separation purifying technique that the rate of recovery and product purity are high, solves in fermentation liquid in the prior art
The problem that Glucosamine recovery rate is lower and the period is long；And provide the aminoglucose hydrochloride of purification anti-pancreas in vivo
The new application of gland cancer.
Specific technical solution is as follows:
A method of the refining amino glucosamine salt hydrochlorate from microbial fermentation solution, comprising:
(1) microbial fermentation solution containing Glucosamine is taken, after removing thallus, supernatant is collected, obtains solution I；
(2) dilute hydrochloric acid is added into solution I, acidolysis is carried out in boiling water bath, obtains solution II；
The concentration of the dilute hydrochloric acid is 0.1~0.4mol/L, and the acidolysis time is 1~6h；
(3) active carbon is added into solution II to be adsorbed, after the completion of absorption, filtering obtains solution III；
In terms of the quality of solution II, the additive amount of active carbon is 2~5%, and the temperature of the absorption is 40~50 DEG C, the time
For 1~2h；
(4) alkali is added into solution III, adjusts pH value to neutrality, hyperfiltration treatment is carried out using polypropylene filter, is obtained molten
Liquid IV；
The molecular weight of the polypropylene filter is 1000~6000D, and the pressure of the ultrafiltration is 0.4~0.8Mpa；
(5) first solution IV is concentrated in vacuo, obtains concentrate, then ethyl alcohol is added into concentrate, consecutive low temperature stirs
Carry out crystallization treatment is mixed, is centrifuged after crystallization, aminoglucose hydrochloride crystallization is obtained；Then, aminoglucose hydrochloride
Washed and vacuum drying treatment is crystallized, the amino acid hydrochloride salt refined.
Further, in step (1), the microbial fermentation solution is the acetyl containing N- obtained by engineering bacteria fermentation
The fermentation liquid of D- Glucosamine and D- Glucosamine；The host strain of the genetic engineering bacterium is Escherichia coli.
The innovation of the invention consists in that: using the process for refining of " acidolysis+activated carbon adsorption+ultrafiltration+condensing crystallizing ", to spy
The fixed microbial fermentation solution containing N- acetyl D-amino glucose and D- Glucosamine is isolated and purified, and the rate of recovery is obtained
The high aminoglucose hydrochloride with product purity solves Glucosamine recovery rate in microbial fermentation solution in the prior art
The problem of length of lower and period.Especially, ultrafiltration membrane treatment step can be cooperated with acidolysis and activated carbon adsorption processing, will
It is difficult to the big molecular impurity removed filtering removal in microbial fermentation solution, significantly improves the recycling of aminoglucose hydrochloride
Rate.
Preferably, the revolving speed of the centrifugation is 10000~12000rpm in step (1), the time is 8~12min.
Further preferably, in step (2), the concentration of the dilute hydrochloric acid is 0.2mol/L, and the acidolysis time is 4h.
Further preferably, in step (3), in terms of the quality of solution II, the additive amount of active carbon is 4%, the absorption
Temperature is 40 DEG C, time 1h.
Further preferably, in step (4), the alkali is NaOH, and concentration is 0.3~0.8mol/L；The polypropylene filter
Molecular weight be 3000D, the pressure of ultrafiltration is 0.5Mpa.
Preferably, the temperature of the vacuum concentration is 50~70 DEG C in step (5), cycles of concentration is 20~30 times；
The temperature of the crystallization is 3-8 DEG C, and the time is 4~8h, and the volume ratio of concentrate and ethyl alcohol is 1:2~6, stirring
Rate is 100-180rpm/min；
The speed of the centrifugation is 5000-8000rpm/min；
When washing, mass fraction is used to be washed for 93%~97% ethanol water, washing times are 1~2 time；
The vacuum drying temperature is 50~80 DEG C, and the time is 4~10h.
The present invention also provides the aminoglucose hydrochlorides that the refining methd obtains.
And further existing, new application of the aminoglucose hydrochloride in anti-pancreatic cancer activity.
By the tumor mass quality and thymus index of measurement tumor-bearing mice, discovery separates pure the present invention from microbial fermentation solution
The aminoglucose hydrochloride of change can improve the immunocompetence of tumor-bearing mice, and by reducing tumor by improving thymus index
Block quality, to inhibit the growth of pancreatic cancer cell.
Compared with prior art, the invention has the following advantages:
(1) present invention uses the process for refining of " acidolysis+activated carbon adsorption+ultrafiltration+concentration+crystallization+washing+drying ", right
Microbial fermentation solution specifically containing N- acetyl D-amino glucose and D- Glucosamine is isolated and purified, and is returned
Yield and the high aminoglucose hydrochloride of product purity；It solves Glucosamine in microbial fermentation solution in the prior art to mention
Take rate lower and the problem of period length.
(2) present invention is equal by N-acetylglucosamine in fermentation liquid and Glucosamine using dilute hydrochloric acid processing fermentation liquid
It is converted into aminoglucose hydrochloride, efficiently separates purifying convenient for subsequent；Utilize the physical absorption principle of active carbon, removal hair
Pigment impurity in zymotic fluid；Utilize remaining protein, nucleic acid, colloidal solid and macromolecular in membrane filtration technique removal fermentation liquid
Impurity；By adding a certain proportion of dehydrated alcohol and low temperature chromatography, the crystalline rate of aminoglucose hydrochloride is improved.This
The method that invention provides reduces purification procedures, significantly improves the recycling of aminoglucose hydrochloride product in fermentation liquid
Efficiency, and have the characteristics that process flow is simple, at low cost, it can get the aminoglucose hydrochloride product of high-purity.
(3) refining methd provided by the invention is remarkably improved the recycling of aminoglucose hydrochloride in microbial fermentation solution
Rate, in activated carbon adsorption elution and membrane filtration process the rate of recovery of aminoglucose hydrochloride be 95.8%, vacuum concentration and
The rate of recovery of aminoglucose hydrochloride is 97.3% in crystallization purifying technique, and aminoglucose hydrochloride always recycles in fermentation liquid
Rate 93.2%, and the purity of aminoglucose hydrochloride sample is 94.6%.
Detailed description of the invention
Fig. 1 is microbial fermentation solution (ie in solution I) in embodiment 1 after the dilute hydrochloric acid of various concentration handles different time,
The changes of contents situation of N-acetylglucosamine in fermentation liquid.
Fig. 2 is to be added in fermentation refined solution (ie in solution IV) after the ethyl alcohol of different proportion in embodiment 1 to Glucosamine
The comparison situation of change of crystal of hydrochloride amount of precipitation.
Fig. 3 analyzes for the HPLC purity detecting of aminoglucose hydrochloride sample obtained in embodiment 1.
Fig. 4 is to grow in the aminoglucose hydrochloride sample body of 2 different dosing amount of embodiment to pancreatic cancer cell Pan02
Influence.
Fig. 5 is change of the aminoglucose hydrochloride sample to the thymus index of tumor-bearing mice of 2 different dosing amount of embodiment
Change situation.
Specific embodiment
The invention will be further described combined with specific embodiments below, and what is be exemplified below is only specific implementation of the invention
Example, but protection scope of the present invention is not limited only to this.Experimental method used in the following example unless otherwise specified, is
Conventional method, material, reagent used in embodiment etc., unless otherwise specified, commercially obtains.
Embodiment 1
1, the acquisition of Glucosamine microbial fermentation solution
The Glucosamine fermentation liquid that the present embodiment uses is fermented by art methods to be obtained, it may be assumed that by Escherichia coli
Engineering strain BL21 (DE3)-pET28a-ES-glmS-GAN1BL21 be seeded in fermentation medium and ferment, obtain
The fermentation liquid containing Glucosamine and N-acetylglucosamine obtained.
Particular content is shown in that publication No. is CN108588049A, entitled " a kind of Glucosamine synzyme, engineering bacteria and its answers
With " patent application text；Wherein, for engineering bacteria preparation process referring to Examples 1 and 2, fermentation prepares the process of Glucosamine
Referring to embodiment 5.
2, the purification of aminoglucose hydrochloride
(1) after taking Glucosamine microbial fermentation solution, 12000rpm to be centrifuged 10min, thallus is removed, collects supernatant,
Obtain solution I；
(2) acidolysis: the hydrochloric acid of 0.2mol/L being added into solution I, and acidolysis handles 4h in 100 DEG C of boiling water baths, obtains molten
Liquid II；
In order to make N-acetylglucosamine deacetylate in fermentation liquid, dilute salt of various concentration is added in solution I
Acid carries out 100 DEG C of boiling water bath processing, and the different disposal time is arranged, and detects the variation feelings of N-acetylglucosamine in fermentation liquid
Condition, shown in result figure 1.
The experimental results showed that the dilute hydrochloric acid of various concentration can reduce the content of N-acetylglucosamine in fermentation liquid,
And with the extension of time, the amount of its conversion is gradually promoted.After the HCl treatment 1h of 0.1mol/L, 44.2% in fermentation liquid
Acetylglucosamine can be converted into Glucosamine, and after handling 2h, conversion ratio is up to 56.7%.When the concentration of hydrochloric acid increases
When adding as 0.2mol/L, the conversion ratio of N-acetylglucosamine is respectively 58.2% and 72.8% when handling 1h and 2h.
Comprehensively consider time cost, transformation efficiency and concentration of hydrochloric acid, the hydrochloric acid that finally determining condition is 0.2mol/L boils
4h is handled in water-bath, with this condition, the conversion ratio of N-acetylglucosamine is up to 98.6% in fermentation liquid.
Wherein, the detection of N-acetylglucosamine is detected using HPLC method, and specific operating procedure is as follows: taking
Fermentation liquid after appropriate acidolysis, after 12000rom is centrifuged 10min, after taking suitable supernatant to be diluted with ultrapure water, by sample
0.22 μM of membrane filtration of dilution.High performance liquid chromatography detects N-acetylglucosamine content and uses amino post detection,
Mobile phase uses acetonitrile-phosphate buffer (65:35, pH 7.5), flow velocity 1.0mL/min, Detection wavelength 195nm, into
Sample volume is 10 μ L.
(3) activated carbon adsorption: at 40 DEG C, into solution II, (in terms of the quality of solution II, active carbon adds addition active carbon
Dosage is that 4%), absorption 1h (is handled primary) at interval of 10min concussion, after the completion of absorption, filters while hot, obtains filtered fluid, then
The ultrapure water elution that 0.6 times of volume of acid hydrolysis solution is added in active carbon filter residue after filtration is primary, and filtering obtains eluent, incited somebody to action
Filtrate and eluent merge, and obtain solution III；
After the completion of acidolysis process processing, in fermentation liquid remaining pigment will affect it is subsequent isolate and purify, and influence final
The quality of product.To reduce the pigment content in Glucosamine, using active carbon, to treated, fermentation liquid is carried out at absorption
Reason.
In 40 DEG C of waters, the activated carbon adsorption 1h (mass fraction 2%, 3%, 4% and 5%) of various concentration is added,
The concussion processing of period 10min is primary, comes into full contact with active carbon with fermentation liquid；It after having handled, filters while hot, work after filtration
Property charcoal filter residue in be added 0.6 times of volume of acid hydrolysis solution ultrapure water elution it is primary, filtering obtain eluent, by filtered fluid and eluent
Merge.
It is final to determine that selecting the amounts of activated carbon being added is 4% (mass ratio), this concentration by observing the color of solution III
Lower solution III is opposite to be clarified after absorption, significantly reduces the pigment content in fermentation liquid.
(4) film filters: the NaOH of 0.5mol/L being added into solution III, adjusts the pH value of solution III to neutrality, uses
The polypropylene filter that molecular cut off is 3000D carries out the hyperfiltration treatment that pressure is 0.5Mpa, obtains solution IV；
PH value to the neutrality, then use retention that the solution III after activated carbon adsorption is adjusted using the NaOH of 0.5mol/L is divided
The polypropylene that son amount is 3000D filters, and carries out hyperfiltration treatment to the fermentation liquid by acidolysis and activated carbon adsorption, removes fermentation liquid
In the small molecular weight impurities such as remaining protein, nucleic acid, colloidal solid and big molecular impurity and pigment, polypeptide.
It is computed, the content of aminoglucose grape sugar hydrochloride is 24.6g/L in the acid hydrolysis solution before absorption and ultrafiltration, by 1L
Acid hydrolysis solution obtains filtered fluid 1.42L after active carbon decolorization adsorption, elution and ultrafiltration membrance filter altogether, through detecting Glucosamine
Hydrochloride content is 16.6g/L, and the rate of recovery of aminoglucose hydrochloride is 95.8%.
(5) concentration and crystallization purifying: being concentrated solution IV first with Rotary Evaporators, and the temperature of concentration is 60 DEG C, will be molten
Liquid IV is concentrated 25 times, obtains concentrate (concentration for guaranteeing aminoglucose hydrochloride in concentrate is 30~50%)；It again will be dense
Contracting liquid is down to room temperature, takes 100mL concentrate that the ethyl alcohol of 5 times of volumes is added, according to 150rpm/min speed in low temperature chromatography cabinet
After stir process is stayed overnight, after 8000rpm is centrifuged 10min, the aminoglucose hydrochloride crystallization of acquisition is utilized to 95% ethyl alcohol
After solution washing is secondary, it is dried in vacuo 6h, the amino acid hydrochloride salt refined.
Solution IV after ultrafiltration membrane treatment is concentrated using Rotary Evaporators, the temperature of concentration is 60 DEG C, will
Filtered fluid is concentrated 25 times, and the Glucosamine concentration after detection concentration in solution is 33.5% (335g/L).
Concentrate is cooled to room temperature, takes 100mL concentrate that the ethanol solution of different volumes is added, chromatographs cabinet in low temperature
After being stayed overnight in (temperature is set as 4 DEG C) according to 150rpm/min speed stir process, after 8000rpm is centrifuged 10min, by acquisition
Aminoglucose hydrochloride crystallization using 95% ethanol solution wash it is secondary after, be dried in vacuo 6h, weighing, HPLC detection obtain
Sample purity.
As a result as shown in Fig. 2, having significant impact to the crystallization parsing of Glucosamine after different concentration of alcohol processing.
The crystallization that the ethyl alcohol of low concentration is unfavorable for aminoglucose hydrochloride is precipitated, after the ethyl alcohol of 1 times of volume is added, the amino Portugal of acquisition
The amount of grape sugar hydrochloride is only 10.6g, and the amount that its crystallization is precipitated with the increase of ethanol concentration steps up, when 4 times of bodies of addition
After long-pending ethyl alcohol, the aminoglucose hydrochloride amount being precipitated reaches 28.5g, when 5 times that the additional amount of ethyl alcohol is mother liquor volume
It is that the aminoglucose hydrochloride amount of precipitation reaches 32.6g, the later period with the promotion of concentration of alcohol, there is not the yield of crystal
The effect of increasing significantly.It is computed, when the ethyl alcohol that 5 times of volumes are added, after low temperature chromatographs crystallised overnight, can get 32.6g's
Aminoglucose hydrochloride crystallization, the rate of recovery of crystallization reach 97.3%.HPLC detects aminoglucose hydrochloride sample
Purity is 94.6%.
Comparative example 1
For this comparative example in addition to step (4) are adsorbed using ion exchange resin, remaining step is same as Example 1.
Specific step is as follows for ion exchange resin absorption:
(1) it selects cation exchange resin 001 × 8 to adsorb resin, after impregnating 12h with deionized water, washes resin repeatedly,
Resin 6h is impregnated using 4% hydrochloric acid solution of twice of resin volume, after deionized water is washed till neutrality, with about twice resin volume
4% or so sodium hydroxide solution impregnate resin 6h, deionized water is washed till neutrality, then with the 4% of about twice resin volume
Hydrochloric acid solution impregnate resin 4h, be washed with deionized water to water outlet be in neutrality it is rear spare.
(2) fermentation liquid after step (3) activated carbon adsorption elution is added in ion exchange column and is adsorbed, used
The ammonium sulfate elution of 1mol/L is eluted as eluent, obtains aminoglucose hydrochloride eluent.It is computed, from
Sub-exchange resin is 84.2% to the adsorption rate of aminoglucose hydrochloride in fermentation liquid, and ammonium sulfate is to glucosamine salt
The eluting rate 81.5% of hydrochlorate.
By the aminoglucose hydrochloride solution by cation exchange resin after purification, carried out using Rotary Evaporators dense
After 25 times of contracting, the solution being computed in aminoglucose hydrochloride is 37.6% (376g/L), by the Glucosamine after concentration
HCI solution is cooled to room temperature, and takes 100mL concentrate that the ethanol solution of 5 times of volumes is added, and (temperature is set in low temperature chromatography cabinet
Be set to 4 DEG C) according to 120rpm/min speed stir process overnight after, 8000rpm be centrifuged 10min after, by the aminoglucose of acquisition
After sugared crystal of hydrochloride washed once using 95% ethanol solution, it is dried in vacuo 4h, is weighed, the detected sample of HPLC is pure
Degree.
It is computed, in the aminoglucose hydrochloride solution of 100mL, obtains aminoglucose hydrochloride sample 35.6g altogether,
The rate of recovery of crystallization purifying is that the purity of 94.68%, HPLC detection aminoglucose hydrochloride sample is 98.3%.Comprehensive meter
It calculates, ion exchange resin isolation and purification method is 64.9% to the comprehensive recovery of aminoglucose hydrochloride in fermentation liquid.
The internal anti-pancreatic cancer activity research of 2 aminoglucose hydrochloride of embodiment
For the aminoglucose hydrochloride prepared using embodiment 1 as bulk pharmaceutical chemicals, vivo system evaluates its effect to cancer of pancreas.
The subcutaneous transplantation knurl model of cancer of pancreas is established in C57BL/6 black rat with the Pan02 pancreatic cancer cell of source of mouse,
Pan02 cell uses the RPMI 1640 culture medium containing 10% fetal calf serum to be resuspended, in 37 DEG C, 5% CO2Cell incubator in
Culture abandons the culture solution in culture bottle after cell adherent growth nearly 80%, and suitable pancreatin digestion is added after being cleaned with PBS,
It is rounded to attached cell, i.e., plus culture solution stops and 800r/min is centrifuged 5min, takes single cell secondary culture of appropriate ratio.
Amplification Pan02 cell digests cell, 800r/min is offline after the cell fusion degree of each culture bottle reaches 90%
5min, PBS are washed twice, and cell is resuspended with 1640 culture medium of RPMI without serum, cell number is made to reach 107/ mL, mixing are equal
It is even.By pallium cell injection into two flank stocks of C57BL/6 black rat (Shanghai western Poole-Bi Kai experimental animal Co., Ltd)
Subcutaneous, every 100 μ L of side of ditch.
After tumour transplatation 7 days, all model successfully, it is about the tumor mass of 5-8mm that groin, which has major diameter, and black rat is random
It is divided into 3 groups, respectively blank control group, low dosage aminoglucose hydrochloride group and high dose aminoglucose hydrochloride group,
Wherein physiological saline is given in the daily stomach-filling of blank control group, and low concentration is given in the daily stomach-filling of low dosage aminoglucose hydrochloride group
Aminoglucose hydrochloride sample, dosage 100mg/kg, the daily stomach-filling of high dose aminoglucose hydrochloride group gives
The aminoglucose hydrochloride sample of high concentration is given, dosage 300mg/kg after successive administration 21 days, carries out black rat
Disconnected neck is put to death, and is completely stripped tumor mass and is weighed, and the tumour inhibiting rate of each group nude mice is calculated, while removing black rat thymic tissue, is weighed
Calculate the situation of change of thymus index in each group.After wherein thymus index is dissection removing thymus gland, fascia and adipose tissue are removed,
It is weighed with electronic analytical balance, calculates and obtain according to following formula: thymus index (mg/g)=thymus gland weight in wet base (mg)/mouse weight
(g)。
There is certain inhibiting effect to the growth of cancer of pancreas using aminoglucose hydrochloride prepared by embodiment 1 in vivo, and
Concentration dependent is presented.The aminoglucose hydrochloride of 100mg/kg is to the growth inhibition ratio of cancer of pancreas Pan02 cell
18.2%, when the dosage of aminoglucose hydrochloride rises to 300mg/kg, the growth inhibition ratio to cell is
32.6%, the dosage of relatively low-dose improves 79.1%, compared with blank control group, low dosage aminoglucose hydrochloride and
High dose aminoglucose hydrochloride group all has significant difference (P < 0.05).
At the same time, the aminoglucose hydrochloride of high dose can significantly improve the thymus index of tumor-bearing mice, empty
The thymus index of white control group tumor-bearing mice is only 1.82, low dosage aminoglucose hydrochloride administration group and high dose amino Portugal
The thymus index of tumor-bearing mice is 1.96 and 2.14 in grape sugar hydrochloride administration group, is improved respectively compared with blank control group tumor-bearing mice
7.69% and 17.58%, and there is significant difference (P < 0.05) compared with blank control group.
This is the result shows that the aminoglucose hydrochloride that embodiment 1 prepares can be by the immunity energy of raising tumor-bearing mice
Power and play the effect of internal anti-pancreatic cancer.
Claims (10)
1. a kind of method of the refining amino glucosamine salt hydrochlorate from microbial fermentation solution characterized by comprising
(1) microbial fermentation solution containing Glucosamine is taken, after removing thallus, supernatant is collected, obtains solution I；
(2) dilute hydrochloric acid is added into solution I, acidolysis is carried out in boiling water bath, obtains solution II；
The concentration of the dilute hydrochloric acid is 0.1~0.4mol/L, and the acidolysis time is 1~6h；
(3) active carbon is added into solution II to be adsorbed, after the completion of absorption, filtering obtains solution III；
In terms of the quality of solution II, the additive amount of active carbon is 2~5%, and the temperature of the absorption is 40~50 DEG C, the time 1
~2h；
(4) alkali is added into solution III, adjusts pH value to neutrality, hyperfiltration treatment is carried out using polypropylene filter, obtains solution
IV；
The molecular weight of the polypropylene filter is 1000~6000D, and the pressure of the ultrafiltration is 0.4~0.8Mpa；
(5) first solution IV is concentrated in vacuo, obtains concentrate, then ethyl alcohol is added into concentrate, consecutive low temperature stir into
Row crystallization treatment is centrifuged after crystallization, obtains aminoglucose hydrochloride crystallization；Then, aminoglucose hydrochloride crystallizes
Washed and vacuum drying treatment, the amino acid hydrochloride salt refined.
2. as described in claim 1 from microbial fermentation solution refining amino glucosamine salt hydrochlorate method, which is characterized in that
In step (1), the microbial fermentation solution is the acetyl D-amino glucose containing N- and D- obtained by engineering bacteria fermentation
The fermentation liquid of Glucosamine；The host strain of the genetic engineering bacterium is Escherichia coli.
3. as described in claim 1 from microbial fermentation solution refining amino glucosamine salt hydrochlorate method, which is characterized in that
In step (1), the revolving speed of the centrifugation is 10000~12000rpm, and the time is 8~12min.
4. as described in claim 1 from microbial fermentation solution refining amino glucosamine salt hydrochlorate method, which is characterized in that
In step (2), the concentration of the dilute hydrochloric acid is 0.2mol/L, and the acidolysis time is 4h.
5. as described in claim 1 from microbial fermentation solution refining amino glucosamine salt hydrochlorate method, which is characterized in that
In step (3), in terms of the quality of solution II, the additive amount of active carbon is 4%, and the temperature of the absorption is 40 DEG C, time 1h.
6. as described in claim 1 from microbial fermentation solution refining amino glucosamine salt hydrochlorate method, which is characterized in that
In step (4), the alkali is NaOH, and concentration is 0.3~0.8mol/L；The molecular weight of the polypropylene filter is 3000D, ultrafiltration
Pressure be 0.5Mpa.
7. as described in claim 1 from microbial fermentation solution refining amino glucosamine salt hydrochlorate method, which is characterized in that
In step (5), the temperature of the vacuum concentration is 50~70 DEG C, and cycles of concentration is 20~30 times；
The temperature of the crystallization is 3-8 DEG C, and the time is 4~8h, and the volume ratio of concentrate and ethyl alcohol is 1:2~6, the rate of stirring
For 100-180rpm/min；
The speed of the centrifugation is 5000-8000rpm/min；
When washing, mass fraction is used to be washed for 93%~97% ethanol water, washing times are 1~2 time；
The vacuum drying temperature is 50~80 DEG C, and the time is 4~10h.
8. the aminoglucose hydrochloride that the refining methd as described in claim 1~7 obtains.
9. application of the aminoglucose hydrochloride as claimed in claim 8 in preparation anti-pancreatic cancer medicament.
10. application as claimed in claim 9, which is characterized in that the aminoglucose hydrochloride is by reducing tumor mass quality
With the growth for improving thymus index inhibition pancreatic cancer cell.
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| CN111018926A (en) * | 2019-12-17 | 2020-04-17 | 大自然生物集团有限公司 | Method for extracting high-purity glucosamine hydrochloride from glucosamine fermentation liquor |
| CN113402572A (en) * | 2021-06-18 | 2021-09-17 | 山东润德生物科技有限公司 | Process for refining glucosamine composite salt prepared by microbial fermentation method |
| CN113956300A (en) * | 2021-11-12 | 2022-01-21 | 莱特莱德(上海)技术有限公司 | Preparation method of glucosamine hydrochloride |
| CN115403638A (en) * | 2022-10-09 | 2022-11-29 | 山东润德生物科技有限公司 | Improved process for preparing glucosamine by fermentation method |
| CN115433244A (en) * | 2022-10-08 | 2022-12-06 | 山东润德生物科技有限公司 | Method for reducing hygroscopicity of glucosamine sulfate product |
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