CN104498564A - Low molecular weight chondroitin sulfate preparation method - Google Patents
Low molecular weight chondroitin sulfate preparation method Download PDFInfo
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Abstract
A low molecular weight chondroitin sulfate preparation method relates to chondroitin sulfate. The method includes the steps of stewing sturgeon to remove flesh and fat on the surface of bone, soaking with petroleum ether, washing with ethanol, drying and pulverizing to obtain cartilage particles; adding a NaOH solution to the cartilage particles, stirring for the first time and adjusting the pH to neutral with acid to obtain a cartilage mother solution; adding malic acid to the cartilage mother solution, adjusting the pH to 3.0 to 3.2, adding pepsin, stirring for the second time, adjusting the pH to 5.0 to 5.5, adding papain and cellulase, stirring for the third time, adding activated carbon, stirring for the fourth time and filtering to obtain clarified enzymatic hydrolysate; subjecting the enzymatic hydrolysate to resin adsorption, gradient elution in a NaCl solution, phloroglucinol spectrophotometry detection and combined collection to obtain an eluent with color reaction; precipitating the eluate with ethanol, collecting precipitate through centrifugation and drying to obtain low molecular weight chondroitin sulfate crystals.
Description
Technical field
The present invention relates to chondroitin sulfate, especially relate to a kind of preparation method of low-molecular weight chondroitin sulfate.
Background technology
Chondroitin sulfate (Chondroitin sulfate, be called for short CS) be from the natural acidic mucopolysaccharide of a class of animal cartilage tissue, its sugar chain is formed by the disaccharide unit replaced (N-glucuronic acid and N-acetylamino galactosamine) and is connected on core protein.Research display, chondroitin sulfate plays an important role in treatment joint injury, corneal injury, neurodynia and antiphlogistic antibacterial etc., have simultaneously and promote coronary artery circulation, reducing blood-fat, anticoagulation, the multiple biological activity such as antiviral and antitumor, be widely used in the raw material of medicine, protective foods and cosmetic industry.
China is most important sturgeon big producing country in the world, and cultured output accounts for the world more than 85%.Sturgeon entire body is cartilage, and its skull, notochord bone and fin bone account for 10% of fish body, can make full use of and prepare chondroitin sulfate.At present, domestic extraction chondroitin sulfate uses more method to be the method that diluted alkaline or concentrated base are combined with enzyme.Wherein, alkaline hydrolysis effectively can interrupt the O type cardohydrata-peptide linkage that CS is connected with core protein, improve yield, but, complete alkaline hydrolysis (being dissolved as terminal completely with cartilage) generally needs 3 ~ 5h even longer time, the easy like this destruction causing chondroitin sulfate disaccharides structural unit, affects quality product and activity.
The chondroitin sulfate of high molecular, because its apparent viscosity is high, the factor such as the selective permeability of complex structure and cytolemma, cause its bioavailability low, oral absorption is poor and curative effect is unstable, and low-molecular weight chondroitin sulfate (low-molecular weight chondroitin sulfate) has the bioavailability high and activity functional performance such as by force, be more and more subject to people's attention.Can predict, the market requirement of low-molecular weight chondroitin sulfate product will increase severely year by year.But existing production technology all first obtains CS finished product by animal cartilage, then obtains low-molecular weight chondroitin sulfate product through degraded, or directly low-molecular weight chondroitin sulfate product prepared by the commercially available CS raw material of purchase.Its technique needs to repeat the operations such as purifying, precipitation, drying, and operation is numerous and diverse and cost is high.In addition, domestic chondroitin sulfate degradation technique research is less, and common degradation method has enzyme process, acid system, hydrogen peroxide method etc., but all has its incomplete place.Publication number is the preparation method that the Chinese patent application of CN101659973A and CN103602711A all relates to low molecular chondroitin sulfate, although enzyme process has, environmental pollution is little, reaction conditions is gentle, product purity advantages of higher, but the enzyme spcificity of degraded CS is high, expensive, source is rare, is not suitable for scale operation.Publication number is a kind of method that the Chinese patent application of CN1940082A discloses salt acid degradation and prepares low-molecular weight chondroitin sulfate, and its production cycle is long, and product purity is not high; Publication number is the preparation method that the Chinese patent application of CN1995071A relates to a kind of low-molecular weight chondroitin sulfate, but, hydrogen peroxide method degradation mechanism still imperfectly understands, within 2011, state food additive uses standard (GB2760-2011) to cancel hydrogen peroxide as the use of auxiliary agent in food-processing, and its security needs to be investigated further.Publication number is a kind of method that the Chinese patent application of CN103554304A relates to sturgeon cartilage and prepares lower molecular weight sturgeon chondroitin sulfate, although irradiation micronizing degraded chondroitin sulfate is simple and easy to do, pollution-free, but it first obtains macromolecule chondroitin sulfate, to obtain the processing step of low-molecular weight chondroitin sulfate product numerous and diverse in degraded again, production cycle is long, equipment requirements is high, and product molecular weight distribution is wider.So far, safe and reliable, there is industrialization potential, low-molecular weight chondroitin sulfate preparation technology that production cost is lower not yet occurs.
Summary of the invention
The object of the present invention is to provide with sturgeon cartilage is raw material, synchronously can carry out extraction and the degraded of chondroitin sulfate, reduce production stage, prevent the decomposition in low-molecular weight chondroitin sulfate production process, improve purity and the yield of product, step is simple, process stabilizing, quality product is high, with short production cycle, production cost is low, is applicable to the preparation method of a kind of low-molecular weight chondroitin sulfate of suitability for industrialized production.
The present invention includes following steps:
1) cartilage pre-treatment: boiled by sturgeon and remove the surface attachments such as muscle, fat, after soaking, with alcohol flushing, dries, pulverizes to obtain cartilage particles with sherwood oil;
2) oxygenation pretreatment: add NaOH solution at cartilage particles, first time with acid for adjusting pH to neutral, obtains cartilage mother liquor after stirring;
3) acid and ferment treatment: first add oxysuccinic acid in cartilage mother liquor, regulate pH to 3.0 ~ 3.2, add stomach en-again, second time stirs, and regulates pH to 5.0 ~ 5.5, then adds papoid and cellulase, after third time stirring, add gac, stir for the 4th time, filter to obtain clarification enzymolysis solution;
4) column purification is crossed: by step 3) gained enzymolysis solution is through resin absorption, and use NaCl solution gradient elution, utilize Phloroglucinol spectrophotometry, the elutriant with color reaction is collected in merging;
5) precipitation is dry: by elutriant alcohol settling, centrifugal collecting precipitation, after drying, obtains low-molecular weight chondroitin sulfate crystal.
In step 1) in, the time that described sherwood oil soaks can be 8 ~ 12h, preferred 10h.
In step 2) in, described cartilage grain and NaOH solution can be 1 ︰ (4 ~ 6) by solid-liquid ratio, preferably 1 ︰ 5, described NaOH solution can adopt mass percentage concentration be 4% ~ 6% NaOH solution; The condition that described first time stirs can stir 30 ~ 45min in 45 ~ 50 DEG C of constant temperature; Described acid can adopt hydrochloric acid, and the mass percentage concentration of described hydrochloric acid can be 10% ~ 30%.
In step 3) in, the condition that described second time stirs can stir 3 ~ 5h in 37 ~ 38 DEG C of constant temperature; Described adjustment pH to 5.0 ~ 5.5 can adopt 2 ~ 4mol/L NaOH solution to regulate; The condition that described third time stirs can stir 3 ~ 5h in 55 ~ 60 DEG C of constant temperature; The add-on of described gac by volume per-cent can be third time stir after gained solution 0.4% ~ 0.6%, preferably 0.5%; The described condition stirred for 4th time can stir 30 ~ 40min in 80 ~ 90 DEG C of constant temperature;
Described pepsic addition can be 0.8% ~ 1% of cartilage by mass percentage; Described papoid and cellulase two kinds of enzymes add total amount and can be 3% ~ 5% of cartilage by mass percentage, and the mass ratio of cellulase and papoid can be 1 ︰ (1 ~ 1.4).
In step 4) in, described column purification of crossing can adopt D202 strong basic type anion-exchange resin to carry out purifying, and the volumetric molar concentration of NaCl solution used can be 0.5 ~ 2.5mol/L, preferably 1 ~ 1.5mol/L; The described Phloroglucinol spectrophotometry that utilizes can utilize Phloroglucinol spectrophotometry at 558nm place.
In step 5) in, described ethanol can adopt final concentration be 60% ~ 70% ethanol; The time of described precipitation can be 6 ~ 8h; The condition of described drying can be placed in vacuum drying oven constant temperature 60 DEG C of drying 4 ~ 6h.
Gained low-molecular weight chondroitin sulfate content detection adopts HPLC method, and design parameter is as follows: with sigma shark chondroitine for reference substance, and HPLC method detects low-molecular weight chondroitin sulfate content.Moving phase: second nitrile ︰ pentanesulfonic acid sodium solution=1 ︰ 9; Chromatographic column: C
18(5 μm × 250mm × 4.6mm); Flow velocity: 0.8mL/min; Determined wavelength: 192nm; Sampling volume: 10 μ L.Precision takes reference substance, be mixed with concentration be 0.1,0.2,0.4,0.6,0.8, the standardized solution of 1.0mg/mL, measure by chromatographic condition.
The present invention is by the improvement to current alkalization process, adopt the pre-treatment of part alkaline hydrolysis in conjunction with complex enzyme hydrolysis technology, interrupt the O type cardohydrata-peptide linkage that part CS is connected with core protein within a short period of time, reduce follow-up complex enzyme hydrolysis difficulty, avoid the destruction of long-time alkaline purification to CS simultaneously, maintain its integrity and biological activity, improve quality product.
Advantage of the present invention and beneficial effect have:
1, the present invention is to propagate sturgeon cartilage artificially for raw material, its abundance, safe and reliable, has ensured the raw material supply that industrialization is produced.
2, the present invention is by the improvement to current alkalization process, adopt the pre-treatment of part alkaline hydrolysis in conjunction with complex enzyme hydrolysis technology, interrupt the O type cardohydrata-peptide linkage that part CS is connected with core protein within a short period of time, reduce follow-up complex enzyme hydrolysis difficulty, avoid the destruction of long-time alkaline purification to CS simultaneously, maintain its integrity and biological activity, improve quality product.Compared to traditional alkaline hydrolysis technique (alkaline hydrolysis time general 3 ~ 5h), its reaction times only needs 30 ~ 45min; Compared to direct combination enzymolysis, its enzymolysis time shortens to some extent, and product yield significantly improves.
3, the present invention first utilizes oxysuccinic acid and stomach en-to act in acid condition and obtains middle low-molecular weight chondroitin sulfate, then obtains low-molecular weight chondroitin sulfate product by papoid and the further enzymolysis of cellulase.For the technique that traditional first extraction macromole chondroitin sulfate is degraded again, the present invention adopts the technique of simultaneous extraction and degraded chondroitin sulfate, substantially reduces the production cycle; reduce production cost; and step is simple, process stabilizing, is applicable to low cost, large-scale production.
4, the low-molecular weight chondroitin sulfate products molecule weight range prepared of the present invention is 3000 ~ 6000 dalton, and content can reach 92.4%, and biological activity is high.
5, the reagent needed for present invention process, preparation all meet state food additive and use standard, and security is high, are applicable to the exploitation of food, medicine and healthcare products.
Accompanying drawing explanation
Fig. 1 is the HPLC collection of illustrative plates of the low-molecular weight chondroitin sulfate product that the embodiment of the present invention 2 obtains.
Fig. 2 is the HPLC collection of illustrative plates of the low-molecular weight chondroitin sulfate product that the embodiment of the present invention 3 obtains.
Fig. 3 is the 3D collection of illustrative plates of low-molecular weight chondroitin sulfate product within the scope of 190 ~ 400nm that the embodiment of the present invention 2 obtains.
Fig. 4 is the 3D collection of illustrative plates of low-molecular weight chondroitin sulfate product within the scope of 190 ~ 400nm that the embodiment of the present invention 3 obtains.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further illustrated.
The embodiment of the present invention comprises the following steps:
(1) cartilage pre-treatment: sturgeon is boiled and removes the surface attachments such as muscle, fat, and sherwood oil soaks, after alcohol flushing, oven dry, pulverizing obtain cartilage particles;
(2) oxygenation pretreatment: cartilage particles adds the NaOH solution of 4% ~ 6% by solid-liquid ratio 1 ︰ 5, after 45 ~ 50 DEG C of constant temperature stir 30 ~ 45min, salt acid for adjusting pH, to neutral, obtains cartilage mother liquor;
(3) acid and ferment treatment: cartilage mother liquor adds oxysuccinic acid and stomach en-, 3 ~ 5h is stirred in 37 ~ 38 DEG C of constant temperature, regulate pH to 5.0 ~ 5.5, add papoid and cellulase, after 55 ~ 60 DEG C of constant temperature stir 3 ~ 5h, add the gac of liquor capacity 0.5%, stir 30 ~ 40min in 80 ~ 90 DEG C of constant temperature, filter to obtain clarification enzymolysis solution;
(4) cross column purification: enzymolysis solution is through resin absorption, and different concns NaCl solution gradient elution, utilizes Phloroglucinol spectrophotometry at 558nm place, merge the elutriant collected and there is color reaction;
(5) precipitation is dry: elutriant final concentration is after the alcohol settling 6 ~ 8h of 60% ~ 70%, centrifugal collecting precipitation, is placed in vacuum drying oven constant temperature 60 DEG C of drying 4 ~ 6h, obtains low-molecular weight chondroitin sulfate crystal.
In step (2), the alkaline purification time is 30 ~ 45min.Adding oxysuccinic acid in step (3) to pH is 3.0 ~ 3.2, pepsic addition is 0.8% ~ 1% of cartilage quality, it is 3% ~ 5% of cartilage quality that papoid and cellulase two kinds of enzymes add total amount, and the mass ratio of cellulase and papoid is 1 ︰ (1 ~ 1.4).D202 strong basic type anion-exchange resin is selected to carry out purifying in step (4), NaCl concentration preferably 1 ~ 1.5mol/L.
Below provide specific embodiment:
Embodiment 1
Get after fresh sturgeon spine tissue is placed in boiling water bath 1h, reject the dirt settlings such as chiropractic table facial muscle meat, fat, sherwood oil soaks, 70 DEG C of oven dry after alcohol wash, pulverizer pulverizing, weighs for subsequent use.Take 30g cartilage particles, add 4%NaOH solution by solid-liquid ratio 1 ︰ 5, stir 30min in 45 DEG C of constant temperature.After salt acid for adjusting pH to 7.0, adding oxysuccinic acid to pH is 3.0, adds the stomach en-of cartilage quality 0.8%, stirs 4h in 37 DEG C of constant temperature, and period regulates pH to be stabilized in 3.0.Adjust pH to 5.0, add papoid and the cellulase of cartilage quality 3%, adding proportion is 1 ︰ 1, stirs 4h in 55 DEG C, and period regulates pH to be stabilized in 5.0.Add the gac of liquor capacity 0.5%, stir 30min in 80 DEG C of constant temperature, cool, filter to obtain enzymolysis solution.Regulate pH to 6.0, regulate NaCl concentration to be 0.25mol/L, adopt the absorption of macroporous strong-base type anionite-exchange resin, NaCl different concns gradient (0.5,1,1.5mol/L) wash-out, merges and collects elutriant.Elutriant final concentration is the alcohol settling 6h of 60% ~ 70%, centrifugal collecting precipitation, is placed in vacuum drying oven constant temperature 60 DEG C of dry 5h, obtains low-molecular weight chondroitin sulfate product.On the basis of the above, change the adding proportion of papoid and cellulase two kinds of enzymes, carry out 6 groups of experiments, thus determine the adding proportion of best enzyme.Experimental data is as shown in table 1.
Table 1
| Sequence number | Pepsin enzyme amount (%) | Xian ties up Su Mei ︰ papoid | Thick extraction yield (%) | Viscosity-average molecular weight (Da) |
| 1 | 0.8 | 1︰0.8 | 26.3 | 5188 |
| 2 | 0.8 | 1︰1 | 32.5 | 4195 |
| 3 | 0.8 | 1︰1.2 | 33.1 | 4670 |
| 4 | 0.8 | 1︰1.4 | 32.9 | 5326 |
| 5 | 0.8 | 1︰1.6 | 33.4 | 8223 |
| 6 | 0.8 | 1︰1.8 | 33.0 | 10174 |
Experimental result shows, the mass ratio of cellulase and papoid is better in 1 ︰ (1 ~ 1.4) scope.When adding proportion is lower than 1 ︰ 1, cannot make full use of the subsequent extracted effect of papoid, the thick extraction yield of low-molecular weight chondroitin sulfate is lower; When adding proportion is higher than 1 ︰ 1.4, the degradation efficiency of enzyme is not good, and viscosity-average molecular weight is higher, and product bioavailability is lower.
Embodiment 2
Get after fresh sturgeon spine tissue is placed in boiling water bath 1h, reject the dirt settlings such as chiropractic table facial muscle meat, fat, sherwood oil soaks, 70 DEG C of oven dry after alcohol wash, pulverizer pulverizing, weighs for subsequent use.Take 30g cartilage particles, add 4%NaOH solution by solid-liquid ratio 1 ︰ 5, stir 30min in 45 DEG C of constant temperature.After salt acid for adjusting pH to 7.0, adding oxysuccinic acid to pH is 3.0, adds the stomach en-of cartilage quality 1%, stirs 4h in 37 DEG C of constant temperature, and period regulates pH to be stabilized in 3.0.Adjust pH to 5.0, add papoid and the cellulase of cartilage quality 4%, adding proportion is 1.2 ︰ 1, stirs 4h in 55 DEG C, and period regulates pH to be stabilized in 5.0.Add the gac of liquor capacity 0.5%, stir 30min in 80 DEG C of constant temperature, cool, filter to obtain enzymolysis solution.Regulate pH to 6.0, regulate NaCl concentration to be 0.25mol/L, adopt the absorption of macroporous strong-base type anionite-exchange resin, NaCl different concns gradient (0.5,1,1.5mol/L) wash-out, merges and collects elutriant.Elutriant final concentration is the alcohol settling 6h of 60% ~ 70%, centrifugal collecting precipitation, is placed in vacuum drying oven constant temperature 60 DEG C of dry 5h, and obtain the low-molecular weight chondroitin sulfate product 9.81g that viscosity-average molecular weight is 4620Da, yield is 32.7%.Detect through HPLC method, its content is 88.9%.
Embodiment 3
Get after fresh sturgeon spine tissue is placed in boiling water bath 1h, reject the dirt settlings such as chiropractic table facial muscle meat, fat, sherwood oil soaks, 70 DEG C of oven dry after alcohol wash, pulverizer pulverizing, weighs for subsequent use.Take 30g cartilage particles, add 6%NaOH solution by solid-liquid ratio 1 ︰ 5, stir 45min in 50 DEG C of constant temperature.After salt acid for adjusting pH to 7.0, adding oxysuccinic acid to pH is 3.0, adds the stomach en-of cartilage quality 1%, stirs 4h in 37 DEG C of constant temperature, and period regulates pH to be stabilized in 3.0.Adjust pH to 5.5, add papoid and the cellulase of cartilage quality 4%, adding proportion is 1.4 ︰ 1, stirs 4h in 60 DEG C of constant temperature, and period regulates pH to be stabilized in 5.5.Add the gac of liquor capacity 0.5%, stir 30min in 90 DEG C, cool, filter to obtain enzymolysis solution.Regulate pH to 6.0, regulate NaCl concentration to be 0.25mol/L, adopt the absorption of macroporous strong-base type anionite-exchange resin, NaCl different concns gradient (0.5,1,1.5mol/L) wash-out, merges and collects elutriant.Elutriant final concentration is the alcohol settling 8h of 60% ~ 70%, centrifugal collecting precipitation, is placed in vacuum drying oven constant temperature 60 DEG C of dry 5h, and obtain the low-molecular weight chondroitin sulfate product 10.53g that viscosity-average molecular weight is 3863Da, yield is 35.1%.Detect through HPLC method, its content is 92.4%.
Above-mentioned obtained low-molecular weight chondroitin sulfate product is analyzed:
(1) with sigma shark chondroitine for reference substance, HPLC method detects low-molecular weight chondroitin sulfate content.Moving phase: second nitrile ︰ pentanesulfonic acid sodium solution=1 ︰ 9; Chromatographic column: C
18(5 μm × 250mm × 4.6mm); Flow velocity: 0.8mL/min; Determined wavelength: 192nm; Sampling volume: 10 μ L.Precision takes reference substance, be mixed with concentration be 0.1,0.2,0.4,0.6,0.8, the standardized solution of 1.0mg/mL, measure by chromatographic condition.With peak area A for X-coordinate, with reference substance concentration C for ordinate zou is mapped, drawing standard curve, obtains regression equation A=7.5 × 10
6c+4.0 × 10
5, R
2=0.9997.The product configuration that embodiment 2 and embodiment 3 obtain is become the solution of 1.0mg/mL, measure by chromatographic condition, as shown in Figure 1 and Figure 2, its 3D collection of illustrative plates within the scope of 190 ~ 400nm as shown in Figure 3, Figure 4 for result.The low-molecular weight chondroitin sulfate product content that known embodiment 2 and embodiment 3 obtain as calculated is respectively 88.9% and 92.4%.From Fig. 3 and 4, products obtained therefrom does not detect the ultraviolet absorption peak of other impurity such as protein, nucleic acid.
(2) viscosity method is adopted to survey low-molecular weight chondroitin sulfate viscosity-average molecular weight.Accurately take 100mg sample and be settled to 100mL, obtain the low-molecular weight chondroitin sulfate mother liquor of 1mg/mL.Take purified water as contrast, measure purified water elution time T0 and sample elution time Ti, get the experimental data that 3 errors are less than 0.2s and average.Through converting, obtaining limiting viscosity [η], [η] is substituted into formula [η]=KM
w α, show that embodiment 2 product and embodiment 3 product viscosity-average molecular weight are respectively 4620Da and 3863Da, wherein K=1.97 × 10
-4, α=0.627, M
wrepresent viscosity-average molecular weight.
To sum up, take sturgeon cartilage as raw material, optimize alkali pretreatment, select oxysuccinic acid in conjunction with the method for stomach en-, papoid and cellulase, synchronously carry out extraction and the degraded of chondroitin sulfate, again through macroporous type anionite-exchange resin purification process, obtain molecular weight ranges at 3000 ~ 6000 daltonian low-molecular weight chondroitin sulfate products, content can reach 92.4%.Present invention improves over traditional technology to need first to extract macromole chondroitin sulfate finished product, degrade again, technique thinking that low-molecular weight chondroitin sulfate is prepared in purifying, drying, acid is adopted synchronously to carry out extraction and the degraded of chondroitin sulfate in conjunction with enzyme process, greatly reduce production stage, reduce production cost, and process stabilizing, cycle is shorter, product safety is reliable, and quality is high, is applicable to suitability for industrialized production.
Claims (10)
1. a preparation method for low-molecular weight chondroitin sulfate, is characterized in that comprising the following steps:
1) cartilage pre-treatment: boiled by sturgeon and remove the surface attachments such as muscle, fat, after soaking, with alcohol flushing, dries, pulverizes to obtain cartilage particles with sherwood oil;
2) oxygenation pretreatment: add NaOH solution at cartilage particles, first time with acid for adjusting pH to neutral, obtains cartilage mother liquor after stirring;
3) acid and ferment treatment: first add oxysuccinic acid in cartilage mother liquor, regulate pH to 3.0 ~ 3.2, add stomach en-again, second time stirs, and regulates pH to 5.0 ~ 5.5, then adds papoid and cellulase, after third time stirring, add gac, stir for the 4th time, filter to obtain clarification enzymolysis solution;
4) column purification is crossed: by step 3) gained enzymolysis solution is through resin absorption, and use NaCl solution gradient elution, utilize Phloroglucinol spectrophotometry, the elutriant with color reaction is collected in merging;
5) precipitation is dry: by elutriant alcohol settling, centrifugal collecting precipitation, after drying, obtains low-molecular weight chondroitin sulfate crystal.
2. the preparation method of a kind of low-molecular weight chondroitin sulfate as claimed in claim 1, is characterized in that in step 1) in, the time that described sherwood oil soaks is 8 ~ 12h, preferred 10h.
3. the preparation method of a kind of low-molecular weight chondroitin sulfate as claimed in claim 1, is characterized in that in step 2) in, described cartilage grain and NaOH solution are 1 ︰ (4 ~ 6) by solid-liquid ratio, preferably 1 ︰ 5.
4. the preparation method of a kind of low-molecular weight chondroitin sulfate as claimed in claim 1, is characterized in that in step 2) in, described NaOH solution adopts mass percentage concentration to be the NaOH solution of 4% ~ 6%; The condition that described first time stirs can stir 30 ~ 45min in 45 ~ 50 DEG C of constant temperature; Described acid can adopt hydrochloric acid, and the mass percentage concentration of described hydrochloric acid can be 10% ~ 30%.
5. the preparation method of a kind of low-molecular weight chondroitin sulfate as claimed in claim 1, is characterized in that in step 3) in, the condition that described second time stirs stirs 3 ~ 5h in 37 ~ 38 DEG C of constant temperature; Described adjustment pH to 5.0 ~ 5.5 are that employing 2 ~ 4mol/L NaOH solution regulates; The condition that described third time stirs stirs 3 ~ 5h in 55 ~ 60 DEG C of constant temperature; The described condition stirred for 4th time stirs 30 ~ 40min in 80 ~ 90 DEG C of constant temperature.
6. the preparation method of a kind of low-molecular weight chondroitin sulfate as claimed in claim 1, is characterized in that in step 3) in, the add-on of described gac by volume per-cent is 0.4% ~ 0.6% of gained solution after stirring third time, preferably 0.5%.
7. the preparation method of a kind of low-molecular weight chondroitin sulfate as claimed in claim 1, is characterized in that in step 3) in, described pepsic addition is 0.8% ~ 1% of cartilage by mass percentage; It is 3% ~ 5% of cartilage by mass percentage that described papoid and cellulase two kinds of enzymes add total amount, and the mass ratio of cellulase and papoid is 1 ︰ (1 ~ 1.4).
8. the preparation method of a kind of low-molecular weight chondroitin sulfate as claimed in claim 1, it is characterized in that in step 4) in, described column purification of crossing adopts D202 strong basic type anion-exchange resin to carry out purifying, the volumetric molar concentration of NaCl solution used can be 0.5 ~ 2.5mol/L, preferably 1 ~ 1.5mol/L.
9. the preparation method of a kind of low-molecular weight chondroitin sulfate as claimed in claim 1, is characterized in that in step 4) in, the described Phloroglucinol spectrophotometry that utilizes utilizes Phloroglucinol spectrophotometry at 558nm place.
10. the preparation method of a kind of low-molecular weight chondroitin sulfate as claimed in claim 1, is characterized in that in step 5) in, described ethanol adopts final concentration to be the ethanol of 60% ~ 70%; The time of described precipitation can be 6 ~ 8h; The condition of described drying can be placed in vacuum drying oven constant temperature 60 DEG C of drying 4 ~ 6h.
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| CN106009068A (en) * | 2016-06-25 | 2016-10-12 | 仇颖超 | Preparation method of hyaluronic acid grafted micromolecular chondroitin sulfate composite |
| CN106397629A (en) * | 2016-08-30 | 2017-02-15 | 集美大学 | Method for extracting chondroitin sulfate from sturgeon bones, chondroitin sulfate extracted through method, and application of chondroitin sulfate |
| CN108610436A (en) * | 2018-03-28 | 2018-10-02 | 上海景峰制药有限公司 | A kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate |
| CN110218756A (en) * | 2019-05-31 | 2019-09-10 | 嫦娥创新（武汉）生物科技有限公司 | A kind of selenium-rich sturgeon bone peptide extracting method and product with Antiageing effect |
| CN111808214A (en) * | 2020-07-02 | 2020-10-23 | 舟山市齐晟水产有限公司 | Method for extracting chondroitin sulfate by using sturgeon cartilage |
| CN112106938A (en) * | 2020-09-24 | 2020-12-22 | 成都维德医疗器械有限责任公司 | Method for preparing hydrolyzed cartilage product |
| CN113293186A (en) * | 2021-05-11 | 2021-08-24 | 中国农业大学 | Low-molecular-weight sturgeon cartilage polysaccharide and preparation method thereof |
| CN114921511A (en) * | 2022-06-29 | 2022-08-19 | 晶昌明科技贸易(上海)有限公司 | Preparation method of fish cartilage hydrolysate rich in small-molecule chondroitin sulfate |
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| CN106009068A (en) * | 2016-06-25 | 2016-10-12 | 仇颖超 | Preparation method of hyaluronic acid grafted micromolecular chondroitin sulfate composite |
| CN106397629A (en) * | 2016-08-30 | 2017-02-15 | 集美大学 | Method for extracting chondroitin sulfate from sturgeon bones, chondroitin sulfate extracted through method, and application of chondroitin sulfate |
| CN106397629B (en) * | 2016-08-30 | 2019-08-20 | 集美大学 | Method, the chondroitin sulfate by this method extraction and the application of chondroitin sulfate are extracted from sturgeon fish-bone |
| CN108610436A (en) * | 2018-03-28 | 2018-10-02 | 上海景峰制药有限公司 | A kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate |
| CN110218756A (en) * | 2019-05-31 | 2019-09-10 | 嫦娥创新（武汉）生物科技有限公司 | A kind of selenium-rich sturgeon bone peptide extracting method and product with Antiageing effect |
| CN111808214A (en) * | 2020-07-02 | 2020-10-23 | 舟山市齐晟水产有限公司 | Method for extracting chondroitin sulfate by using sturgeon cartilage |
| CN112106938A (en) * | 2020-09-24 | 2020-12-22 | 成都维德医疗器械有限责任公司 | Method for preparing hydrolyzed cartilage product |
| CN113293186A (en) * | 2021-05-11 | 2021-08-24 | 中国农业大学 | Low-molecular-weight sturgeon cartilage polysaccharide and preparation method thereof |
| CN113293186B (en) * | 2021-05-11 | 2022-04-19 | 中国农业大学 | Low-molecular-weight sturgeon cartilage polysaccharide and preparation method thereof |
| CN114921511A (en) * | 2022-06-29 | 2022-08-19 | 晶昌明科技贸易(上海)有限公司 | Preparation method of fish cartilage hydrolysate rich in small-molecule chondroitin sulfate |
| CN114921511B (en) * | 2022-06-29 | 2023-12-22 | 晶昌明科技贸易(上海)有限公司 | Preparation method of fish cartilage hydrolysate rich in small molecular chondroitin sulfate |
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