CN104664454B - A kind of anti-trioxypurine peptide chelating calcium and its production and use - Google Patents
A kind of anti-trioxypurine peptide chelating calcium and its production and use Download PDFInfo
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Abstract
The invention discloses a kind of anti-trioxypurine peptide chelating calcium and its production and use, its preparation method comprises the following steps：Saury is rubbed, then heating stirring, then adjust pH value to 4.2, centrifugation takes precipitation, added protease hydrolyzed, supernatant, i.e. saury anti-trioxypurine peptide liquid are taken after enzymolysis product centrifugation；Calcium chloride solid is added, absolute ethyl alcohol and stirring is then added, centrifuged, taking precipitate vacuum drying obtains anti-trioxypurine peptide chelating calcium product.The present invention discharges the active fragment in saury albumen by modern biotechnology zymolysis technique, then prepares protein peptides chelating calcium product using wet method chelating.The bioactivity peptide fragment discharged from saury albumen has obvious anti-trioxypurine effect, simultaneously, the protein peptides have preferable chelated calcium effect, the anti-trioxypurine peptide chelating calcium product prepared not only there is significant anti-trioxypurine to act on, while solving the problems, such as to maintaining bone, the normal absorption of the vital calcium of muscle function and the loss utilized.
Description
Technical field
The invention belongs to the intensive processing field of saury, and in particular to a kind of anti-trioxypurine peptide from saury is chelated
Calcium, and its production and use.
Background technology
Uric acid is the metabolite of purine in endogenous purine and diet, the nucleic acid mainly decomposed by cell metabolism and other
Purine compound is decomposed through enzyme effect.Uric acid 20%, which is derived from, in human body is rich in purine or nucleic acid-protein food, and category is outer
Source property；And 80% is metabolized from endogenous purine.There is nearly 70% to pass through kidney excretion, one in the uric acid that human body is produced daily
The generation of denier uric acid is excessive and/or excretion is reduced, and will cause the rising of serum uric acid level, when blood uric acid persistently rises, then can draw
Deposition of the lithate in soft tissue is played, and then induces hyperuricemia and gout or even involves life.
With expanding economy, the living standard of people is greatly enhanced, the dietary structure and life style of people also with
Changed, the incidence of disease of high lithemia disease is in the trend that substantially rises in recent years, and is easily sent out in the elderly.Although at present
Having some has the medicine of obvious anti-trioxypurine or antigout, but all there is toxic side effect in various degree so that people are to health
The demand of medicine and health food is more urgent.
Biologically active peptide refers to the polypeptide thing with some special physiologicals activity connected by amino acid by peptide bond
Matter, its peptide chain length differs, and structure is different, causes it to have a different physiologically actives, such as hypotensive, norcholesterol, anti-
Thrombus, anticancer, anti-oxidant etc..By the unremitting effort of researcher, increasing biologically active peptide is constantly found.
In addition, hyperuricemia can cause the breaking-out of ventilation disease, saturation is reached mainly due to when uric acid in blood concentration
When, the continuing of uric acid level, which increases, to be formed ventilation stone and simultaneously be deposited in IA synovia, not only so that uric acid is crystallized
So that synovia loses the effect in lubrication protection joint, on the contrary can as mortar with joint-friction, feel bad Principle of Pain to be formed
Gout, but also the calcareous of articular surface that can grind away so that the weight capacity in joint is worse, are deformed more serious.In China, height urine
Sour patient is common in the elderly, and they natively have different degrees of calcium deficiency, causes to be worse off.Therefore, treatment or
While preventing hyperuricemia, it should also pay attention to supplement and the absorption of calcium.
Saury is a kind of marine fish, and it survives in the particular surroundings of ocean for a long time, there will necessarily be some uniquenesses inside it
Bioactive substance, if it is possible to these bioactive substances are discharged, and with calcareous combination, for treating and pre-
Anti- high lithemia disease, can reach the effect of anti-trioxypurine, can play a part of replenishing the calcium again, will have greatly economic and society's meaning
Justice.
The content of the invention
The primary and foremost purpose of the present invention is to provide a kind of chelated calcium preparation method of anti-trioxypurine peptide, and this method is using biology
Zymolysis technique discharges the anti-trioxypurine active fragment inside saury, and it into chelatropic reaction occur with calcium.
Another object of the present invention is to provide the anti-trioxypurine peptide as made from the above method to chelate calcium, the product has drop urine concurrently
Acid and calcium supplementing effect.
It is still another object of the present invention to provide the above-mentioned chelated calcium purposes of anti-trioxypurine peptide.
The purpose of the present invention is achieved through the following technical solutions：
A kind of chelated calcium preparation method of anti-trioxypurine peptide, comprises the following steps：
(1) saury pre-processes：Saury decaptitating, internal organ are removed, cleaned up, crossed meat grinder and rub, add saury broken
The water that 3~5 times of meat quality, heating stirring 1~2 hour at 40~50 DEG C, then regulation system pH value is to 4.2, and continuation is stirred
Heating 1~1.5 hour, is centrifuged, reject supernatant and upper strata grease, taking precipitate；
(2) preparation of anti-trioxypurine peptide：The water of 1~1.5 times of its quality, regulation system pH value are added into saury sediment
To 7.0, protease is then added, after insulation is hydrolyzed 6~9 hours at 50~55 DEG C, go out enzyme, centrifugation takes supernatant, obtains the autumn
Hairtail anti-trioxypurine peptide liquid；Using the quality of saury sediment as calculating benchmark, total addition of protease accounts for 1.5~3.0%；
(3) saury peptide is chelated calcium prepares：The pH value of saury anti-trioxypurine peptide liquid is adjusted to 7.0~8.5, by saury
Anti-trioxypurine peptide liquid solid quality 30~50% adds calcium chloride solid, and 30~60min is incubated at 30~35 DEG C, is then added
15~30min of absolute ethyl alcohol and stirring；Herein, the chelating calcium of generation does not dissolve in absolute ethyl alcohol, and adding absolute ethyl alcohol can reach point
From chelated calcium purpose；Reactant is centrifuged, taking precipitate vacuum drying, obtain anti-trioxypurine peptide chelating calcium product；
Described regulation system pH value is adjusted with 0.1~0.5mol/L NaOH solution or HCl solution；
Described centrifugation is that 5000r/min centrifuges 15~20min；
Protease described in step (2) is the alkali protease and flavor protease of business, preferably Novozymes Company
Alkali protease (Alcalase 2.4L) and flavor protease (Flavourzyme 500MG)；The addition of alkali protease is accounted for
The 0.6~0.8% of saury sediment quality, the addition of flavor protease account for saury sediment quality 1.0~
1.5%；
In step (3), absolute ethyl alcohol quality is 8~12 times of saury anti-trioxypurine peptide liquid quality.
Anti-trioxypurine peptide chelating calcium can be used for preparing the medicine with anti-trioxypurine and/or effect of replenishing the calcium as made from the above method
And health products, in use, combined Chinese herb that can be by it with being acted on anti-trioxypurine.
The present invention has the following advantages and effect relative to prior art：
(1) present invention discharges the active fragment in saury albumen by modern biotechnology zymolysis technique first, then uses
Wet method chelating prepares protein peptides chelating calcium product.The bioactivity peptide fragment discharged from saury albumen has substantially drop urine
Sour effect, meanwhile, the protein peptides have the calcareous effect of good chelating, and the anti-trioxypurine peptide chelating calcium product prepared is not only
With the effect of significant anti-trioxypurine, while solving to maintaining bone, the normal absorption of the vital calcium of muscle function and utilization
Loss problem, can turn into a kind of and outstanding have both anti-trioxypurine and the product of supplement calcium.
(2) present invention process is simple to operate, production cost is low, without any pollution, gained anti-trioxypurine peptide chelating calcium product
Anti-trioxypurine activity it is strong, and the absorptivity of calcium is high.
Brief description of the drawings
Fig. 1 is the dissolubility block diagram of each embodiment and comparative example product under different pH condition.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
In following embodiment, each sample is as follows to the experimental method of xanthine oxidase inhibiting rate：
(1) configuration of solution
0.2mol/L (pH 7.5) phosphate buffer (PBS)：Accurately weigh 30.0838g Na2HPO4·12H2O and
2.4962g NaH2PO4·2H2O, with deionized water dissolving, is settled to 500ml.
Xanthine solution：6.4mg xanthine accurately is weighed, is first dissolved with 1ml 1M NaOH, adds 100ml
PBS, pH value is adjusted to 7.5 with 1M HCl.
Xanthine oxidase：120 μ l enzyme liquids are taken, 8ml is diluted to PBS.
Uric acid mark song：It is accurate to weigh 10mg uric acid, 10ml water is added, then be diluted to 0.1~0.9mg/ml.
Ammoniom-Acetate-glacial acetic acid：Accurately weighing Ammoniom-Acetate 3.85g is settled to 1000ml, then adds 4ml glacial acetic acid.
(2) sample pretreatment：
Sample is diluted to 40mg/ml, 50 μ l samples and 50 μ l xanthine, each sample are sequentially added in 96 hole elisa Plates
Product do 3 it is parallel, add 150 μ l xanthine oxidases after 37 DEG C of insulation 10min, 37 DEG C are continued to add 80 μ l after being incubated 20min
1M HCl terminating reactions, cross 0.25 μm of aqueous film, to be measured.
Chromatographic column：Zorbax Eclipse XDB-C18 posts (5 μm, 4.6 × 250mm, Agilent),
Liquid-phase condition：Eluent is 10%+90% Ammoniom-Acetate of methanol-glacial acetic acid solution, and sampling volume is 20 μ L, flow velocity
1ml/min, Detection wavelength is 290nm, run time 10min.
(3) calculation formula
In formula：
A0--- it is not added with peptide sample and carries out efficient liquid phase chromatographic analysis, the peak area at uric acid peak
A --- addition peptide sample carries out efficient liquid phase chromatographic analysis, the peak area at uric acid peak
Embodiment 1
A kind of chelated calcium preparation method of anti-trioxypurine peptide, comprises the following steps：
(1) saury pre-processes：Saury decaptitating, internal organ are removed, cleaned up, crossed meat grinder and rub, add saury broken
The water that 3 times of meat quality, then heating stirring 1.0 hours at 50 DEG C add HCl solution (0.5mol/L) adjustment system pH extremely
4.2, continue agitating and heating 1.5 hours, centrifugation (5000r/min, 15~20min) is separated, reject supernatant and upper strata grease take
Sediment.
(2) preparation of anti-trioxypurine peptide：The water of 1 times of its quality is added into saury sediment, using 0.5mol/L's
System pH is adjusted to 7.0 by NaOH solution, and saury slurry temperature is risen into 55 DEG C, then adds saury sediment quality
The flavor protease of the Alcalase 2.4L and 1.2% of 0.6% Novozymes Company Novozymes Company, is incubated at 55 DEG C
After hydrolysis 6 hours, 15min is heated at 95 DEG C, 15~20min is then centrifuged under 5000r/min, supernatant is taken, obtains the autumn
Hairtail peptide liquid；
(3) saury peptide is chelated calcium prepares：With 0.1mol/L NaOH solution by the pH in saury anti-trioxypurine peptide liquid
Then regulation adds calcium chloride by 50% of solid quality in saury anti-trioxypurine peptide liquid, is incubated at 30 DEG C to 7.0
40min, insulation adds 10 times of absolute ethyl alcohol and stirring 20min of saury fish anti-trioxypurine peptide liquid quality after terminating, it is heavy to be taken after centrifugation
Starch is dried in vacuo, and obtains anti-trioxypurine peptide chelating calcium product A.
Anti-trioxypurine peptide chelating calcium product A physical and chemical index, dissolubility and XOD inhibiting rates such as Tables 1 and 2.
The dissolubility such as Fig. 1 of anti-trioxypurine peptide chelating calcium product A at various ph values.
Embodiment 2
A kind of chelated calcium preparation method of anti-trioxypurine peptide, comprises the following steps：
(1) saury pre-processes：Saury decaptitating, internal organ are removed, cleaned up, crossed meat grinder and rub, add saury broken
The water that 4 times of meat quality, then heating stirring 2.0 hours at 40 DEG C add HCl solution (0.5mol/L) adjustment system pH extremely
4.2, continue agitating and heating 1.0 hours, centrifugation (5000r/min, 15~20min) is separated, reject supernatant and upper strata grease take
Sediment.
(2) preparation of anti-trioxypurine peptide：The water of 1.5 times of its quality is added into saury sediment, using 0.5mol/L's
System pH is adjusted to 7.0 by NaOH solution, and saury slurry temperature is risen into 50 DEG C, then adds saury sediment quality
The flavor protease of the Alcalase 2.4L and 1.5% of 0.8% Novozymes Company Novozymes Company, is incubated at 50 DEG C
After hydrolysis 9 hours, 15min is heated at 95 DEG C, 15~20min is then centrifuged under 5000r/min, supernatant is taken, obtains the autumn
Hairtail peptide liquid；
(3) saury peptide is chelated calcium prepares：With 0.1mol/L NaOH solution by the pH in saury anti-trioxypurine peptide liquid
Then regulation adds calcium chloride by 40% of solid quality in saury anti-trioxypurine peptide liquid, is incubated at 35 DEG C to 8.5
60min, insulation adds 8 times of absolute ethyl alcohol and stirring 30min of saury anti-trioxypurine peptide liquid quality, taking precipitate after centrifugation after terminating
It is dried in vacuo, obtains anti-trioxypurine peptide chelating calcium product B.
Anti-trioxypurine peptide chelating calcium product B physical and chemical index, dissolubility and XOD inhibiting rates such as Tables 1 and 2.
The dissolubility such as Fig. 1 of anti-trioxypurine peptide chelating calcium product B at various ph values.
Embodiment 3
A kind of chelated calcium preparation method of anti-trioxypurine peptide, comprises the following steps：
(1) saury pre-processes：Saury decaptitating, internal organ are removed, cleaned up, crossed meat grinder and rub, add saury broken
The water that 5 times of meat quality, then heating stirring 1.5 hours at 45 DEG C add HCl solution (0.5mol/L) adjustment system pH extremely
4.2, continue agitating and heating 1.2 hours, centrifugation (5000r/min, 15-20min) is separated, reject supernatant and upper strata grease take
Sediment.
(2) preparation of anti-trioxypurine peptide：The water of 1.2 times of its quality is added into saury sediment, using 0.5mol/L's
System pH is adjusted to 7.0 by NaOH solution, and saury slurry temperature is risen into 53 DEG C, then adds saury sediment quality
The flavor protease of the Alcalase 2.4L and 1.0% of 0.6% Novozymes Company Novozymes Company, is incubated at 53 DEG C
After hydrolysis 8 hours, 15min is heated at 95 DEG C, 15~20min is then centrifuged under 5000r/min, supernatant is taken, obtains the autumn
Hairtail peptide liquid；
(3) saury peptide is chelated calcium prepares：With 0.1mol/L NaOH solution by the pH in saury anti-trioxypurine peptide liquid
Then regulation adds calcium chloride by 30% of solid quality in saury anti-trioxypurine peptide liquid, is incubated at 30 DEG C to 8.0
60min, insulation adds 12 times of absolute ethyl alcohol and stirring 15min of saury anti-trioxypurine peptide liquid quality after terminating, precipitation is taken after centrifugation
Thing is dried in vacuo, and obtains anti-trioxypurine peptide chelating calcium product C.
Anti-trioxypurine peptide chelating calcium product C physical and chemical index, dissolubility and XOD inhibiting rates such as Tables 1 and 2.
The dissolubility such as Fig. 1 of anti-trioxypurine peptide chelating calcium product C at various ph values.
Comparative example
A kind of preparation method of saury anti-trioxypurine peptide, comprises the following steps：
(1) saury pre-processes：Saury decaptitating, internal organ are removed, cleaned up, crossed meat grinder and rub, add saury broken
The water that 5 times of meat quality, then heating stirring 1.5 hours at 45 DEG C add HCl solution (0.5mol/L) adjustment system pH extremely
4.2, continue agitating and heating 1.2 hours, centrifugation (5000r/min, 15~20min) is separated, reject supernatant and upper strata grease take
Sediment.
(2) preparation of anti-trioxypurine peptide：The water of 1.2 times of its quality is added into saury sediment, using 0.5mol/L's
System pH is adjusted to 7.0 by NaOH solution, and saury slurry temperature is risen into 53 DEG C, then adds saury sediment quality
The flavor protease of the Alcalase 2.4L and 1.0% of 0.6% Novozymes Company Novozymes Company, is incubated at 53 DEG C
After hydrolysis 8 hours, 15min is heated at 95 DEG C, 15~20min is then centrifuged under 5000r/min, supernatant is taken, obtains the autumn
Hairtail peptide liquid；Solid content more than 30% is concentrated in vacuo to, is spray-dried, saury peptide is obtained.
Physical and chemical index, dissolubility and the XOD inhibiting rates such as Tables 1 and 2 of saury peptide product.
The dissolubility of saury peptide product at various ph values such as Fig. 1.
The physical and chemical index and dissolubility of each embodiment of table 1 and comparative example product
| Moisture/% | Albumen/% | Calcium/% | |
| Anti-trioxypurine peptide chelating calcium product A | 7.02 | 68.39 | 16.61 |
| Anti-trioxypurine peptide chelating calcium product B | 6.78 | 69.04 | 15.83 |
| Anti-trioxypurine peptide chelating calcium product C | 7.16 | 72.38 | 16.47 |
| Saury peptide | 5.52 | 90.52 | 0 |
The XOD inhibiting rates of each embodiment of table 2 and comparative example product
| XOD inhibiting rates (40mg/ml)/% | |
| Saury peptide | 21.68 |
| Anti-trioxypurine peptide chelating calcium product A | 13.81 |
| Anti-trioxypurine peptide chelating calcium product B | 14.06 |
| Anti-trioxypurine peptide chelating calcium product C | 12.96 |
Calcium is distributed in whole body everywhere as a kind of extremely important macroelement, in the metabolism and life of body
Significant role is played in maintenance process.Calcium has a variety of functions in human body：Constitute bone and tooth；Maintenance is nervimuscular just
Often activity；Promote the activity of some enzymes in vivo；Participate in blood clotting process etc..But calcium enters in human body, is generally more difficult to directly be absorbed
Utilize, be primarily due to calcium and could only be absorbed in soluble ionic condition in small enteral, the pH value of small intestine epimere is relatively low,
Calcium can exist with ionic condition, and small intestine hypomere is in neutral to alkalescence, and calcium ion is easily combined generation phosphoric acid with phosphate anion
Calcium precipitate, causes human body to be greatly reduced the absorptivity of calcium.Therefore, only calcium can be absorbed, and can be only achieved the effect replenished the calcium.
As shown in Table 1, the product prepared using the inventive method is a kind of joint product rich in protein peptides and calcium,
Particularly a kind of protein peptides chelating calcium product, and protein peptides and calcium mass ratio between 4.0~4.5, illustrate the product
Middle calcium content enriches very much, meanwhile, the dissolubility of such protein peptides chelating calcium product preferably, is easy to be dissolved in water as shown in Figure 1,
Make it have wider array of application.
As seen from Table 2, protein peptides (the saury peptide of comparative example) prepared by saury are hydrolyzed using modern biotechnology
Effect with stronger suppression xanthine oxidase activity (XOD), and XOD is the key enzyme in human body in purine metabolism, its
Main function is catalysis hypoxanthine generation xanthine, and then generates uric acid, or directly catalysis xanthine generation uric acid, is suppressed
The activity of xanthine oxidase, also can just suppress the generation of internal uric acid to a certain extent, illustrate saury peptide have compared with
The potentiality of strong anti-trioxypurine.
Saury peptide with calcium after being chelated, and the reduction of its XOD inhibiting rate is primarily due to caused by the reduction of the concentration of protein peptides,
Illustrate that saury protein peptides chelating calcium product also has larger anti-trioxypurine potentiality.
Fig. 1 shows that the anti-trioxypurine peptide prepared using the inventive method chelates calcium product in the range of pH value 2~10
With preferable dissolubility, it is ensured that it enters after human body, it is to avoid calcium is by plant acid, oxalic acid and hydrochloric acid in gastric juice, alkaline shadow in food
Ring, can directly by intestinal mucosa, will not in stomach and small intestine condition Direct precipitation, being additionally, since protein peptides can preferable quilt
Intestinal absorption, so as to further promote the absorption for the calcium being sequestered in protein peptides.
Therefore, the anti-trioxypurine peptide prepared using the inventive method chelates calcium product can be as treatment and prevention high lithemia disease
Medicine or healthy food material, while can reach the purpose replenished the calcium again, reduce high lithemia disease because the friction of calculus urate causes
The harm of the calcareous missing in joint, with preferable application prospect.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (5)
1. a kind of chelated calcium preparation method of anti-trioxypurine peptide, it is characterised in that comprise the following steps：
（1）Saury pre-processes：Saury decaptitating, internal organ are removed, cleaned up, crossed meat grinder and rub, add saury meat mincing matter
3 ~ 5 times of water of amount, heating stirring 1 ~ 2 hour at 40 ~ 50 DEG C, then regulation system pH value is to 4.2, continue agitating and heating 1 ~
1.5 hours, centrifuge, reject supernatant and upper strata grease, taking precipitate；
（2）The preparation of anti-trioxypurine peptide：The water of 1 ~ 1.5 times of its quality is added into saury sediment, regulation system pH value is extremely
7.0, protease is then added, after insulation is hydrolyzed 6 ~ 9 hours at 50 ~ 55 DEG C, go out enzyme, centrifugation takes supernatant, obtains saury
Anti-trioxypurine peptide liquid；
（3）Saury peptide is chelated calcium to be prepared：The pH value of saury anti-trioxypurine peptide liquid is adjusted to 7.0 ~ 8.5, drops and urinates by saury
Sour peptide liquid solid quality 30 ~ 50% adds calcium chloride solid, and 30 ~ 60min is incubated at 30 ~ 35 DEG C, absolute ethyl alcohol is then added
15 ~ 30 min are stirred, centrifugation, taking precipitate vacuum drying obtains anti-trioxypurine peptide chelating calcium product；
Step（2）Described protease is alkali protease and flavor protease, and the addition of alkali protease accounts for saury and sunk
The 0.6-0.8% of starch quality, the addition of flavor protease accounts for the 1.0-1.5% of saury sediment quality；
Described alkali protease is the Alcalase 2.4L of Novozymes Company, and flavor protease is Novozymes Company
Flavourzyme 500MG。
2. the chelated calcium preparation method of anti-trioxypurine peptide according to claim 1, it is characterised in that：Step（3）In, anhydrous second
Alcohol quality is 8 ~ 12 times of saury anti-trioxypurine peptide liquid quality.
3. a kind of anti-trioxypurine peptide chelates calcium, it is characterised in that：It is to be prepared as the method described in claim 1 or 2.
4. the anti-trioxypurine peptide chelating calcium described in claim 3 is preparing medicine and health care with anti-trioxypurine and/or effect of replenishing the calcium
Application in product.
5. anti-trioxypurine peptide according to claim 4 chelating calcium prepare medicine with anti-trioxypurine and/or effect of replenishing the calcium and
Application in health products, it is characterised in that：By described anti-trioxypurine peptide chelating calcium and the combined Chinese herb with anti-trioxypurine effect.
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