CN101972004B - Preparation method and application of oyster zymolyte - Google Patents

Preparation method and application of oyster zymolyte Download PDF

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CN101972004B
CN101972004B CN2010102687049A CN201010268704A CN101972004B CN 101972004 B CN101972004 B CN 101972004B CN 2010102687049 A CN2010102687049 A CN 2010102687049A CN 201010268704 A CN201010268704 A CN 201010268704A CN 101972004 B CN101972004 B CN 101972004B
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zymolyte
oyster
enzyme
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activity
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CN101972004A (en
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董英
周越
孙芳芳
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Nanning Haiwang Health Biotechnology Co., Ltd.
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Jiangsu University
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Abstract

The invention discloses a method for preparing oyster zymolyte with antioxidation and antiaging activity and application thereof. The method includes the following steps: dry oyster powder is taken as raw material, aqueous solution with protein mass fraction of 2% is prepared, water bath is carried out for 30min at 52.5-55 DEG C, then combinative protease is used for successive or simultaneous hydrolysis; enzyme adding amount of each protease is 6000-8000U/g substrate protein, pH is regulated to be 6.0-7.0, hydrolysis is carried out for 2 hours at the temperature of 52.5-55 DEG C, enzymatic hydrolysate is centrifuged, and then supernate is subject to ultrafilteration by virtue of ultrafilteration membrane with the cut-off molecular weight of 1kDa; and retentate is subject to spray drying, thus obtaining oyster zymolyte. The oyster zymolyte can be used for preparing food additive with antioxidation activity or nutritional food with antiaging activity. The invention has the advantages of available raw materials, simple preparation process, high efficiency and no pollution; and zymolyte has strong antioxidation activity and obvious antiaging effect.

Description

The preparation method of oyster zymolyte and application
Technical field
The present invention relates to prepare a kind of method that possesses the active Oyster Protein zymolyte that delays senility, can be used as anti-oxidant, delay senility nutrient or medicine interpolation, belong to the marine biotechnology application.
Background technology
Oyster is commonly called as oyster, oyster, is first cultivated shellfish in the world, is also one of China's four large cultivated shellfishes, is distributed widely in the ground such as China Liaoning, Shandong, Fujian and Coast of Guangdong Province.Oyster meat is tender delicious, and dietotherapeutic in China's history of existing centuries, have the laudatory title of " marine milk ", is that China classifies one of health care remedy diet of integration of drinking and medicinal herbs in the first batch as.The protein content of oyster can be up to 50%, and amino acid forms fully.Also contain the mineral matters such as unrighted acid, vitamin and the selenium such as abundant glycogen, DHA (DHA) and eicosapentaenoic acid (EPA) and zinc in oyster meat.Over past ten years, enzyme preparation has obtained general application in the seashells recourse processing process, and the research of shellfish protein enzymolysis technique and zymolyte effect thereof constantly makes progress.At present, the important means that zymolysis technique has become that the seashells protein resource is high-valued, resource, ecology develop.But, how to use for reference and to break through traditional zymolysis technique and technique, to the green high-valued exploitation of seashells protein resource, be still important field of research in China's Mollusca Resource comprehensive utilization.
Summary of the invention
The present invention adopts the combination zymolysis technique to obtain oyster zymolyte take ostreae testa pulverata as raw material, significantly is better than the effect of various single enzyme hydrolysis.Prove conclusively oyster combination zymolyte by various anti-oxidant detection architecture and had significant antioxidation activity; And adopting the mensuration of the index such as LPO and activities of antioxidant enzymes in drosophila survival experiment, murine liver tissue, conclusive evidence oyster combination zymolyte has the activity that delays senility significantly.The notable biological activity that the zymolyte of selecting this new technology to prepare has can be realized the higher value application of oyster.
The present invention realizes by following technological means, combination enzymolysis process: be made into the aqueous solution of albumen quality mark as 2% take ostreae testa pulverata in water, in reactor, 52.5 ℃ of-55 ℃ of water-baths were soaked 30 minutes, then successively or add simultaneously the combined protein enzyme to be hydrolyzed; The enzyme concentration of each protease is the 6000-8000U/g substrate protein, regulates pH 6.0-7.0 with phosphate buffer, and reaction temperature is the Water Under solution 2 hours of 52.5-55 ℃, boils the 10min enzyme that goes out; Centrifugal each enzymolysis liquid, getting the supernatant molecular cut off is the milipore filter ultrafiltration of 1kDa, trapped fluid carries out spray-drying, obtains oyster zymolyte; Wherein said combined protein enzyme is neutral proteinase and composite flavor enzyme.Preferably 55 ℃ of temperature, pH6.0, neutral proteinase and composite flavor enzyme that to add simultaneously two kinds of enzyme amounts be the 6000U/g substrate protein.
One of application of above-mentioned oyster zymolyte, remove DPPH, OH, superoxide anion radical effect and reducing power thereof as foundation take it, measure in conjunction with the mouse liver even slurry detection architecture, proved that oyster zymolyte has stronger antioxidation activity in vitro, can prepare food, food ingredient or health products with antioxidation activity.
Two of the application of above-mentioned oyster zymolyte through the evaluation that delays senility, is measured three aspects as evaluation index take existence test, LPO mensuration and the antioxidase activity of fruit bat, has proved conclusively oyster zymolyte and has improved average life span and the maximum life span of fruit bat; Improve SOD activity in Mice Body, reduce the content of MDA in Mice Body, have the effect that delays senility, can prepare the nutraceutical with delaying senility function.
Beneficial effect of the present invention: ostreae testa pulverata is after combined protein enzyme enzymolysis, and its peptide yield and antioxidation activity all are improved, and possesses the effect that significantly delays senility.
The present invention has the advantages such as raw material sources are extensive, cost of manufacture is low, preparation technology is simple, efficient is high, pollution-free, meets the large-scale production requirement, easily implements.Made oyster zymolyte both can be used as that to have the nutraceutical that delays senility directly edible, also can be used as food ingredient or health products.
Description of drawings
Fig. 1 is process chart of the present invention.
The specific embodiment
Below in conjunction with embodiment, the present invention is further elaborated:
The protease that uses in the present invention comprises: neutral proteinase (enzyme activity is 16.0 ten thousand U/g), alkali protease (enzyme activity is 154.64 ten thousand U/mL), composite flavor enzyme (enzyme activity is 6.9 ten thousand U/g) can enzyme preparation Co., Ltd of section provide by the Wuxi is outstanding; Trypsase (enzyme activity is 26.26 ten thousand U/g), papain (enzyme activity is 4.96 ten thousand U/g) and pepsin (enzyme activity is 1.6 ten thousand U/g) are provided by Solution on Chemical Reagents in Shanghai company.
Comparative Examples 1
The preparation of oyster neutral protease enzymolysis thing
Accurately take dry ostreae testa pulverata, adding water, to be made into the albumen quality mark be 2% the aqueous solution, puts into reactor, and 55 ℃ of water-baths were soaked 30 minutes, than adding neutral proteinase for the amount of 8000U/g, use phosphate buffer regulator solution pH to 7.0 according to the enzyme-to-substrate albumen quality; Be placed in 55 ℃ of lower enzymolysis of thermostat water bath 2 hours, 10 minutes enzymes that go out are boiled in the zymolyte heating, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 46.8%, and Scavenging action to hydroxyl free radical is 90.9%.
Comparative Examples 2
The preparation of oyster composite flavor enzyme zymolyte
Accurately take dry ostreae testa pulverata, adding water, to be made into the albumen quality mark be 2% the aqueous solution, puts into reactor, and 50 ℃ of water-baths were soaked 30 minutes, than adding the composite flavor enzyme for the amount of 8000U/g, use phosphate buffer regulator solution pH to 6.0 according to the enzyme-to-substrate albumen quality; Be placed in 50 ℃ of lower enzymolysis of thermostat water bath 2 hours, 10 minutes enzymes that go out are boiled in the enzymolysis liquid heating, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 42.2%, and Scavenging action to hydroxyl free radical is 88.6%.
Embodiment 3
The preparation of oyster combination zymolyte
Accurately take dry ostreae testa pulverata, adding water, to be made into albumen quality concentration be 2% aqueous solution, and 55 ℃ of water-baths were soaked 30 minutes, first by the enzyme-to-substrate albumen quality than adding neutral proteinase for 8000U/g, regulate pH 7.0 with phosphate buffer, be placed in 55 ℃ of lower enzymolysis hydrolysis 120min of thermostat water bath; The composite flavor enzyme that adds again 8000U/g, at temperature 50 C, pH 6.0, hydrolysis 120min.10 minutes enzymes that go out are boiled in the zymolyte heating, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 53.6%, and Scavenging action to hydroxyl free radical is 98.2%.
Embodiment 4
The preparation of oyster combination zymolyte
Accurately take dry ostreae testa pulverata, adding water, to be made into albumen quality concentration be 2% aqueous solution, and 55 ℃ of water-baths were soaked 30 minutes, first by the enzyme-to-substrate albumen quality than adding the composite flavor enzyme for 8000U/g, regulate pH 6.0 with phosphate buffer, be placed in 50 ℃ of lower enzymolysis hydrolysis 120min of thermostat water bath; The neutral proteinase that adds again 8000U/g, at 55 ℃ of temperature, pH 7.0, hydrolysis 120min.10 minutes enzymes that go out are boiled in the zymolyte heating, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 49.1%, and Scavenging action to hydroxyl free radical is 98.4%.
Embodiment 5
The preparation of oyster combination zymolyte
Accurately take dry ostreae testa pulverata, add water and be made into albumen quality concentration 2% aqueous solution, put into reactor, 52.5 ℃ water-bath was soaked 30 minutes, press every kind of enzyme-to-substrate albumen quality than being the enzyme concentration of 6000U/g substrate protein, add simultaneously neutral proteinase and composite flavor enzyme, with phosphate buffer regulator solution pH to 6.0; Be placed in 52.5 ℃ of lower enzymolysis of thermostat water bath 2 hours, zymolyte boils the enzyme that went out in 10 minutes lives, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 92.7%, and Scavenging action to hydroxyl free radical is 99.8%.
Embodiment 6
The preparation of oyster combination zymolyte
Accurately take dry ostreae testa pulverata, add water and be made into concentration 2% aqueous solution, put into reactor, 52.5 ℃ water-bath was soaked 30 minutes, press every kind of enzyme-to-substrate albumen quality than being the enzyme concentration of 6000U/g substrate protein, add simultaneously neutral proteinase and composite flavor enzyme, with phosphate buffer regulator solution pH to 7.0; Be placed in 52.5 ℃ of lower enzymolysis of thermostat water bath 2 hours, zymolyte boils the enzyme that went out in 10 minutes lives, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 90.3%, and Scavenging action to hydroxyl free radical is 91.8%.
The zymolyte activity test is (take the material of measuring preparation in embodiment 5 as example) for example.
Test 1: remove DPPH free radical (DPPH) test
Get the preparation-obtained oyster zymolyte of embodiment 5, compound concentration is the solution of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL, and the DPPH solution concentration is 0.04g/L, and solvent is absolute ethyl alcohol, and the detection wavelength is 517nm.The clearance rate computing formula is:
K=[1-(A i-A j)/A 0]×100
In formula: K is the clearance rate to the DPPH free radical, %; A 0The light absorption value that adds the 2mL absolute ethyl alcohol for the DPPH ethanol solution of 2mL; A iThe light absorption value that adds 2mL protein hydrolysate solution for the DPPH ethanol solution of 2mL; A jThe light absorption value that adds 2mL protein hydrolysate solution for the 2mL absolute ethyl alcohol.Each sample replication is averaged for three times.
By result of the test (seeing Table 1) as can be known, when oyster zymolyte concentration was 6.0mg/mL, its clearance rate to DPPH had reached 92.3%, illustrated that zymolyte has stronger removing ability to the DPPH free radical.
Test 2: the test of removing hydroxy radical (OH)
Get the preparation-obtained oyster zymolyte of embodiment 5, compound concentration is respectively 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL.Respectively get the hydrating solution of the above-mentioned concentration of 2mL, add successively the FeSO of 2mL 6mmol/L 4, 2mL 6mmol/L H 2O 2, standing 10min after mixing, then add 2mL 6mmol/L salicylic acid, mixing, standing 30min is designated as A at its light absorption value of 510nm place's survey i, the light absorption value when replacing salicylic acid with distilled water is designated as A jThe blank group replaces protein hydrolysate solution with distilled water, and light absorption value is designated as A 0Calculate according to the following formula protein hydrolysate to the clearance rate K of hydroxy radical:
K=[1-(A i-A j)/A 0]×100
As shown in Table 1, oyster zymolyte has the ability of stronger removing hydroxy radical, and its maximal clearance to the OH free radical can reach 95.4%.
Test 3: remove superoxide anion (O 2 -) test
Get the preparation-obtained oyster zymolyte of embodiment 5, compound concentration is respectively the sample solution of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL.Get 4.50ml concentration and be the Tris-HCl cushioning liquid (pH8.2) of 0.1mol/L in test tube, preheating 20min in 25 ℃ of water-baths, add the 1ml testing sample solution, 0.5mL concentration is the pyrogallol solution of 2.5mmol/L, after mixing, after accurate response 4min, add immediately the HCl solution 0.1mL cessation reaction of 10mol/L in 25 ℃ of water-baths, with the Tris-HCl buffer solution zeroing of pH 8.2, measure light absorption value under wavelength 325nm.Control group replaces pyrogallol solution with 0.5mL distilled water, sets up simultaneously the reagent blank pipe.The clearance rate computing formula is:
SA ( % ) = ( 1 - A i - A j A 0 ) × 100 %
In formula: A 0The light absorption value of-blank; The light absorption value of Ai-sample
The background light absorption value of the sample when Aj-distilled water replaces pyrogallol
Result (seeing Table 1): oyster zymolyte has the ability of stronger removing superoxide anion, and its maximal clearance has reached 72.3%.
The removing ability of table 1 oyster zymolyte to various free radicals
Test 4: reducing power test
Get the preparation-obtained oyster zymolyte of embodiment 5, compound concentration is respectively the sample solution of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL.The FRAP working solution of the fresh configuration of 2.5ml is added the pre-temperature of oyster sample solution of each concentration of 1ml to 37 ℃, read absorbance in 593nm.Light absorption value substitution calibration curve Solving Equations is got corresponding Fe 2+Concentration.
The TAC measurement result of oyster enzymolysis liquid sees Table 2.Enzymolysis liquid is along with the increase of concentration, Fe 2+Concentration larger, the expression Fe 3+Be reduced to Fe 2+Ability stronger, show that oyster zymolyte has stronger reduction.
Test 5: the Lipid peroxidation test in the mouse liver even slurry system
Get the preparation-obtained oyster zymolyte of embodiment 5, compound concentration is respectively the sample solution of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL.The oyster sample liquid of 0.1ml LH, 0.2ml, the H of 0.1ml 10mmol/L get in the liver tissue homogenate of preparation 10% (w/v) 2O 2Solution and 0.1ml Tris-HCl buffer solution (pH7.4) mix, add the thiobarbituricacidα-(TBA) of 1ml 0.67% (w/v) and the trichloroacetic acid (TCA) of 1ml 10% after 37 ℃ of placement 1h, and boiled 15 minutes in 100 ℃ of water-baths, then put ice bath centrifugal (6000rpm after cooling 10 minutes, 10min), get supernatant in 532nm place's survey light absorption value, calculate the lipid peroxidation inhibiting rate.Blank group replaces sample with the Tris-HCl buffer solution.All samples is done three repetitions.
Lipid peroxidation inhibiting rate (%)=(A Sample-A Blank) * 100/A Blank
H when as can be seen from Table 2, oyster zymolyte is to LH system incubation 2O 2The generation of bringing out lipid peroxidation has certain inhibitory action, when the concentration of 6.0mg/ml, zymolyte to the lipid peroxidation inhibiting rate up to 40%.
The reduction of table 2 oyster zymolyte and Lipid peroxidation
Through above-mentioned evidence, the oyster zymolyte that obtains than conventional method according to method provided by the invention has significant anti-oxidation efficacy, can for the preparation of corresponding product, be applied to the aspects such as food, health products.
Test 6: drosophila survival experiment
(1) choose female, the male drosophila of the not mating of hatching in 10h, use etherization, then be divided at random control group, oyster zymolyte interpolation high dose group (6mg/mL) and oyster zymolyte by sex and add low dose group (1mg/mL), each 100 of every group of male and female, be placed in temperature (25 ± 1) ℃, cultivate in the incubator that the illumination in 12 hours of humidity 55-65% replace.Every pipe is put the 2ml culture medium, seals with double gauze.
(2) the last fortnight is all fed basal medium, and after 14 days, each test group begins to add oyster zymolyte.
(3) dead fly number is regularly observed and recorded to every morning, changed a subculture in 4 days, till observing the extremely complete death of fruit bat.Reject the fruit bat number that accidentally flies away in improper former cause death and operating process in experimentation.
(4) statistics is respectively organized drosphila metanogaster and MaLS at last.
Table 3 oyster zymolyte is on the impact of life span of drosophila melanogaster (x ± s)
As can be seen from Table 3: average life span and maximum life span that oyster zymolyte can the significant prolongation fruit bat.
Test 7: on the impact test of murine liver tissue MDA and SOD
Set up D-gal Aging mouse model, set up at random sampling test group, model group and control group, test group gavage oyster zymolyte (6mg/ml, 1mg/ml), preparation 10% LH after 8 weeks carries out MDA content and SOD determination of activity in hepatic tissue.
Table 4 is respectively organized SOD in Mice, MDA measurement result (x ± s)
As can be seen from Table 4, add the oyster zymolyte SOD vigor in Mice Body that can significantly raise, reduce the content of MDA in Mice Body.
Through above-mentioned evidence, oyster zymolyte prepared according to the methods of the invention has significant function of delaying senility, can for the preparation of corresponding product, be applied in food and health products.

Claims (3)

1. the preparation method of oyster zymolyte, it is characterized in that adopting the combination enzymolysis process, comprise the following steps: be made into the aqueous solution of albumen quality mark as 2% in water take ostreae testa pulverata, in reactor, 52.5 ℃ of-55 ℃ of water-baths were soaked 30 minutes, then added simultaneously the combined protein enzyme to be hydrolyzed; Wherein said combined protein enzyme is neutral proteinase and composite flavor enzyme; Than being the enzyme concentration of 6000 U/g substrate proteins, add simultaneously neutral proteinase and composite flavor enzyme by every kind of enzyme-to-substrate albumen quality, regulate pH6.0 with phosphate buffer, reaction temperature is the Water Under solution 2 hours of 55 ℃, boils the 10min enzyme that goes out; Centrifugal enzymolysis liquid, getting the supernatant molecular cut off is the milipore filter ultrafiltration of 1kDa, trapped fluid carries out spray-drying, obtains oyster zymolyte.
2. the application of the oyster zymolyte for preparing of a preparation method who adopts the described oyster zymolyte of claim 1, preparation has food, food ingredient or the health products of antioxidation activity.
3. the application of the oyster zymolyte for preparing of a preparation method who adopts the described oyster zymolyte of claim 1, preparation has nutraceutical or the health products that delay senility.
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