CN102286589B - The preparation method of turtle oligopeptide - Google Patents
The preparation method of turtle oligopeptide Download PDFInfo
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Abstract
The object of the present invention is to provide a kind of extracting method of turtle oligopeptide, be after raw material Sumizyme MP enzymolysis with soft-shelled turtle, and adjustment local flavor circumscribed with flavor protease again, enzymolysis solution carries out ultrafiltration, Sumizyme MP add-on is the 0.2-5% of raw material soft-shelled turtle weight, flavor protease add-on is the 0.1-2% of raw material soft-shelled turtle weight, and retaining molecular weight is that the daltonian ultra-filtration membrane of 0.5-30 ten thousand carries out ultrafiltration, and thin film concentration spraying dry obtains turtle oligopeptide; Present method is easy and simple to handle, and mouthfeel is good, and cost is low, environment-friendly high-efficiency, is very applicable to industrialized production.
Description
Technical field
The present invention relates to the deep process technology field of soft-shelled turtle, being specifically related to a kind of is raw material with soft-shelled turtle, utilizes the method for compound bio-enzyme enzyme-squash techniqued turtle oligopeptide.
Background technology
Soft-shelled turtle has another name called soft-shelled turtle, the soft-shelled turtle, have enrich blood, strong bone, antifatigue, the effect of promoting longevity, being China's traditional food, is also the very good material of exploitation high-class healthy food; The main nutrient composition of soft-shelled turtle is protein, fat, iron, calcium, gelatin, cutin bletilla multivitamin etc.Soft-shelled turtle contains very high protein, about per kilogram has 165 grams of protein, and the protein in food or medicine generally wants digested liquid enzymolysis be oligopeptide or amino acid and absorbed by body and utilize, therefore absorbs slowly, utilization ratio is low, so also do not reach due drug effect.
Utilize modern biotechnology zymolysis technique, ultrafiltration and thin film concentration, the small molecules turtle oligopeptide that spray drying technology process obtains, can overcome the problems referred to above, make turtle oligopeptide oral can more completely and direct digested road absorb, at present, the method extracting turtle oligopeptide mainly contains alkali liquor extraction method and biologic enzymolysis method, and the former adds a large amount of alkaline purifications in leaching process, has both destroyed the composition of protein, cause the bad mouthfeels such as finished product is bitter salty, and to environment; The preparation method of small molecular peptides of soft-shelled turtle is disclosed: with pancreatin at 55-60 ° of C enzymolysis 1-4h in application number 201010103157.9 patent documentation, 10h is incubated at 90-95 ° of C, be cooled to 60 ° of C, add compound protease enzymolysis 1-4h, then through degrease, activated carbon decolorizing, vacuum concentration, alcohol precipitation, high-temperature sterilization, spraying dry obtains finished product.But the method trypsinase cost is high, and operating process is oversize, complex steps is not suitable for suitability for industrialized production.
Summary of the invention
There is fishy smell in the enzymolysis solution for prior art, saline taste and molecular weight large, molecular weight distribution is not concentrated, the defect that cost is high, the object of the present invention is to provide a kind of extracting method of turtle oligopeptide, take soft-shelled turtle as raw material Sumizyme MP and flavor protease enzymolysis, retaining molecular weight is that the daltonian ultra-filtration membrane of 0.5-30 ten thousand carries out ultrafiltration and thin film concentration purification techniques and spraying dry and obtains turtle oligopeptide; Present method is easy and simple to handle, and mouthfeel is good, and cost is low, environment-friendly high-efficiency, is applicable to industrialized production.
In order to realize above-mentioned technical purpose, the present invention takes following technical measures:
A preparation method for turtle oligopeptide, its step is as follows:
1. add Sumizyme MP by the ratio of the 0.2-5% of the weight of raw material soft-shelled turtle; 40-70 DEG C of constant temperature stirs; enzymolysis 1-10h; flavor protease is added again by the ratio of the 0.1-2% of raw material soft-shelled turtle weight; 40-70 DEG C of constant temperature enzymolysis 0.2-4h; the enzymolysis solution obtained is warming up to 85-120 DEG C, and go out enzyme 10-60min;
2. by centrifugal for enzymolysis solution rear filtration, supernatant liquor is through hollow fiber ultrafiltration film, rolled film or tubular membrane refining spearation, retaining molecular weight is the daltonian ultra-filtration membrane of 0.5-30 ten thousand, intake pressure is 1.8-3bar, top hole pressure 1-2bar, and enzymolysis solution temperature is 20-30 DEG C and carries out ultra-filtration and separation, ultrafiltrated is through thin film concentration, and namely spraying dry obtains turtle oligopeptide.
The present invention also first can blend raw material soft-shelled turtle, decocts 1-10h, carries out enzymolysis again after being cooled to 45-65 DEG C.
Preferred preparation method:
1. add Sumizyme MP by the ratio of the 0.5-3% of the weight of raw material soft-shelled turtle; 50-65 DEG C of constant temperature enzymolysis 2-4h, then the flavor protease of 0.25-1% pressing raw material soft-shelled turtle weight, 50-65 DEG C of constant temperature enzymolysis 0.5-2h; the enzymolysis solution obtained is warming up to 90-110 ° of C, go out enzyme 20-40min;
2. by centrifugal for enzymolysis solution rear filtration, supernatant liquor is through hollow fiber ultrafiltration film, rolled film or tubular membrane refining spearation, retaining molecular weight is the daltonian ultra-filtration membrane of 5-15 ten thousand, intake pressure is 1.2-2.5bar, top hole pressure 1-1.8bar, and enzymolysis solution temperature is that 20-30 ° of C carries out ultra-filtration and separation, ultrafiltrated is through thin film concentration, and namely spraying dry obtains turtle oligopeptide finished product.
Also first raw material soft-shelled turtle can be blended in above-mentioned steps, decoct 2-4h, after being cooled to 50-62 ° of C, carry out enzymolysis again.
More excellent method:
1. by raw material soft-shelled turtle weight 2% ratio add Sumizyme MP, 60 DEG C of constant temperature enzymolysis 3h, then press respectively raw material soft-shelled turtle weight 0.5% flavor protease, 60 DEG C of constant temperature enzymolysis 1h, are warming up to 100 ° of C by the enzymolysis solution obtained, go out enzyme 30min;
2. by centrifugal for enzymolysis solution rear filtration, supernatant liquor through hollow fiber ultrafiltration film, rolled film or tubular membrane refining spearation, retaining molecular weight is 100,000 daltonian ultra-filtration membranes, and intake pressure is 1.5-2bar, top hole pressure 1.5bar; Enzymolysis solution temperature is that 25 ° of C carry out ultra-filtration and separation, and ultrafiltrated is through thin film concentration, and namely spraying dry obtains turtle oligopeptide finished product.
Also first raw material soft-shelled turtle can be blended in above-mentioned steps, decoct 4h, after being cooled to 60 ° of C, carry out enzymolysis again.
This product is the entirety of Trionychidae animal soft-shelled turtle Trionyx sinensis Wiegmann.
Compared with prior art, the advantage of the inventive method and beneficial effect as follows:
1. the turtle oligopeptide purity of the inventive method extraction is high, and molecular weight is concentrated, and molecular weight accounts for more than 50% 1000-140 dalton.
2. the human body of turtle oligopeptide that the present invention produces easily absorbs.
3. this method avoids and regulate PH, nanofiltration desalination operation, decreases industrial cost, improves the local flavor of finished product, improves the yield of oligopeptide.
4. of the present invention with short production cycle, easy and simple to handle, cost is low, reliable product quality, safe without toxic side effect, can be widely used in the field such as healthcare products, medicine.
Embodiment
Embodiment 1
One, preparation method, its step is as follows:
1. be raw material with Trionyx sinensis (Wiegmann), select fresh and alive soft-shelled turtle 100kg, gill and grease, blend, add 1000L pure water, 100 ° of C decoct 2h;
2. be cooled to 60 ° of C, by raw material soft-shelled turtle weight 2% ratio add Sumizyme MP 2kg, 60 DEG C of constant temperature enzymolysis 3h, add 0.5kg flavor protease, 60 ° of C constant temperature enzymolysis 1h, and be then warming up to 100 ° of C, go out enzyme 30min;
3. be cooled to room temperature, the centrifugal 20min of 4000r/min, collect supernatant liquor, supernatant liquor is 100,000 daltonian rolled film ultrafiltration through molecular weight, and intake pressure is 1.8-3bar, top hole pressure 1-2bar, collects ultrafiltrated;
4. ultrafiltrated is between thin film concentration to density 1.05-1.1, and namely spraying dry obtains 13.55g powder turtle oligopeptide.
Two, turtle oligopeptide assay
1. method summary
Low-molecular-weight protein hydrolystate (comprising peptide class and total free aminoacids) dissolves in solution of trichloroacetic acid; The protein of high molecular easily precipitates in solution of trichloroacetic acid.Sample is after solution of trichloroacetic acid dissolves, and centrifugation goes out protein precipitation metallic substance, determines the acid-soluble protein content in centrifugal clear liquid, and the acid-soluble protein content in clear liquid deducts the content that free aminoacid content is oligopeptide.
2. analytical procedure
The mensuration of 2.1 acid-soluble protein contents
Take 2g(and be accurate to 1mg) sample, be added in 10mL volumetric flask, with 15% solution of trichloroacetic acid constant volume, mix, leave standstill 10min.Sample solution after centrifugal 10min, is got whole clear liquid under 4000rpm, and the method specified by GB/T 5009.5 measures the acid-soluble protein in clear liquid, and protein reduction factor is 6.25.Assay weight loss on drying per sample, converts as butt.
The mensuration of 2.2 free aminoacid contents
Sample pre-treatments: take 20 ~ 30mg sample, be accurate to 0.0001g is even with 3% sulphosalicylic acid solubilize.Sample solution is transferred in 50ml volumetric flask, constant volume.Be that on 4000r/min whizzer, centrifugal 5min obtains clear liquid by sample solution at rotating speed, then use 0.45 μm of filtering with microporous membrane clear liquid, filtrate is transferred in 50ml volumetric flask, as instrument detection sample after constant volume.The method that all the other operations specify with the mensuration of GB 12292 fruit, vegetables juice free aminoacid content.
The statement of 2.3 results
The content of oligopeptide
calculate by formula (1):
……………………………………………………（1）
In formula:
---oligopeptide content (in butt) in sample, %;
---acid-soluble protein content (in butt) in sample, %;
---sample Determination of Free Amino Acids (in butt), %.
2.4 measurement result
Measure the content of oligopeptide in finished product, the results are shown in Table 1.
The assay result of oligopeptide in table 1 Gly-His-Lys
| Sample | Turtle oligopeptide |
| Oligopeptide content | 81.06% |
3. relative molecular mass is less than 1000 daltonian oligopeptide proportions (high performance gel filtration chromatography)
3.1 method summaries
Employing high performance gel filtration chromatography measures.Namely be stationary phase with porous filler, difference according to sample component molecular volume size is separated, detect under the uv-absorbing wavelength 220nm condition of peptide bond, gel chromatography is used to measure the exclusive data process software (i.e. GPC software) of relative molecular mass distribution, color atlas and data thereof are processed, calculates relative molecular mass size and the distribution range of oligopeptide.
3.2 reagent
Acetonitrile: chromatographically pure; Trifluoracetic acid: analytical pure; Water: ultrapure water or redistilled water.
Relative molecular mass calibration curve standard substance used: cytochrome C (cyyochrome, MW12500); Press down phthalein enzyme (aprotinin, MW6500); Bacillus enzyme (bacitracin, MW1450); Glycocoll-glycocoll-Tyr-Arg (MW451); Glycocoll-glycocoll-glycocoll (MW189).
3.3 instrument and equipment
High performance liquid chromatograph: be furnished with UV-detector and containing the chromatographic working station of GPC data processing software or totalizing instrument; Moving phase vacuum filtration de-gassing vessel; Ultrasonic oscillator; Analytical balance: sensibility reciprocal 0.0001g.
3.4 chromatographic conditions and system flexibility are tested
Chromatographic column: of the same type other that TSKgel G2000 SWXL 300mm × 7.8mm or performance are close are therewith applicable to the gel column measuring proteins and peptides; Moving phase: acetonitrile: water: trifluoroacetic acid, 45:55:0.1(volume ratio) determined wavelength: UV220nm; Flow velocity: 0.5ml/min; Column temperature: 30 DEG C; Sampling volume: 10 μ l.
Testing requirement is met for making chromatographic system, under being defined in above-mentioned chromatographic condition, the post effect of gel chromatographic columns and theoretical plate number (N) calculate by three poly saccharide peptide standard products (glycocoll-glycocoll-glycocoll) peak and are not less than 5000, and the partition ratio (Kd) of oligopeptide should between 0 ~ 1.
3.5 relative molecular mass calibration curves make
Be mixed with 0.1%(W/V by moving phase respectively) the poly saccharide peptide standard product solution of above-mentioned different relative molecular mass, be sample introduction respectively after 0.2-0.5 μm of tetrafluoroethylene or nylon filter membrane filtration with aperture, obtain the color atlas of serial standards.With the logarithm of relative molecular mass (lgMW) to retention time mapping or obtain relative molecular mass calibration curve and equation thereof do linear regression.
3.6 sample preparation
Take sample 20.0mg in 10mL volumetric flask, be settled to scale by moving phase, sonic oscillation 10min, make sample fully dissolve mixing, after being 0.2-0.5 μm of tetrafluoroethylene or nylon filter membrane filtration with aperture, upper machine sample introduction.
The calculating of 3.7 relative molecular masses
The sample solution that 3.6 prepare is analyzed under above-mentioned chromatographic condition.Then use GPC data processing software, the chromatographic data of sample is substituted in calibration curve equation and calculates, the relative molecular mass and its distribution scope of peptide in sample can be obtained.The peak area relative percentage sum of the oligopeptide of relative molecular mass scope below 1000 dalton is calculated with areas of peak normalization method.
3.8 measurement result
Turtle oligopeptide range of molecular weight distributions measurement result in table 6,7.
Table 2 turtle oligopeptide result
| Molecular weight ranges | Time opening min | End time min | Weight-average molecular weight | Peak area %(λ 220nm) |
| 1000-10000 | 14.050 | 19.056 | 1428 | 8.20 |
| 500-1000 | 19.056 | 20.621 | 556 | 20.56 |
| 140-500 | 20.509 | 23.249 | 306 | 60.50 |
| 70-140 | 23.249 | 24.812 | 108 | 10.74 |
Above result shows, turtle oligopeptide molecular weight mainly concentrates on 140-1000 dalton, accounts for more than 80%.
Embodiment 2
One, the preparation method of turtle oligopeptide, its step is as follows:
1. be raw material with Trionyx sinensis (Wiegmann), select fresh and alive soft-shelled turtle 100kg, gill and grease, blend, add 1000L pure water, 100 ° of C decoct 2h;
2. be cooled to 45 ° of C, by raw material soft-shelled turtle weight 3% ratio add Sumizyme MP 2kg, 50 DEG C of constant temperature enzymolysis 5h, add 0.05kg flavor protease, and 50 DEG C of constant temperature enzymolysis 1h, are then warming up to 85 ° of C, and go out enzyme 10min;
3. be cooled to room temperature, the centrifugal 20min of 4000r/min, collect supernatant liquor, supernatant liquor is 0.5 ten thousand daltonian tubular membrane ultrafiltration through molecular weight, and intake pressure is 1.2-2.5bar, top hole pressure 1-1.8bar, collects ultrafiltrated;
4. ultrafiltrated is concentrated between density 1.05-1.1 through thin, and namely spraying dry obtains 11.8. kg powder turtle oligopeptide.
Two, turtle oligopeptide assay:
According to the method for embodiment 1, turtle oligopeptide molecular weight mainly concentrates on 140-1000 dalton, accounts for more than 50%.
Embodiment 3
One, the preparation method of turtle oligopeptide, its step is as follows:
1. be raw material with Trionyx sinensis (Wiegmann), select fresh and alive soft-shelled turtle 100kg, gill and grease, blend, add 1000L pure water,
100 ° of C decoct 2h;
2. be cooled to 65 ° of C, by raw material soft-shelled turtle weight 5% ratio add Sumizyme MP 5kg, the permanent enzymolysis 2h of 65 DEG C of constant temperature, add 2kg flavor protease, 65 DEG C of constant temperature enzymolysis 0.5h, are then warming up to 120 ° of C, and go out enzyme 60min;
3. be cooled to room temperature, the centrifugal 20min of 4000r/min, collect supernatant liquor, supernatant liquor is 300,000 daltonian tubular membrane ultrafiltration through molecular weight, and intake pressure is 1.5-2bar, top hole pressure 1.5bar, collects ultrafiltrated;
4. ultrafiltrated is between thin film concentration to density 1.05-1.1, and namely spraying dry obtains 12.55kg powder turtle oligopeptide.
Two, turtle oligopeptide assay:
According to the method for embodiment 1, turtle oligopeptide molecular weight mainly concentrates on below 1000 dalton, accounts for more than 75%.
Embodiment 4
One, the preparation method of turtle oligopeptide, its step is as follows:
1. be raw material with Trionyx sinensis (Wiegmann), select fresh and alive soft-shelled turtle 100kg, gill and grease, blend, add 1000L pure water, 100 ° of C decoct 2h;
2. be cooled to 55 ° of C, by raw material soft-shelled turtle weight 0.5% ratio add Sumizyme MP 0.5kg, 70 DEG C of constant temperature perseverance separates 4h, adds 0.25kg flavor protease, and 70 ° of C constant temperature stir enzymolysis 2h, and be then warming up to 110 ° of C, go out enzyme 40min;
3. be cooled to room temperature, the centrifugal 20min of 4000r/min, collect supernatant liquor, supernatant liquor is 150,000 daltonian hollow fiber ultrafiltration membrane ultrafiltration through molecular weight, and intake pressure is 1.5-2bar, top hole pressure 1.5bar, collects ultrafiltrated;
4. ultrafiltrated is between thin film concentration to density 1.05-1.1, and namely spraying dry obtains 11.05kg powder turtle oligopeptide.
Two, turtle oligopeptide assay:
According to the method for embodiment 1, turtle oligopeptide molecular weight mainly concentrates on 300-700 dalton, accounts for more than 20%.
Embodiment 5
One, the preparation method of turtle oligopeptide, its step is as follows:
1. be raw material with Trionyx sinensis (Wiegmann), select fresh and alive soft-shelled turtle 100kg, gill and grease, blend, add 1000L pure water, 100 ° of C decoct 2h;
2. be cooled to 60 ° of C, by raw material soft-shelled turtle weight 0.2% ratio add Sumizyme MP 2kg, 40 DEG C of constant temperature enzymolysis 4h, add 0. 1kg flavor protease, and 40 DEG C of constant temperature enzymolysis 0.5h, are then warming up to 90 ° of C, and go out enzyme 10-20min;
3. be cooled to room temperature, the centrifugal 20min of 4000r/min, collect supernatant liquor, supernatant liquor is 50,000 daltonian tubular membrane ultrafiltration through molecular weight, and intake pressure is 1.5-2bar, top hole pressure 1.5bar, collects ultrafiltrated;
4. ultrafiltrated is concentrated between density 1.05-1.1, and namely spraying dry obtains 13.26 kg powder turtle oligopeptides.
Two, turtle oligopeptide assay:
According to the method for embodiment 1, turtle oligopeptide molecular weight mainly concentrates on 300-700 dalton, accounts for more than 50%.
Claims (5)
1. the preparation method of a turtle oligopeptide, comprise raw material soft-shelled turtle enzymolysis, concentrated, dry, it is characterized in that: with Sumizyme MP and flavor protease enzymolysis, Sumizyme MP add-on is the 0.2-5% of raw material soft-shelled turtle weight, constant temperature enzymolysis 1-10h, add the flavor protease of raw material soft-shelled turtle weight 0.1-2% again, after constant temperature enzymolysis 0.2-4h, enzymolysis solution is warming up to 85-120 DEG C, go out enzyme 10-60min, and enzymolysis solution selects retaining molecular weight to be the daltonian hollow fiber ultrafiltration film of 0.5-30 ten thousand or rolled film or tubular membrane ultrafiltration.
2. the preparation method of turtle oligopeptide according to claim 1, it is characterized in that: Sumizyme MP add-on is the 0.5-3% of raw material soft-shelled turtle weight, constant temperature enzymolysis 2-4h, add the flavor protease of raw material soft-shelled turtle weight 0.25-1% again, after constant temperature enzymolysis 0.5-2h, enzymolysis solution is warming up to 90-110 DEG C, and go out enzyme 20-40min.
3. the preparation method of turtle oligopeptide according to claim 2, is characterized in that: Sumizyme MP add-on is 2% of raw material soft-shelled turtle weight, constant temperature enzymolysis 3h, add the flavor protease of raw material soft-shelled turtle weight 0.5% again, enzymolysis 1h, enzymolysis solution is warming up to 100 DEG C, and go out enzyme 30min.
4. the preparation method of turtle oligopeptide according to claim 1, is characterized in that: ultrafiltration selects retaining molecular weight to be the daltonian hollow fiber ultrafiltration film of 5-15 ten thousand or rolled film or tubular membrane.
5. the preparation method of turtle oligopeptide according to claim 4, is characterized in that: ultrafiltration selects retaining molecular weight to be 100,000 daltonian hollow fiber ultrafiltration films or rolled film or tubular membrane.
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| CN103845721A (en) * | 2012-11-29 | 2014-06-11 | 江中药业股份有限公司 | Composition for control of radiation damage or chemotherapy damage and preparation method thereof |
| CN103845723A (en) * | 2012-11-29 | 2014-06-11 | 江中药业股份有限公司 | Composition for control of radiation damage or chemotherapy damage and preparation method thereof |
| CN103845720A (en) * | 2012-11-29 | 2014-06-11 | 江中药业股份有限公司 | Composition for control of radiation damage or chemotherapy damage and preparation method thereof |
| CN103845722A (en) * | 2012-11-29 | 2014-06-11 | 江中药业股份有限公司 | Oligopeptide composition for control of radiation damage or chemotherapy damage and preparation method thereof |
| CN103397066B (en) * | 2013-07-29 | 2015-02-25 | 广西还珠海洋生物科技有限公司 | Method for extracting active peptides from Qinzhou yellow pond turtle |
| CN105341767A (en) * | 2015-12-09 | 2016-02-24 | 哈尔滨神守生物科技有限公司 | Preparing method for soft-shelled turtle protein peptide |
| CN108771244A (en) * | 2018-05-24 | 2018-11-09 | 南京中生生物科技有限公司 | The preparation method of the oligomeric peptide extract of abalone, method, abalone oligopeptide and its application for preparing abalone oligopeptide and abalone powder |
| CN110393727B (en) * | 2019-08-08 | 2021-06-08 | 江西神农氏生态农业开发有限公司 | Soft-shelled turtle extract and preparation method and application thereof |
| CN113197315B (en) * | 2021-03-23 | 2023-03-17 | 宁波御坊堂生物科技有限公司 | A food composition for improving male sexual function, and its preparation method |
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