CN102277406A - Method for preparing donkey-hide gelatin peptide - Google Patents
Method for preparing donkey-hide gelatin peptide Download PDFInfo
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- CN102277406A CN102277406A CN2011102363237A CN201110236323A CN102277406A CN 102277406 A CN102277406 A CN 102277406A CN 2011102363237 A CN2011102363237 A CN 2011102363237A CN 201110236323 A CN201110236323 A CN 201110236323A CN 102277406 A CN102277406 A CN 102277406A
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Abstract
The invention aims to provide a method for extracting donkey-hide gelatin peptide for overcoming the drawbacks of bitter enzymolysis solution, strong salt taste, high molecular weight, non-centralized molecular weight distribution and high cost of the prior art. The method is to perform the enzymolysis of donkey-hide gelatin serving as a raw material in presence of an alkaline protease or neutral protease or trypsin, treat the enzymolysis solution by concentrating and refining technology and subject concentrated and refined enzymolysis solution to spray drying to obtain the donkey-hide gelatin peptide product. The method is simple and convenient in operation, the donkey-hide gelatin peptide has a good taste, and the method is low in cost, environment-friendly and very suitable for industrial mass production.
Description
Technical field
The present invention relates to the deep process technology field of donkey-hide gelatin, be specifically related to utilize the biological enzyme enzymolysis to prepare the donkey-hide gelatin peptide
The preparation method.
Background technology
The main collagen protein (80%) that contains in the donkey-hide gelatin, instructions of taking is generally oral, and the protein in food or the medicine will be oligopeptide or amino acid and absorbed by body and utilize by the Digestive system enzymolysis generally, and above-mentioned indication crowd is common weakness of the spleen and the stomach or gastrointestinal reaction arranged, therefore digestive enzyme is active low, digestion ability is poor, and therefore oral donkey-hide gelatin not only can cause the burden of patient's Digestive tract, and does not reach due drug effect.
At present, the method for extracting the donkey-hide gelatin oligopeptide mainly contains alkali liquor extraction method and biologic enzymolysis method, and the former adds a large amount of alkaline purifications in leaching process, both destroyed proteinic composition, causes bad mouthfeels such as finished product is bitter salty, and environment is polluted.Publication number is the Chinese patent application " a kind of extracting method of donkey-hide gelatin peptide " of CN 102021215A, disclose 100 ℃ and decocted 6-8h, use Sumizyme MP and trypsin digestion 6-10h then, use flavor protease enzymolysis 8h again, enzymolysis process is regulated PH7-8 with sodium hydroxide, again through activated carbon decolorizing, the nanofiltration desalination and concentration, spray-dired preparation method, this method operating process is oversize, complex steps is not suitable for suitability for industrialized production; Trypsinase cost height; Though the PH of control enzymolysis process can slightly improve yield, need the nanofiltration desalination, and the nanofiltration desalting effect is not remarkable yet, has influenced the yield and the local flavor of finished product.Publication number is the Chinese patent application " preparation method of donkey-hide gelatin oligopeptide " of CN101294186A, stomach en-is disclosed and trypsinase is hydrolyzed, process again, ultrafiltration, macroporous resin separation and purification, decompression, cryoconcentration, the exsiccant preparation method, this method steps is loaded down with trivial details, be not suitable for suitability for industrialized production, and adopt cryoconcentration to influence the quality of product.
Summary of the invention
Purpose of the present invention is at the enzymolysis solution hardship of prior art, saline taste and molecular weight are big, molecular weight distribution is not concentrated, defect of high cost, a kind of extracting method of donkey-hide gelatin peptide is provided, being raw material with the donkey-hide gelatin carries out enzymolysis with Sumizyme MP enzymolysis or neutral protease or trypsinase, and enzymolysis solution concentrates the donkey-hide gelatin peptide product that purification techniques and spraying drying obtain.Present method is easy and simple to handle, and mouthfeel is good, and cost is low, and environment-friendly high-efficiency is fit to industrialized production very much.
In order to realize above-mentioned technical purpose, the present invention takes following technical measures:
A kind of preparation method of donkey-hide gelatin peptide, its step is as follows:
1. be raw material with the donkey-hide gelatin, pulverized the 60-100 mesh sieve, add raw material weight 6-15 pure water doubly and stir 85-120 ℃ of passivation lipase 30min-60min;
2. be cooled to 40-70 ℃, the ratio of 0.5-5% of pressing the weight of raw material donkey-hide gelatin adds Sumizyme MP, 40-70 ℃ of constant temperature enzymolysis 1-10h, or the ratio of pressing the 0.5-5% of raw material donkey-hide gelatin weight adds neutral protease, 40-70 ℃ of constant temperature enzymolysis 1-10h, or the ratio of pressing the 0.5-5% of raw material donkey-hide gelatin weight adds trypsinase, 40-70 ℃ of constant temperature enzymolysis 1-10h, the enzymolysis solution that obtains is warming up to 85-110 ℃, and enzyme 10-50min goes out;
3. filter, supernatant concentration density 1.05-1.1, spraying drying promptly obtains donkey-hide gelatin peptide finished product.
Preferred manufacturing procedure is as follows:
1. be raw material with authentic Donga donkey-hide gelatin piece, pulverized the 80-100 mesh sieve, add raw material weight 8-12 pure water doubly and stir 85-100 ℃ of passivation lipase 30min-60min;
2. be cooled to 45-60 ℃, the ratio of 1-4% of pressing the weight of raw material donkey-hide gelatin adds Sumizyme MP, 45-65 ℃ of constant temperature enzymolysis 2-5h, or the ratio of pressing the 1-4% of raw material donkey-hide gelatin weight adds neutral protease, 45-60 ℃ of constant temperature enzymolysis 2-6h, or the ratio of pressing the 1-3% of raw material donkey-hide gelatin weight adds trypsinase, 45-60 ℃ of constant temperature enzymolysis 2-5h, the enzymolysis solution that obtains is warming up to 90-105 ℃, and enzyme 15-40min goes out;
3. filter, supernatant concentration density 1.05-1.1, spraying drying promptly obtains donkey-hide gelatin oligopeptide finished product.
More excellent method:
1. be raw material with authentic Donga donkey-hide gelatin piece, pulverized 80 mesh sieves, the pure water that adds 10 times of raw material weights stirs 100 ℃ of passivation lipase 30min;
2. be cooled to 60 ℃, 2% the ratio of pressing the weight of raw material donkey-hide gelatin adds Sumizyme MP, 60 ℃ of constant temperature enzymolysis 4h, or 3% the ratio of pressing raw material donkey-hide gelatin weight adds neutral protease, 55 ℃ of constant temperature enzymolysis 5h, or 2% the ratio of pressing raw material donkey-hide gelatin weight adds trypsinase, 60 ℃ of constant temperature enzymolysis 3h, the enzymolysis solution that obtains is warming up to 100 ℃, and enzyme 30min goes out;
3. filter, filtrate concentrates density 1.05-1.1, and spraying drying promptly obtains donkey-hide gelatin peptide finished product.
Raw material donkey-hide gelatin of the present invention be equine species donkey Equus asinus L. and other donkey hides through decocting, concentrating the solid gums of making, nature and flavor are sweet, and are flat, return lung, liver, kidney channel, the function enriching yin of enriching blood is moisturized, hemostasis, and it is sallow to cure mainly the deficiency of blood, dizzy, palpitaition multiplely goes out blood trouble, yin deficiency syndrome and dry card.
The advantage and the beneficial effect of the inventive method are embodied in:
1. the purity height of the donkey-hide gelatin oligopeptide product produced of the inventive method, molecular weight is concentrated, and wherein molecular weight reaches more than 99% 10000-140 dalton in the enzymolysis solution, and wherein 1000-140 dalton reaches more than 50%;
2. the molecular weight of the donkey-hide gelatin oligopeptide produced of the present invention is little and concentrated, and human body absorbs easily;
3. the present invention has effectively avoided chemical means and has handled the environmental pollution that brought promptly to the problem of human body harm;
4. present method has been avoided adjusting PH, and nanofiltration desalination operation has reduced industrial cost, has improved the local flavor of finished product, has improved the yield of oligopeptide;
5. of the present invention with short production cycle, easy and simple to handle, cost is low, reliable product quality, and safe without toxic side effect can be widely used in fields such as healthcare products, medicine.
Embodiment
Embodiment 1
1. take by weighing the Donga donkey-hide gelatin 20kg that pulverizes and cross 80 mesh sieves,, add distilled water 160kg, after stirring, be warming up to 85 ℃ of passivation lipase 30min;
2. be cooled to 60 ℃, add Sumizyme MP 400g, stir enzymolysis 4h, be warming up to 100 ℃ then, enzyme 30min goes out;
3. filter, filtrate concentrates density 1.05-1.1, and spraying drying promptly obtains donkey-hide gelatin peptide finished product, obtains the 9.70kg powder.
Donkey-hide gelatin oligopeptide assay
1. method summary
Low-molecular-weight protein hydrolystate (comprising peptide class and total free aminoacids) dissolves in trichoroacetic acid(TCA) solution; High-molecular weight protein easily precipitates in trichoroacetic acid(TCA) solution.Sample is after the dissolving of trichoroacetic acid(TCA) solution, and centrifugation goes out the protein precipitation metallic substance, determines the sour molten protein content in the centrifugal clear liquid, and the sour molten protein content in the clear liquid deducts the content that free aminoacid content is oligopeptide.
2. analytical procedure
2.1 the mensuration of sour molten protein content
Take by weighing 2g(and be accurate to 1mg) sample, be added in the 10mL volumetric flask, with 15% trichoroacetic acid(TCA) solution constant volume, mix, leave standstill 10min.Sample solution behind centrifugal 10min under the 4000rpm, is got whole clear liquids, and the method for pressing GB/T 5009.5 regulation is measured the sour molten protein in the clear liquid, and the protein reduction factor is 6.25.Assay weight loss on drying is per sample converted and is butt.
2.2 the mensuration of free aminoacid content
Sample pre-treatments: take by weighing 20～30mg sample, be accurate to 0.0001g, evenly with the dissolving of 3% sulphosalicylic acid solution.Sample solution is transferred in the 50ml volumetric flask constant volume.Is that centrifugal 5min gets clear liquid on the 4000r/min whizzer with sample solution at rotating speed, uses 0.45 μ m filtering with microporous membrane clear liquid again, filtrate is transferred in the 50ml volumetric flask, behind the constant volume as the instrument detecting sample.All the other operations are with the method for the mensuration regulation of GB 12292 fruit, vegetables juice free aminoacid content.
2.3 result's statement
The content of oligopeptide
Calculate by formula (1):
In the formula:
2.4 measurement result
Measure the content of oligopeptide in the finished product, the results are shown in Table 1.
The assay result of oligopeptide in the table 1 peptide powder
| Sample | The donkey-hide gelatin oligopeptide |
| Oligopeptide content | 60.46% |
3. relative molecular mass is less than 1000 peptide proportion (high performance gel filtration chromatography)
3.1 method summary
The employing high performance gel filtration chromatography is measured.It promptly is stationary phase with the porous filler, difference according to sample component molecular volume size is separated, under the uv-absorbing wavelength 220nm of peptide bond condition, detect, use gel chromatography to measure the exclusive data process software (being GPC software) that relative molecular mass distributes, color atlas and data thereof are handled, calculated the 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.
The used standard substance of relative molecular mass calibration curve: cytochrome C (cyyochrome, MW12500); But the phthalein enzyme (aprotinin, MW6500); The bacillus enzyme (bacitracin, MW1450); Glycocoll-glycocoll-tyrosine-arginine (MW451); Glycocoll-glycocoll-glycocoll (MW189).
3.3 instrument and equipment
High performance liquid chromatograph: be furnished with UV-detector and the chromatographic working station or the totalizing instrument that contain the GPC data processing software; Moving phase vacuum filtration de-gassing vessel; Ultrasonic oscillator; Analytical balance: sensibility reciprocal 0.0001g.
3.4 chromatographic condition and system flexibility experiment
Chromatographic column: of the same type other that TSKgel G2000 SWXL 300mm * 7.8mm or performance are close therewith are applicable to the gel column of measuring protein and polypeptide; Moving phase: acetonitrile: water: trifluoroacetic acid, 45:55:0.1(volume ratio) detect wavelength: UV220nm; Flow velocity: 0.5ml/min; Column temperature: 30 ℃; Sampling volume: 10 μ l.
For making the requirement of chromatographic system coincidence detection, be defined under the above-mentioned chromatographic condition, it is that theoretical plate number (N) is not less than 5000 by the calculating of three poly saccharide peptide standard products (glycocoll-glycocoll-glycocoll) peak that the post of gel chromatographic columns is imitated, and the partition ratio of oligopeptide (Kd) should be between 0～1.
3.5 the relative molecular mass calibration curve is made
Be mixed with 0.1%(W/V with moving phase respectively) the poly saccharide peptide standard product solution of above-mentioned different relative molecular masses, be that 0.2-0.5 μ m tetrafluoroethylene or nylon filtering membrane filter back sample introduction respectively with the aperture, obtain the color atlas of series standard product.Logarithm (lgMW) with relative molecular mass obtains relative molecular mass calibration curve and equation thereof to the retention time mapping or do linear regression.
3.6 specimen preparation
Take by weighing sample 20.0mg in the 10mL volumetric flask, be settled to scale with moving phase, sonic oscillation 10min makes sample fully dissolve mixing, is after 0.2-0.5 μ m tetrafluoroethylene or nylon filtering membrane filter with the aperture, last machine sample introduction.
3.7 the calculating of relative molecular mass
The sample solution of 3.6 preparations is analyzed under above-mentioned chromatographic condition.Use the GPC data processing software then, calculate in the chromatographic data substitution calibration curve equation with sample, can obtain the relative molecular mass and the distribution range thereof of peptide in the sample.Calculate the peak area relative percentage sum of relative molecular mass scope with the peak area normalization method at the peptide below 1000.
3.8 measurement result
Donkey-hide gelatin peptide molecular weight distribution range measurement result sees Table 6,7.
Table 2, donkey-hide gelatin peptide molecular weight distribution results
| Molecular weight ranges | Time opening min | Concluding time min | Weight-average molecular weight | Peak area %(λ 220nm) |
| 1000-10000 | 13.656 | 18.744 | 2346 | 15.45 |
| 500-1000 | 18.744 | 20.673 | 680 | 30.55 |
| 140-500 | 20.673 | 23.289 | 310 | 35.42 |
| 70-140 | 23.289 | 24.852 | 94 | 18.58 |
Above result shows that the donkey-hide gelatin peptide molecular weight mainly concentrates on 140-1000, accounts for more than 50%.
Embodiment 2
1. take by weighing the Donga donkey-hide gelatin 20kg that pulverizes and cross 80 mesh sieves, add distilled water 120kg, after stirring, be warming up to 85 ℃ of passivation lipase 30min;
2. be cooled to 70 ℃, add trypsinase 10g, stir enzymolysis 4h, be warming up to 85 ℃ then, enzyme 15min goes out;
3. filter, filtrate concentrates density 1.05-1.1, and spraying drying promptly obtains donkey-hide gelatin peptide finished product, obtains the 10.20kg powder.
Embodiment 3
1. take by weighing the Donga donkey-hide gelatin 20kg that pulverizes and cross 100 mesh sieves, add distilled water 300kg, after stirring, be warming up to 120 ℃ of passivation lipase 60min;
2. be cooled to 40 ℃, add neutral protease 100g, stir enzymolysis 4h, be warming up to 110 ℃ then, enzyme 10min goes out;
3. filter, filtrate concentrates density 1.05-1.1, and spraying drying promptly obtains donkey-hide gelatin peptide finished product, obtains the 11.30kg powder.
Claims (4)
1. the preparation method of a donkey-hide gelatin peptide comprises raw material donkey-hide gelatin enzymolysis, concentrated, dry, it is characterized in that: with Sumizyme MP or neutral protease or trypsin digestion.
2. the preparation method of donkey-hide gelatin peptide according to claim 1 is characterized in that: the Sumizyme MP add-on is the 0.5-5% of raw material donkey-hide gelatin weight, enzymolysis 1-10h; Or the neutral protease of adding raw material donkey-hide gelatin weight 0.5-5%, constant temperature enzymolysis 1-10h; Or the trypsinase of adding raw material donkey-hide gelatin weight 0.5-5%, enzymolysis solution is warming up to 85-110 ° of C, and enzyme 10-50min goes out.
3. the preparation method of donkey-hide gelatin peptide according to claim 2 is characterized in that: the Sumizyme MP add-on is the 1-4% of raw material donkey-hide gelatin weight, enzymolysis 2-5h, or the neutral protease of adding raw material donkey-hide gelatin weight 1-4%, constant temperature enzymolysis 2-5h; Or the trypsinase of adding raw material donkey-hide gelatin weight 1-4%, enzymolysis 2-6h, enzymolysis solution are warming up to 90-105 ° of C, and enzyme 15-45min goes out.
4. the preparation method of donkey-hide gelatin peptide according to claim 3 is characterized in that: the Sumizyme MP add-on is 2% of a raw material donkey-hide gelatin weight, enzymolysis 4h, or the neutral protease of adding raw material donkey-hide gelatin weight 3%, constant temperature enzymolysis 5h; Or the trypsinase of adding raw material donkey-hide gelatin weight 2%, enzymolysis 3h, enzymolysis solution are warming up to 100 ° of C, and enzyme 30min goes out.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106107398A (en) * | 2016-08-01 | 2016-11-16 | 广州美贝贸易有限公司 | A kind of solid beverage rich in donkey-hide gelatin peptide and preparation method thereof |
| CN107998333A (en) * | 2017-12-13 | 2018-05-08 | 国药肽谷有限公司 | A kind of preparation method of donkey-hide gelatin peptides products |
| CN109394672A (en) * | 2018-12-21 | 2019-03-01 | 山东人面桃花阿胶养生有限公司 | A kind of donkey-hide gelatin collagen micro mist, its preparation method and the application in skin care item |
| CN109453204A (en) * | 2018-12-21 | 2019-03-12 | 山东人面桃花阿胶养生有限公司 | A kind of donkey-hide gelatin collagen micro mist and preparation method thereof |
| CN110172490A (en) * | 2019-03-13 | 2019-08-27 | 济南大学 | A method of except grease in donkey-hide gelatin peptide |
| CN110643660A (en) * | 2019-08-29 | 2020-01-03 | 北京化工大学 | Method for preparing antioxidant peptide by donkey hide protease hydrolysis under assistance of ultrasound |
| CN112353828A (en) * | 2020-10-29 | 2021-02-12 | 光亚生物科技(广州)有限公司 | Ginseng paste for replenishing blood and activating meridians and preparation method thereof |
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| CN102021215A (en) * | 2010-11-11 | 2011-04-20 | 湖北远成药业有限公司 | Method for extracting gelatin peptides |
| CN102101884A (en) * | 2009-12-18 | 2011-06-22 | 济南瑞安药业发展有限公司 | Preparation method and use of donkey-hide gelatin polypeptide |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106107398A (en) * | 2016-08-01 | 2016-11-16 | 广州美贝贸易有限公司 | A kind of solid beverage rich in donkey-hide gelatin peptide and preparation method thereof |
| CN107998333A (en) * | 2017-12-13 | 2018-05-08 | 国药肽谷有限公司 | A kind of preparation method of donkey-hide gelatin peptides products |
| CN107998333B (en) * | 2017-12-13 | 2021-01-29 | 国药肽谷有限公司 | Preparation method of donkey-hide gelatin peptide product |
| CN109394672A (en) * | 2018-12-21 | 2019-03-01 | 山东人面桃花阿胶养生有限公司 | A kind of donkey-hide gelatin collagen micro mist, its preparation method and the application in skin care item |
| CN109453204A (en) * | 2018-12-21 | 2019-03-12 | 山东人面桃花阿胶养生有限公司 | A kind of donkey-hide gelatin collagen micro mist and preparation method thereof |
| CN109453204B (en) * | 2018-12-21 | 2021-05-18 | 山东聚胶之元生物科技有限公司 | Donkey-hide gelatin collagen micro powder and preparation method thereof |
| CN110172490A (en) * | 2019-03-13 | 2019-08-27 | 济南大学 | A method of except grease in donkey-hide gelatin peptide |
| CN110643660A (en) * | 2019-08-29 | 2020-01-03 | 北京化工大学 | Method for preparing antioxidant peptide by donkey hide protease hydrolysis under assistance of ultrasound |
| CN112353828A (en) * | 2020-10-29 | 2021-02-12 | 光亚生物科技(广州)有限公司 | Ginseng paste for replenishing blood and activating meridians and preparation method thereof |
| CN112353828B (en) * | 2020-10-29 | 2022-08-16 | 光亚生物科技(广州)有限公司 | Ginseng cream for enriching blood and activating meridians and preparation method thereof |
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