CN105132503A - Refining process of pond turtle bioactive peptides and application of pond turtle bioactive peptides in preventing liver cirrhosis and resisting fatigue - Google Patents
Refining process of pond turtle bioactive peptides and application of pond turtle bioactive peptides in preventing liver cirrhosis and resisting fatigue Download PDFInfo
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Abstract
The invention discloses a refining process of pond turtle bioactive peptides. Acetic acid immersion pretreatment is firstly adopted, expanding tissue is softened, part of impurity protein is removed, an enzymolysis method is adopted for treatment, citric acid is added to be used as an enzymolysis medium, the extraction rate of the bioactive peptides is higher than 25% while the extraction rate in the prior art is only about 10%, and defects of time consumption and energy dissipation with a conventional processing method, damage to amino acid with a general acid hydrolyzation method, high probability of racemization with an alkali hydrolysis method and the like are overcome. Besides, a multilevel membrane separation technology is combined, the selectivity is high, few by-products are produced, the adaptability is high, the purity of the separated bioactive peptides is higher than 75%, the molecular weight is smaller than 10 KD, the bioactive peptides can enter an organism circulation system completely and are easy to absorb, and however, molecular weight is not uniformly distributed and the absorption effect is poor with a commonly used centrifugal and precipitation filtering method. Desalination and debitterization are also completed in the enzymolysis, microfiltration and multilevel ultrafiltration processes, and the prepared pond turtle bioactive peptides have shallower color, don't have foreign smell, have uniform molecular weight, are safe and non-toxic, and have wide application prospect in health care of liver cirrhosis prevention, fatigue resistance, immunity enhancing and the like.
Description
Technical field
The present invention relates to biological health-care food technical field, is stone tortoise bioactive peptide method for refining and the application in prevention liver cirrhosis, antifatigue specifically.
Background technology
Society, rhythm of life is fast, dog-eat-dog, greatly, many people are busy with work for work, studying and living pressure, and overdraw is healthy, take food irregular, diet is uncontrolled, causes the disorder of liver, kidney system, direct or indirect damage liver kidney, immunizing power reduces, and resistance against diseases declines, in China, about have the people of 15% to be healthy, the people of 15% is in non-health, and the people of 70% is in " subhealth state " state.The scale of " subhealth state " crowd and severity allow of no optimist at present, and research and develop protection hepatic and renal tissue, prevention liver cirrhosis, antifatigue, strengthening immunity, improves the quality of life of people and the healthcare products of body constitution have been very urgent.
Stone tortoise, namely terrapin is intended in Guangxi, also known as stone small water turtle, Vietnam stone tortoise, Guangxi terrapin, pond turtle with yellow throat, be loaded in Han dynasty Shennong's Herbal as medicinal head, be classified as top grade, after this how on the books book on Chinese herbal medicine is, wherein " book on Chinese herbal medicine is looked for the truth " speech " specially enter kidney, double enter the heart, Gan Xian; be slightly cold ", Compendium of Material Medica record road " (tortoise) can lead to conception vessel, thus get its first with bushing, kidney tonifying, remove hemostasis; stop blooding dysentery; continue muscles and bones, control overstrain, subduing inflammation ".Since ancient times, the health-care effect that China just has food tortoise to improve the health, modern medicine study shows, tortoise contains abundant essential amino acid, protein, cutin, osso-albumin, Ca, P, K and multivitamin, blood sugar, the nutritive ingredients such as lipid acid, human body easily absorbs, add the resistance to famine of tortoise, yearningly resistance to, cold-resistant, the life habit that life-span is long, constitute the multiple long-lived cell factor of its body, have nourishing and suppressing Yang, the strong bone of kidney-nourishing, consolidating channel for hemostasis, heart tonifying and tranquilizing, can be used for deficiency of kidney yin, the deficiency of Yin is partially high, night sweat, seminal emission etc., especially for hepatic fibrosis, antifatigues etc. show good health-care effect, also studies have found that stone tortoise bioactive peptide can promote cancer patients's Leukocyte Growth in body after chemicotherapy, recover healthy, reach anti-cancer efficacy.
Qiezhou, be located in Subtropical Coastal district, benign climate, because geographical natural condition is richly endowed by nature, Qiezhou stone small water turtle is closer to natural, ecological product, excellent quality, become Chinese geography famous special product, with the nutritive health-care that it is exclusive, economic worth becomes the up-and-coming youngster of famous-brand and high-quality cultivation, current stone tortoise raiser crosses ten thousand, annual value of production is more than 3.5 hundred million, but, although ample supply and prompt delivery, but manyly also stay in single cultivation sales mode, the product category of having gone on the market is less as fresh and alive quick frozen product, tinned food, and often technique is coarse, mouthfeel is poor, lack the market competitiveness, intensive processing is still in space state.How to extract from stone tortoise, separation and purification goes out the large difficult point that bioactive peptide is still research, traditional concocted method effective constituent is fried needs the long period, energy consumption is large, general acid extraction method partial amino-acid is damaged, product yield is low, easily there is racemization in the amino acid containing hydroxyl and sulfydryl that potass extraction method extracts, peptide class inactivation, and the separation and purification of peptide class is a systematic engineering of business, general sedimentation filtration, centrifugation, solvent extraction is difficult to reach good separation effect, therefore the present invention develops a kind of stone tortoise deep processing method on great many of experiments in early stage basis, extraction yield is high, separation and purification is effective, active high, be intended to make full use of Qiezhou local resources advantage, to the nutrition of stone tortoise, health value gos deep into systematic study, form scientific research, produce, deep processing, sell the integral framework be integrated, the stone tortoise bioactive peptide developed has and prevents liver cirrhosis preferably, antifatigue, strengthening immunity health-care effect.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of simple and safe, easy to operate, stone tortoise bioactive peptide method for refining that product purity is high is provided, additionally provide it in prevention liver cirrhosis, anti-fatigue health-caring application, be intended to make full use of ground ashlar tortoise resource, develop the deep processing healthcare products had a extensive future more.
For achieving the above object, the invention provides stone tortoise bioactive peptide method for refining, comprise the steps:
1) raw materials pretreatment: put to death by stone tortoise, decaptitate, rejects internal organ, washs 3-4 time, pulverizes, adjust pH2-3, less than 10 DEG C are soaked 24h, take out after expansion evenly, repeatedly clean 2-3 time extremely without tart flavour with water, drain, the 8-10 that adds water doubly pulls an oar, and to homogenate, adds water and supplies weightlessness;
2) enzymolysis: to step 1) add 1% (w/w) citric acid as enzymolysis process Extraction medium in the homogenate that obtains, regulate PH2-4, add the stomach en-of 0.5-2% (w/w), enzymolysis 5-7h at 4 DEG C, stirring 10-15min per hour, heating, the enzyme that goes out is lived, and obtains stone tortoise enzymolysis solution;
3) Using Multistage Membranes separation and purification:
1. micro-filtration: get step 2) the stone tortoise enzymolysis solution that obtains, being diluted with water to massfraction is 10%, and with 0.3 μm of membrane microfiltration, working pressure is 0.2MPa;
2. one-level ultrafiltration: the filtrate that 1. step is obtained by the ultrafiltration of 20kD ultra-filtration membrane, working pressure 0.2-0.25MPa, ultrafiltration 2-3 time;
3. two-stage ultrafiltering: by step 2. gained filtrate adopt the ultrafiltration 2 times under 0.8MPa working pressure of 10kD ultra-filtration membrane again, obtain permeate, autoclaving, concentrating under reduced pressure is dry;
4. regular Membrane cleaning: after reaction for some time, ultra-filtration membrane has gel coat, and membrane flux can reduce, carry out Membrane cleaning, first rinse about 15min with water, to permeate colorless and odorless, add 0.5% (w/w) sodium hydroxide and repeatedly rinse 15min, be washed to filtered solution for neutral;
4) sterilizing, drying, obtain stone tortoise bioactive peptide powder.
The preferred pure water of the present invention's water used.
Step 1) described in the preferred 1:20 of addition (w/v) of acetic acid.
Step 2) described in stirring, preferably adopt magnetic stirrer to stir.
Step 4) described in sterilizing, drying, preferably adopt spraying dry, inlet temperature 120-150 DEG C, air outlet temperature 70-90 DEG C.
The invention still further relates to the stone tortoise bioactive peptide adopting above-mentioned method for refining to obtain, and it is preventing the application in liver cirrhosis, antifatigue, also can be according to actual needs, add pure water, essence is appropriate, adopts existing preparation technique and processing method, makes oral preparations with conventional auxiliary material, comprise oral liquid, granule, tablet, capsule etc.
Compared to the prior art, the invention has the advantages that:
1, in prior art, general acid extraction method partial amino-acid is damaged, and product yield is low, and the amino acid containing hydroxyl and sulfydryl that potass extraction method extracts easily the shortcomings such as racemization occurs.The present invention adds 1% citric acid as enzymolysis and extraction medium, regulate PH2-4, there is provided acid-reaction environment, make raw material swelling, structure is more fluffy, be beneficial to stomach en-infiltration, and be that stomach en-is in optimum pH in such circumstances, enzymic activity improves, and bioactive peptide extraction yield is more than 25%, overcome traditional concocted method and easily occur precipitation at leaching process, the shortcoming of time consumption and energy consumption.
2, present invention contemplates stone tortoise enzymolysis solution and contain a large amount of protein matters, after operation certain hour, ultrafiltration membrane surface can adsorb a certain amount of protein substance, forms gel coat, makes flux depression, affect separating effect, disclose a kind of easy and simple to handle, fast, feasible film method for cleaning, after reaction for some time, adopt 0.5% sodium hydroxide circulation to wash Membrane cleaning, when measuring filtered solution and being neutral, this step can ensure the penetrating amount of film that system held one is ideal.
3, the present invention before carrying out multi-ultrafiltration through the microfiltration membrane pre-treatment of 0.3 μm, prevent macromole suspended impurity and protein contamination ultra-filtration membrane, macromole impurity protein is removed again by the ultrafiltration of 20kD ultra-filtration membrane, obtain filtrate and remove some inorganic salt and small molecular weight impurity through 10kD ultra-filtration membrane again, finally obtain stone tortoise bioactive peptide purity more than 75%, molecular weight is less than 10KD, can complete form ingress engine systemic circulatory system, close with normal people's body physiological product, be beneficial to body to absorb, and it is general centrifugal, sedimentation filtration method molecular weight distribution heterogeneity, difficult absorption, this micro-filtration, complete desalination in Using Multistage Membranes sepn process simultaneously, debitterize, obtained stone tortoise bioactive peptide color is thin out, mouthfeel is good.
4, the present invention adopts micro-filtration and Using Multistage Membranes to be separated enzymolysis solution, and achieve effective control of enzymolysis product molecular weight distribution, molecular weight is less than 10KD, composition is tending towards homogeneous, stable in properties, and carry out pharmacodynamics with enzymolysis solution obtained by common centrifugal separation and compare, found that for CCl
4caused Liver Damage in Rats model; the liver of common centrifugal separation group rat shows faint yellow; there is slight swelling; show in particle nodositas; and Using Multistage Membranes discrete group; rat liver between sorrel and faint yellow between; do not find obvious particle tubercle thing, this is because oligopeptides molecular weight distribution is wide in the bioactive peptide of common separation gained, not easily absorb; and oligopeptides molecular weight is concentrated in Using Multistage Membranes isolating active peptide; small-molecular peptides content is high, active strong, and hepatic fibrosis is taken a turn for the worse; at protection hepatic tissue, the effect of prevention liver cirrhosis is more obvious.
Embodiment
With embodiment, the present invention will be described below, but the present invention is not limited to these embodiments.
Embodiment 1
Stone tortoise bioactive peptide method for refining, comprises the steps:
1) raw materials pretreatment: put to death by stone tortoise, decaptitate, rejects internal organ, clear water washs 3 times, just pulverizes, and adds the acetic acid (1:20W/V) of about PH2.5,5 DEG C are soaked 24h, take out after expansion evenly, add water and repeatedly clean 2 times to without tart flavour, drain, add water 8 times and pull an oar, to homogenate, add water and supply weightlessness, for subsequent use;
2) enzymolysis: add 1% citric acid and regulate PH to 3 as enzymolysis process Extraction medium in homogenate, add the stomach en-of 1%, enzymolysis 5h at 4 DEG C, every 1h adopts magnetic stirrer to stir 15min, heating, and the enzyme that goes out is lived, and obtains stone tortoise enzymolysis solution;
3) Using Multistage Membranes separation and purification:
1. micro-filtration: get stone tortoise enzymolysis solution, be diluted with water to massfraction and be about 10%, with 0.3 μm of membrane microfiltration, working pressure is 0.2MPa; Until micro-filtration to add-on 80% time, in trapped fluid, add suitable quantity of water, stir, continue micro-filtration, until micro-filtrate be collected into volume equal liquid add-on time, stop micro-filtration, obtain micro-filtrate for subsequent use;
2. one-level ultrafiltration: filtrate passes through the ultrafiltration of 20kD ultra-filtration membrane, working pressure 0.2MPa, ultrafiltration 2 times;
3. two-stage ultrafiltering: one-level ultrafiltration gained filtrate adopts the ultrafiltration 2 times under 0.8MPa working pressure of 10kD ultra-filtration membrane again, obtains permeate;
4. regular Membrane cleaning: after reaction for some time, if find, ultra-filtration membrane has gel coat, when membrane flux reduces, need Membrane cleaning be carried out, first rinse about 15min with water, to permeate colorless and odorless, repeatedly rinse 15min with 0.5% sodium hydroxide, be washed to filtered solution for neutral;
4) sterilizing, dry: sterilizing, adopt spray-drying process dry, inlet temperature 120 DEG C, air outlet temperature 90 DEG C, obtains stone tortoise bioactive peptide powder.
Embodiment 2
Stone tortoise bioactive peptide method for refining, comprises the steps:
1) raw materials pretreatment: put to death by stone tortoise, decaptitate, rejects internal organ, clear water washs 4 times, just pulverizes, and adds the acetic acid (1:20W/V) of about PH2.5,10 DEG C are soaked 24h, take out after expansion evenly, add water and repeatedly clean 3 times to without tart flavour, drain, add water 9 times and pull an oar, to homogenate, add water and supply weightlessness, for subsequent use;
2) enzymolysis: add 1% citric acid and regulate PH to 3 as enzymolysis process Extraction medium in homogenate, add the stomach en-of 2%, enzymolysis 7h at 4 DEG C, every 1h adopts magnetic stirrer to stir 15min, heating, and the enzyme that goes out is lived, and obtains stone tortoise enzymolysis solution;
3) Using Multistage Membranes separation and purification:
1. micro-filtration: get stone tortoise enzymolysis solution, be diluted with water to massfraction and be about 10%, with 0.3 μm of membrane microfiltration, working pressure is 0.2MPa; Until micro-filtration to add-on 80% time, in trapped fluid, add suitable quantity of water, stir, continue micro-filtration, until micro-filtrate be collected into volume equal liquid add-on time, stop micro-filtration, obtain micro-filtrate for subsequent use;
2. one-level ultrafiltration: filtrate passes through the ultrafiltration of 20kD ultra-filtration membrane, working pressure 0.25MPa, ultrafiltration 3 times;
3. two-stage ultrafiltering: one-level ultrafiltration gained filtrate adopts the ultrafiltration 2 times under 0.8MPa working pressure of 10kD ultra-filtration membrane again, obtains permeate;
Regular Membrane cleaning: after reaction for some time, if find, ultra-filtration membrane has gel coat, when membrane flux reduces, need carry out Membrane cleaning, first rinse about 15min with water, to permeate colorless and odorless, repeatedly rinse 15min with 0.5% sodium hydroxide, is washed to filtered solution for neutral;
4) sterilizing, dry: sterilizing, adopt spray method dry, inlet temperature 150 DEG C, air outlet temperature 80 DEG C, obtains stone tortoise bioactive peptide powder.
Embodiment 3
Stone tortoise bioactive peptide method for refining, comprises the steps:
1) raw materials pretreatment: put to death by stone tortoise, decaptitate, rejects internal organ, clear water washs 3 times, just pulverizes, and adds the acetic acid (1:20W/V) of about PH2.5,10 DEG C are soaked 24h, take out after expansion evenly, add water and repeatedly clean 3 times to without tart flavour, drain, add water 10 times and pull an oar, to homogenate, add water and supply weightlessness, for subsequent use;
2) enzymolysis: add 1% citric acid and regulate PH to 3 as enzymolysis process Extraction medium in homogenate, add the stomach en-of 2%, enzymolysis 6h at 4 DEG C, every 1h adopts magnetic stirrer to stir 15min, heating, and the enzyme that goes out is lived, and obtains stone tortoise enzymolysis solution;
3) Using Multistage Membranes separation and purification:
1. micro-filtration: get stone tortoise enzymolysis solution, be diluted with water to massfraction and be about 10%, with 0.3 μm of membrane microfiltration, working pressure is 0.2MPa; Until micro-filtration to add-on 80% time, in trapped fluid, add suitable quantity of water, stir, continue micro-filtration, until micro-filtrate be collected into volume equal liquid add-on time, stop micro-filtration, obtain micro-filtrate for subsequent use;
2. one-level ultrafiltration: filtrate passes through the ultrafiltration of 20kD ultra-filtration membrane, working pressure 0.2MPa, ultrafiltration 3 times;
3. two-stage ultrafiltering: one-level ultrafiltration gained filtrate adopts the ultrafiltration 2 times under 0.8MPa working pressure of 10kD ultra-filtration membrane again, obtains permeate;
4. regular Membrane cleaning: after reaction for some time, if find, ultra-filtration membrane has gel coat, when membrane flux reduces, need Membrane cleaning be carried out, first rinse about 15min with water, to permeate colorless and odorless, add 0.5% sodium hydroxide and repeatedly rinse 15min, be washed to filtered solution for neutral;
4) sterilizing, dry: sterilizing, adopt spray method dry, inlet temperature 150 DEG C, air outlet temperature 70 DEG C, obtains stone tortoise bioactive peptide powder.
Result:
Experimental example: pharmacodynamic experiment
Test 1 stone tortoise bioactive peptide anti hepatic fibrosis
1, need testing solution
The stone tortoise bioactive peptide powder that embodiment 3 is made, add water ultrasonic dissolution, and be mixed with every milliliter contained 2g.
2, laboratory animal
SD rat, 60, male, SPF level, body weight 160-200g.
3, method
SD rat conforms and is divided into 5 groups at random afterwards in 5 days, be respectively blank group, model group, positive controls, stone tortoise bioactive peptide high dose group, stone tortoise bioactive peptide low dose group, often organize 10, model group and each test administration group rat back subcutaneous injection 40%CCl
4, 2 times weekly, 5ml/kg subcutaneous injection is pressed in modeling first, presses 3ml/kg subcutaneous injection later, amounts to 12 weeks.Normal group subcutaneous injection normal saline.Continuous 12 weeks backward, normal group and model group gave gavage physiological saline, and positive controls gives colchicine 0.2mg/kg/d, and stone tortoise bioactive peptide high and low dose group gives test trial-product medication amount respectively and is respectively: 12mg/kg/d, 4mg/kg/d.After experiment terminates, all Rat Fast can't help water 12h, 10% chloral hydrate anesthesia, and broken end gets blood, separation of serum, and radioimmunology surveys HA, LN, PC III, IV-C.
4, data processing
Experimental data represents with (X ± s), and data statistics SPSS11.0 software carries out, and between group, contrast adopts onewayanova to carry out.
Each test group is on the impact of hepatic fibrosis index
Note: ## compares P<0.01* with blank group, and * * compares P<0.05 with model control group, P<0.01
5, analysis and thinking
From upper experimental result, model group compared with normal group in rat blood serum the equal significance of HA, LN, PC III, IV-C raise (P<0.01), show modeling success.Positive controls and each dosage group of stone tortoise bioactive peptide are compared with model group, and the equal significance of HA, LN, PC III, IV-C reduces (P < 0.05), has statistical significance.There are some researches show that hepatic fibrosis is the pathologic basis that multiple chronic hepatopathy develops into liver cirrhosis; long-term life is irregular; to drink etc. and stimulate liver all can cause hepatic fibrosis; experiment shows that stone tortoise bioactive peptide can alleviate degree of hepatic fibrosis; reverse hepatic fibrosis; there is protection liver, the health-care effect of prevention liver cirrhosis.
Test 2 stone tortoise bioactive peptide antifatigue researchs
1, test drug
The stone tortoise bioactive peptide powder that embodiment 3 is made, add water ultrasonic dissolution, and be mixed with every milliliter contained 0.5g.
2, animal
Kunming mouse, male, SPF level, body weight (20 ± 2) g
3, method
Get Kunming mouse and be divided into 4 groups, often organize 10, be respectively blank group, the high, medium and low dosage group of stone tortoise bioactive peptide.The high, medium and low dosage component of stone tortoise bioactive peptide does not give test trial-product medication amount and is respectively 50mg/kg/d, 25mg/kg/d, 10mg/kg/d, blank group gavage respective volume physiological saline.
Loaned swimming test: after last administration 30min, puts mouse at water tank went swimming, depth of water 30cm, water temperature about 25 DEG C, the plasticine of mouse root of the tail portion load 5% body weight.Record mouse starts to the dead time, as swimming time from swimming.
Serum urea nitrogen determination: after last administration 30min, at temperature 25 DEG C of water went swimming 90min, rest 60min posterior orbit gets blood, and colorimetry surveys serum urea nitrogen.
4, result
The impact of stone tortoise Active Peptide in Mice swimming time and serum urea nitrogen
Note: *, * * organizes control group with blank and compares P<0.05, P<0.01
5, analysis and thinking
From experimental result, the stone tortoise bioactive peptide high, medium and low dosage group mouse more blank group of time of swimming time is longer, and prompting stone tortoise bioactive peptide makes body can tolerate strenuous exercise for more time, the generation of delay fatigue or the elimination accelerated fatigue.Serum urea nitrogen is the sensitive indexes evaluating body load ability to bear.Body serum urea nitrogen content with work and exercise load increase and increase, body is poorer to load performance, serum urea nitrogen increase is more obvious, during tranquillization, each group difference of serum urea nitrogen is little, after 90min swimming, stone tortoise bioactive peptide high, middle dosage group mice serum blood urea nitrogen, lower than control group (P<0.05), has statistical significance, prompting stone tortoise bioactive peptide can improve the adaptive faculty of body to load, has good anti-fatigue health efficacy.
Claims (5)
1. stone tortoise bioactive peptide method for refining, is characterized in that, comprise the steps:
1) raw materials pretreatment: put to death by stone tortoise, decaptitate, rejects internal organ, washs 3-4 time, pulverizes, adjust pH2-3, less than 10 DEG C are soaked 24h, take out after expansion evenly, repeatedly clean 2-3 time extremely without tart flavour with water, drain, the 8-10 that adds water doubly pulls an oar, and to homogenate, adds water and supplies weightlessness;
2) enzymolysis: to step 1) add 1% citric acid as enzymolysis process Extraction medium in the homogenate that obtains, regulate PH2-4, add the stomach en-of 0.5-2%, enzymolysis 5-7h at 4 DEG C, stirring 10-15min per hour, heating, the enzyme that goes out is lived, and obtains stone tortoise enzymolysis solution;
3) Using Multistage Membranes separation and purification:
1. micro-filtration: get step 2) the stone tortoise enzymolysis solution that obtains, being diluted with water to massfraction is 10%, and with 0.3 μm of membrane microfiltration, working pressure is 0.2MPa;
2. one-level ultrafiltration: the filtrate that 1. step is obtained by the ultrafiltration of 20kD ultra-filtration membrane, working pressure 0.2-0.25MPa, ultrafiltration 2-3 time;
3. two-stage ultrafiltering: by step 2. gained filtrate adopt the ultrafiltration 2 times under 0.8MPa working pressure of 10kD ultra-filtration membrane again, obtain permeate, autoclaving, concentrating under reduced pressure is dry;
4. regular Membrane cleaning: after reaction for some time, ultra-filtration membrane has gel coat, and membrane flux can reduce, and carries out Membrane cleaning, first rinses about 15min with water, to permeate colorless and odorless, adds 0.5% sodium hydroxide and repeatedly rinses 15min, is washed to filtered solution for neutral;
4) sterilizing, drying, obtain stone tortoise bioactive peptide powder.
2. stone tortoise bioactive peptide method for refining according to claim 1, is characterized in that: step 1) described in the addition of acetic acid be 1:20.
3. stone tortoise bioactive peptide method for refining according to claim 1, is characterized in that: step 2) described in stirring, adopt magnetic stirrer stir.
4. the stone tortoise bioactive peptide that in claim 1-3, arbitrary described method for refining obtains.
5. the application of stone tortoise bioactive peptide according to claim 4 in prevention liver cirrhosis, antifatigue.
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| CN112941137A (en) * | 2021-03-25 | 2021-06-11 | 海南金龟岛生物科技有限公司 | Production method of tortoise peptide |
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