CN101703259B - Method for deeply processing deer placenta - Google Patents
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- CN101703259B CN101703259B CN2009102196754A CN200910219675A CN101703259B CN 101703259 B CN101703259 B CN 101703259B CN 2009102196754 A CN2009102196754 A CN 2009102196754A CN 200910219675 A CN200910219675 A CN 200910219675A CN 101703259 B CN101703259 B CN 101703259B
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Abstract
The invention relates to a method for deeply processing deer placenta. The method comprises the technical scheme of selecting deer placenta and normal saline in a weight ratio of 1:1.5-2.5, grinding and homogenizing the mixture; refrigerating the mixture at the temperature of between 40 and 20 DEG C below zero, melting the mixture at room temperature, performing refrigeration and melting repeatedly, and centrifuging the mixture to collect supernatant, wherein the supernatant is placenta extract and precipitate is for later use; adding acetone into the placenta extract, evenly mixing, and centrifuging the mixture to collect the supernatant; adding acetone into the supernatant in the same ratio, evenly mixing and centrifuging the mixture, distilling the supernatant under reduced pressure, and sterilizing and ultrafiltering the supernatant to prepare active deer placenta extract; selecting the active deer placenta extract to collect component comprising molecular weight of between 3,000 and 8,000 dal through ultrafiltration and dialysis, desalinizing the supernatant through gel-filtration chromatography, and sterilizing the supernatant through membranes to prepare deer placenta immunoregulatory factor extract; and preparing compound amino acid by using microwave to assist acid hydrolysis of precipitate. The method can extract more bioactivator from the deer placenta, not only ensures that waste protein resources can be utilized sufficiently, but also can reduce the treatment cost for the protein, and avoids causing environmental pollution at the same time.
Description
Technical field
The invention belongs to Food Science and bioengineering field, relate to a kind of method of utilizing biotechnology deep processing deer placenta particularly, to obtain to have the new product of BA.
Background technology
Placenta (Placenta) has another name called " dried human placenta (Placenta hominid) ".Clinical practice is extensive, and it is thin and weak to be usually used in consumptive disease, hectic fever due to yin night sweat, the cough and asthma of deficiency of both the lung and kidney, deficiency of kidney-QI, essence and blood decline few impotence and seminal emission, sterile, infertile, few breast etc.It as the gestational period parent and fetus carry out the tie of mass exchange; Has the important physical function; Can secrete multiple hormone, enzyme, contain multiple proteins, antibody, phosphatide, growth factor, cell factor and can stimulate the small-molecule active substance of antibody mediated immunity system.Placenta has the effect of building up resistance with immunity on immunology.Modern biology and medical research prove; Though the structure of animal and human's class placenta is different; But all containing bioactivators such as panimmunity globulin, active peptide, hormone and amino acid, mineral matter and other components in the placenta, is a kind of comparatively desirable immunomodulator and nutrition cosmetic health product.
The deer tire means the band afterbirth in the doe abdomen, the fetus of fetal fluids, or body surface do not have hair, on an empty stomach, in the body each organ grown complete, but fetus or the postpartum of causing miscarriage owing to reasons such as machineries are because of the spitter of former cause deaths such as hypogalactia, doe are refused to feed, pernicious habit.First year Compendium of Material Medica of the pharmacological action of deer tire sayed the effect of its useful kidney yang, replenishing vital essence and blood.
Though China is utilizing that animal tissue's organ and metabolite are cured the disease, long history is being arranged aspect the diseases prevention.But, comparatively deep for the research of sheep placenta, ox tire, pig tire etc. both at home and abroad, and less for the research of deer tire.China is very original for the application of deer tire, and the processing of deer tire product is coarse, and product is elementary, kind is single, and curative effect is not remarkable, causes the significant wastage of deer tire resource.In fact, utilizing modern biotechnology, is that raw material can be developed the diversification product with the deer tire, helps perfect, the upgrading of deer industrial chain.In addition, in original deer tire processing process, directly the residue that produces is abandoned, promptly cause environmental pollution, wasted a lot of beneficiating ingredients in the deer tire again, cause the great wasting of resources.
Summary of the invention
To the problems referred to above, the present invention provides a kind of and economizes on resources through biotechnology, can from the deer tire, extract the method for deeply processing deer placenta of more bioactivator.The present invention changes deer product processing mode in the past, through biotechnology, carries out the deep processing of deer tire scientifically and rationally, realizes the suitability for industrialized production and the new product development of deer product.
In order to achieve the above object, the present invention adopts following technical scheme: a kind of method for deeply processing deer placenta is characterized in that: get fresh deer placenta and remove muscle, film, shred.By weight, deer placenta: physiological saline is 1: 1.5～2.5 to get material, grinding machine for grinding, fully homogenate;-40～-20 ℃ of freezing, room temperatures are melted, freezing repeatedly with melt 3～5 times, centrifugal, supernatant is placenta extracting liquid, sediment is subsequent use.Get placenta extracting liquid, press placenta extracting liquid and acetone volume ratio 2～4: 1 adding acetone, fully behind the mixing, centrifugal, collect supernatant liquor; Be again to add acetone at 2～4: 1 by collected supernatant liquor and acetone volume ratio again, fully behind the mixing, centrifugal, collect the supernatant liquor decompression distillation, through 0.45 μ m film and 0.22 μ m film degerming, at 0.5kg/cm
2Following ultrafiltration, the ultrafiltration template is selected 10000NMWL for use, gets active deer's fetus extract extract.
Used physiological saline is conventional sodium chloride solution.
The active deer's fetus extract extract that obtains further can be made active deer's fetus extract dry powder through freeze drying.
Get above-mentioned active deer's fetus extract extract, at 0.5kg/cm
2Under carry out ultrafiltration (ultrafiltration template 10000NMWL), dialyse afterwards, collect the active component of molecular weight 3000～8000dal, after utilizing the desalination of Sephadex G25 gel permeation chromatography, through 0.22 μ m film degerming, deer tire immune-regulating factor extract.
With the pure article of the further obtained by freeze drying deer of deer tire immune-regulating factor extract tire immune-regulating factor that obtain.
Take by weighing the above-mentioned sediment that obtains, every kilogram of sediment adds the acid of 2～4mol/L of 4～6 liters, is under the 500W at microwave power, and hydrolysis 10～50 minutes is filtered and obtained filtrating, regulates and filtrates to pH6.0, promptly obtains compound amino acid;
Described acid is sulfuric acid, hydrochloric acid or nitric acid.
The invention has the beneficial effects as follows: the invention solves problems such as deer tire product processing in the past is coarse, product is elementary, product category is single.The present invention has made full use of the deer tire, has reached to obtain the beneficiating ingredient in the deer tire to greatest extent, can once produce active deer's fetus extract, deer tire immune-regulating factor and three kinds of new products of compound amino acid, the product category variation.The present invention utilizes deer tire leftover bits and pieces to prepare compound amino acid, has not only saved industrial cost, has also improved utilization ratio of raw materials greatly, the waste protein resource is fully used, and has reduced its processing cost, avoids causing environmental pollution.The active deer's fetus extract that the present invention makes has extraordinary anti-inflammatory, anti-ageing, enhancing body immunity, multiple efficacies such as prophylaxis of tumours, beauty treatment, beauty treatment.Deer tire immune-regulating factor has tangible antitumor activity, and colorectal cancer, lung cancer, breast cancer etc. are had extraordinary inhibition activity.Contain 17 seed amino acids in the compound amino acid.These amino acid can be used as flavour enhancer, additive and attractant and are applied in field of feed processing.Comprehensive utilization and deep processing have been realized through the present invention, for further new product development lays the foundation to the deer tire.
The specific embodiment
Embodiment 1, the preparation of active deer's fetus extract dry powder
1) gets fresh deer placenta and remove muscle, film, shred; By weight, deer placenta: physiological saline is 1: 1.5～2.5 to get material, grinding machine for grinding, fully homogenate;-40～-20 ℃ are complete freezing, take out under room temperature to melt fully, and multigelation 3～5 times, the centrifugal 10min of 5000rpm, supernatant is a placenta extracting liquid, sediment is subsequent use; Wherein, physiological saline is 0.9% sodium chloride solution.
2) getting placenta extracting liquid, is 3: 1 by the volume ratio of placenta extracting liquid and acetone, adds acetone, and behind the mixing, the centrifugal 10min of 8000rpm gets supernatant liquor.Volume ratio by collected supernatant liquor and acetone is 3: 1 again, adds acetone, and fully behind the mixing, the centrifugal 20min of 8000rpm collects the supernatant liquor decompression distillation, again through 0.45 μ m film and 0.22 μ m film degerming, at 0.5kg/cm
2Ultrafiltration under the condition (the ultrafiltration template is selected 10000NMWL for use) gets little yellow liquid, is active deer's fetus extract extract.
3) the active deer's fetus extract dry powder of the further obtained by freeze drying of the active deer's fetus extract extract of gained.
Active deer's fetus extract productive rate is: per kilogram deer tire can obtain the pure article of the active deer's fetus extract of 10g.
Testing result:
For the BA of active deer's fetus extract, adopt the cell in vitro cultivation.Separate healthy human peripheral blood T cell, measuring active deer's fetus extract with mtt assay stimulates T cell proliferation situation.The result shows that active deer's fetus extract gets final product significant stimulation T cell proliferation when 50ng, and can significantly suppress the growth of colorectal cancer Cx-1 cell, shows that active deer's fetus extract has extraordinary tumor-inhibiting action, and tumour inhibiting rate can reach 18.6%.
Adopt flat band method, detected in the active deer's fetus extract sample whether have aerobic bacteria, anaerobic bacteria, fungi and mycoplasma, the result shows to have no infected by microbes in the active deer's fetus extract, and result of study meets the regulation of " Chinese biological goods rules ".
Adopt the rabbit method; Carried out necessary detection to whether there being pyrogen in the active deer's fetus extract; Behind every tame rabbit ear vein injection active deer's fetus extract sample of 1ml (100mg/ml); 3 rabbit body temperatures measuring do not see to have and surpass 0.6 ℃, and 3 rabbit body temperature intensification summations do not surpass 1.6 ℃ yet, and result of study meets the regulation of " Chinese biological goods rules ".The abnormal toxicity test result shows that unusual death does not appear in test mice, meets the regulation of " Chinese biological goods rules ".Adopting methods such as HPLC, is to have female hormone in the detection of active deer's fetus extract sample, and testing result proves that active deer's fetus extract sample does not contain female hormone such as pregnanedione common in the deer tire, pregnant triketone, meets the regulation of " Chinese biological goods rules ".
Embodiment 2, the preparation of the pure article of deer tire immune-regulating factor
1) gets the active deer's fetus extract extract that embodiment 1 makes, through ultrafiltration (ultrafiltration template 10000NMWL, ultrafiltration pressure: 0.5kg/cm
2) and dialysis (molecular cut off 8000dal), collecting molecular weight is the active component of 3000～8000dal, utilizes gel permeation chromatography (Sephadex G25) desalination, makes deer tire immune-regulating factor extract through degerming (0.22 μ m film).
2) will make deer tire immune-regulating factor extract, further the pure article of obtained by freeze drying deer tire immune-regulating factor.
Deer tire immune-regulating factor productive rate is: per kilogram deer tire can obtain the pure article of 3.5g deer tire immune-regulating factor.
Testing result:
Measure the antitumor activity and the lymphocyte proliferation activity of deer tire immune-regulating factor with mtt assay.Find that through vitro lymphocyte proliferation assay deer tire immune-regulating factor has significantly short lymphopoietic function, and shows certain dosage effect, on average drenching rate of rotation is 20%, and E-rosette test result shows that the bow structure recovery rate reaches 95%.Along with its IA of concentration rising strengthens gradually, immunobiologic activity reaches the top in the time of near 4ng/ml concentration, weakens gradually afterwards.Press down the knurl result of experiment and show that deer tire immune-regulating factor has the effect that suppresses growth of tumour cell, and dose dependent is arranged; Along with its IA of concentration rising strengthens gradually; Reach optimum efficiency at 4ng/ml, tumour inhibiting rate is 72.43%, weakens gradually afterwards.
Adopt flat band method to detect in the deer tire immune-regulating factor sample whether have aerobic bacteria, anaerobic bacteria, fungi and mycoplasma etc.Flat band method is cultivated 24h, no bacterial growth.Increase bacterium and cultivate 144h, no bacterial growth, result of study meets the regulation of " Chinese biological goods rules ".
Adopt the rabbit method to detect in the deer tire immune-regulating factor whether have pyrogen.Behind every tame rabbit ear vein injection 1ml deer tire immune-regulating factor sample (100mg/ml); 3 rabbit body temperatures measuring are not seen to have and are surpassed 0.6 ℃; 3 rabbit body temperature intensification summations are no more than 1.6 ℃, and result of study meets the regulation of " Chinese biological goods rules ".
Acute toxicity test in mice is got 20 of male and female mouse of the same age, is divided into 2 groups at random.Reaction group lumbar injection 1ml deer tire immune-regulating factor (50mg/ml), the control group injecting normal saline was injected non-toxic reaction after the medication continuously 10 days.40 of male and female mouse of the same age are got in the mouse chronic toxicity test, are divided into 2 groups at random.Reaction group lumbar injection 1ml deer tire immune-regulating factor (50mg/ml), the control group injecting normal saline was injected 30 days continuously, did not see toxic reaction, and the result meets the regulation of " Chinese biological goods rules ".
The preparation of embodiment 3 compound amino acids
1) with the step 1) of embodiment 1.
2) take by weighing sediment, every kilogram of sediment adds the sulfuric acid of 6 liters 3mol/L, is under the 500W at microwave power, and hydrolysis 30 minutes is filtered and obtained filtrating, regulates with NaOH and filtrates to pH6.0, promptly obtains compound amino acid.Per kilogram deer tire can produce about 0.5-0.6kg leftover bits and pieces deposition after preparing deer tire extract.Every gram leftover bits and pieces deposition can produce compound amino acid 730mg through the microwave assist acid hydrolysis, and the content of every seed amino acid sees table 1 for details.
Used sulfuric acid also can replace to hydrochloric acid or nitric acid.
Testing result:
Folin-phenol method is measured protein content in the compound amino acid, and formol titration and Kjeldahl are measured degree of hydrolysis, and compound amino acid adopts the 835-50 of Hitachi type automatic amino acid analyzer to measure amino acid content.
Deer tire sediment hydrolyzate protein content increases along with the time increase reduces afterwards earlier, is 0.661mg/ml when protein content is minimum.Formol titration is measured the compound amino acid amino nitrogen, and test is illustrated in the certain hour scope, and amino nitrogen changes more stable.Kjeldahl is measured total nitrogen content, and the compound amino acid total nitrogen content is along with the increase of time, and reducing afterwards earlier increases.The total nitrogen content minimum is 1.197mg/ml.Total nitrogen content changes more obvious than amino nitrogen in time.According to the degree of hydrolysis computing formula: total amino nitrogen/raw material total nitrogen * 100% calculates degree of hydrolysis in degree of hydrolysis (%)=hydrolyzate, and degree of hydrolysis reduces along with the increase of time increases afterwards earlier, and degree of hydrolysis is 8.27% to the maximum.Degree of hydrolysis is directly proportional with amino nitrogen content, is inversely proportional to total nitrogen content.Therefore confirm hydrolysis time between hydrolysis 10～50min, the 30min optimum.Compound amino acid is measured amino acid composition content with a day 835-50 type automatic amino acid analyzer, and the result sees table 1.
Amino acid composition content in table 1 compound amino acid
4 one kinds of method for deeply processing deer placenta of embodiment
1) gets fresh deer placenta and remove muscle, film, shred; By weight, deer placenta: physiological saline is to get material at 1: 2, in grinding machine for grinding, homogenate;-40～-20 ℃ of complete freezing, room temperatures are melted fully, multigelation 3～5 times, and 5000rpm is centrifugal, and supernatant is a placenta extracting liquid, and sediment is subsequent use; Wherein, physiological saline is 0.9% sodium chloride solution.
2) get placenta extracting liquid, add acetone (volume ratio of placenta extracting liquid and acetone is 3: 1), behind the mixing, the centrifugal 10min of 8000rpm gets supernatant liquor.Add the acetone volume ratio of the supernatant liquor of getting and acetone (be 3: 1) again, fully behind the mixing, the centrifugal 10min of 8000rpm collects supernatant liquor, obtains light yellow transparent liquid through decompression distillation.Pass through film (0.45 μ m film utilizes 0.22 μ m film more earlier) degerming, ultrafiltration (ultrafiltration template 10000NMWL, ultrafiltration pressure: 0.5kg/cm again
2) obtain little yellow liquid, be active deer's fetus extract extract.The active deer's fetus extract dry powder of further obtained by freeze drying.Active deer's fetus extract productive rate is: per kilogram deer tire can obtain the pure article of the active deer's fetus extract of 10g.
3) get active deer's fetus extract extract, through ultrafiltration (ultrafiltration template 10000NMWL, ultrafiltration pressure: 0.5kg/cm
2) and dialysis (molecular cut off 8000dal), collecting molecular weight is the active component of 3000～8000dal, utilizes gel permeation chromatography (Sephadex G25) desalination, makes deer tire immune-regulating factor extract through degerming (0.22 μ m film).The further pure article of obtained by freeze drying deer tire immune-regulating factor.Deer tire immune-regulating factor productive rate is: per kilogram deer tire can obtain the pure article of 3.5g deer tire immune-regulating factor.
4) take by weighing the sediment that makes in the step 1), every kilogram of sediment adds the sulfuric acid of 5 liters 3mol/L, is under the 500W at microwave power, and hydrolysis 30 minutes is filtered and obtained filtrating, regulates with NaOH and filtrates to pH6.0, promptly obtains compound amino acid.Per kilogram deer tire can produce about 0.5-0.6kg leftover bits and pieces deposition after preparing deer tire extract.Every gram leftover bits and pieces deposition can produce compound amino acid 730mg through the microwave assist acid hydrolysis, and the content of every seed amino acid sees table 1 for details.
Also replaceable one-tenth hydrochloric acid of used sulfuric acid or nitric acid.
Claims (3)
1. method for deeply processing deer placenta is characterized in that step is following:
1) gets fresh deer placenta and remove muscle, film, shred; By weight, deer placenta: physiological saline is 1: 1.5～2.5 to get material, grinding machine for grinding, fully homogenate;-40～-20 ℃ are freezing, melt under the room temperature, freezing repeatedly with melt 3～5 times, centrifugal, supernatant is a placenta extracting liquid, sediment is subsequent use; Getting placenta extracting liquid, is 2～4: 1 by the volume ratio of placenta extracting liquid and acetone, adds acetone, centrifugal behind the mixing, gets supernatant liquor; Volume ratio by collected supernatant liquor and acetone is 2～4: 1 again, adds acetone, and is fully behind the mixing, centrifugal, collects the supernatant liquor decompression distillation, again through 0.45 μ m film and 0.22 μ m film degerming, at 0.5kg/cm
2Ultrafiltration under the condition, the ultrafiltration template is selected 10000NMWL for use, gets active deer's fetus extract extract;
2) get active deer's fetus extract extract, through ultrafiltration template 10000NMWL, ultrafiltration pressure 0.5kg/cm
2Following ultrafiltration, molecular weight 3000～8000dal active component is collected in dialysis, utilizes after the desalination of Sephadex G25 gel permeation chromatography with 0.22 μ m film degerming, must deer tire immune-regulating factor extract.
2. according to the described method for deeply processing deer placenta of claim 1, it is characterized in that comprising the steps: the pure article of deer tire immune-regulating factor extract obtained by freeze drying deer tire immune-regulating factor.
3. according to the described method for deeply processing deer placenta of claim 1; It is characterized in that comprising the steps: to take by weighing sediment, every kilogram of sediment adds the acid of 2～4mol/L of 4～6 liters, is under the 500W at microwave power; Hydrolysis 10～50 minutes; Filtration obtains filtrating, regulates and filtrates to pH6.0, promptly obtains compound amino acid;
Described acid is sulfuric acid, hydrochloric acid or nitric acid.
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| CN102925523B (en) * | 2012-11-15 | 2014-09-10 | 辽宁大学 | Enzyme hydrolysis method for preparing fetus cervi active peptide |
| JP7011135B2 (en) * | 2016-02-19 | 2022-02-10 | 佳秀工業株式会社 | A method for producing a vascular endothelial growth factor production promoter and a hair growth agent, and a vascular endothelial growth factor production promoter and a hair growth agent. |
| CN112852718B (en) * | 2021-03-15 | 2023-04-21 | 广州赛莱拉生物基因工程有限公司 | Preparation method of bovine placenta exosome |
| AU2022419516A1 (en) * | 2021-12-24 | 2024-02-15 | Ehj Ip Limited | Placental composition |
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| CN1235026A (en) * | 1999-05-28 | 1999-11-17 | 孙月英 | Preparing method for injection of placenta peptide and products thereof |
| CN1298709A (en) * | 2000-11-03 | 2001-06-13 | 内蒙古草原兴发股份有限公司 | Process for preparing polypeptide-nucleic acid nutritive liquid of sheep placenta |
| CN1367178A (en) * | 2001-01-22 | 2002-09-04 | 张建军 | Technological process for extracting and purifying deer's fetus extract |
| CN1640328A (en) * | 2003-03-25 | 2005-07-20 | 于靖波 | Bioactive drink and its preparing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1235026A (en) * | 1999-05-28 | 1999-11-17 | 孙月英 | Preparing method for injection of placenta peptide and products thereof |
| CN1298709A (en) * | 2000-11-03 | 2001-06-13 | 内蒙古草原兴发股份有限公司 | Process for preparing polypeptide-nucleic acid nutritive liquid of sheep placenta |
| CN1367178A (en) * | 2001-01-22 | 2002-09-04 | 张建军 | Technological process for extracting and purifying deer's fetus extract |
| CN1640328A (en) * | 2003-03-25 | 2005-07-20 | 于靖波 | Bioactive drink and its preparing method |
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