CN103882083A - Method for preparing antioxidant collagen peptide - Google Patents
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Abstract
The invention belongs to the technical field of food biology and in particular relates to a method for preparing an antioxidant collagen peptide. The main technical method comprises the following steps: mixing collagen and artificial gastric juice (according to United States Pharmacopeia preparation) under the condition of 37 DEG C, simulating gastric digestion under oscillating conditions, regulating the pH to 6.8 by using NaOH, adding pancreatin and continuously simulating intestinal digestion, ending the reaction after a boiling water bath, centrifuging and taking the supernatant; performing ultrafiltration separation on the digestion fluid by utilizing a membrane with the molecular cut off of 1kDa, collecting the filtrate until the light absorption value at 220nm is close to 0, concentrating, drying, dissolving, loading to a ToyopearlSP-650M column, and performing linear elution by using a NaCl-containing HAc-NaAc buffer solution; collecting and eluting active ingredients, concentrating and loading to a SephadexG10 column, eluting by using distilled water, collecting the eluted active ingredients, concentrating and drying to obtain odorless, tasteless and white powder, namely the target antioxidant collagen peptide.
Description
Technical field
The invention belongs to technical field of food biotechnology, be specifically related to a kind of preparation method of antioxidant collagen peptide.
Technical background
China is a fishery big country, occupies first place in the world in continuous 23 years positions of fishery products ultimate production.But processing of aquatic products state of the art is lower, a large amount of processing fents is used to feed and fertilizer, even directly buries and abandons as rubbish, has caused the serious wasting of resources and environmental pollution.Therefore, develop these tankage, for extend industrial chain, improve added value, reduce environmental pollution, increase economic benefit significant.
In food-processing and storage, oxygenizement can be brought a series of sense organ and nutrition problem, even produces poisonous and harmful substances.The free radical that organism metabolism produces can cause the oxidative damage of the biomacromolecules such as protein, lipid, nucleic acid, with disease and old and feeble closely related.At present, using antioxidant is to suppress Food Oxidation reaction, the most frequently used method of removing interior free yl, but synthetic antioxidant, as BHA, BHT, TBHQ etc. may exist potential potential safety hazard and health risk, become the study hotspot of food and association area so find better natural antioxidants.
In recent years, anti-oxidation peptide is little with its molecule, the superperformance of high reactivity, low-viscosity, soluble, easy absorption, becomes gradually a kind of novel antioxidant and is praised highly.But at present, the preparation of anti-oxidation peptide adopts the method for industrial enzymolysis mostly, and lack further effectively separation and purification, cause that prepared antioxidant peptide active is low, color and luster is dark, bitter road taste, fishy smell weight, thus seriously limit its practical application.
Summary of the invention
The present invention is taking the collagen (collagen protein, gelatin) that exists in a large number in disposal from fishery product processing as raw material, the method digesting by in-vitro simulated human gastrointestinal tract, utilize membrane sepn and column chromatography technology, prepare the collagen peptide that a kind of organoleptic quality is good, anti-oxidant activity is high, can be widely used in the fields such as food, medicine, makeup.
The technical solution used in the present invention:
(1) the external film of collagen is intended pipe intestinal digesting
According to American Pharmacopeia (USP30-NF25) preparation simulated gastric fluid.Under 37 ° of C conditions, collagen and gastric juice are mixed, in the peptic digestion of oscillating condition Imitating, then regulate pH with NaOH, add pancreatin to mixed solution, continue the little intestinal digestion of simulation; After digestion finishes, carry out boiling water bath, centrifuging and taking supernatant after termination reaction.
(2) separation and purification of antioxidant collagen peptide
1. membrane sepn: utilizing molecular weight cut-off is that the membrane ultrafiltration of 1 kDa separates above-mentioned Digestive system, collects filtrate until it is close to 0 at 220 nm place light absorption values, then rotary evaporation concentrate, lyophilize.
2. column chromatography: above-mentioned above-mentioned membrane sepn obtained component membrane sepn obtained component is dissolved in damping fluid, is then splined on the ion exchange column of HAc-NaAc damping fluid balance, use the same buffer that contains NaCl to carry out linear elution; With 1,1-phenylbenzene-2-trinitrophenyl-hydrazine (DPPH) free radical scavenging activity is that index is collected active elution fraction, after concentrated, be splined on again size-exclusion post, distilled water wash-out, collect active elution fraction concentrated, dry after gained odorless, tasteless, white powder, be target antioxidant collagen peptide.
Wherein described in step (1), collagen and gastric juice ratio are 1g:10-25mL; Described oscillation frequency is 120-180 rpm; The described simulation peptic digestion time is 2-4 h; Described pH is 6.8; The ratio of described pancreatin and mixed solution is 1g:100mL; Described simulation small intestine digestion time is 4-6h; The described boiling water bath time is 10-15 min; Described centrifugal be the above centrifugal 10-20 min of 10000 g.
Wherein described in step (2), HAc-NaAc buffer concentration is 0.05 mol/L, and pH is 4.5; The concentration that is dissolved in the membrane sepn obtained component in damping fluid is 50-200 mg/mL; In the described damping fluid that carries out linear elution, the concentration of NaCl is 0-1.0 mol/L.Described ion exchange column is Toyopearl SP-650M ion exchange column; Described size-exclusion post is Sephadex G10 size-exclusion post.
Collagen described in technique scheme comprises all taking processing of aquatic products waste (skin, squama, bone, fin) as raw material, with salt method, acid system, enzyme process, prepared collagen protein and the gelatin of hot-water process.
This technique is applicable to batch production, only need amplify in proportion.
beneficial effect of the present invention:
1. the annual a large amount of disposal from fishery product processing of China is used to feed or fertilizer, even abandons as garbage bury, has caused the serious wasting of resources and environmental pollution.The present invention prepares antioxidant collagen peptide taking processing of aquatic products waste (skin, squama, bone, fin) as raw material, and both extensible industrial chains improve added value of product, can reduce again environmental pollution, increase economic and social benefit.
2. the preparation of anti-oxidation peptide at present adopts the method for industrial enzymolysis mostly, and after prepared anti-oxidation peptide is edible, through the GI digestion again of human body, anti-oxidant activity significantly reduces, even completely dissolve.The method that the present invention digests by in-vitro simulated human gi-tract is prepared anti-oxidation peptide, after eating, digests and assimilates through human gastrointestinal tract, still can effectively keep its higher anti-oxidant activity.
3. current anti-oxidation peptide lacks effective separation purifying technique mostly, thereby causes the finished product color and luster dark, bitter road taste, and fishy smell weight, thus seriously limit its practical application.The present invention utilizes the method separation and purification of membrane sepn and column chromatography to go out a kind of odorless, tasteless, white, pulverulent anti-oxidation peptide, has significantly improved the organoleptic quality of product, can be widely used in the fields such as food, medicine, makeup.
Brief description of the drawings
Fig. 1: chromatography and the anti-oxidant activity collection of illustrative plates of collagen simulation digestion product ultrafiltration component.A, Toyopearl SP-650M column chromatography; B, Sephadex G10 column chromatography; Wherein:
, NaCl concentration (0 – 1.0 mol/L);
, voltage;
, the light absorption value DPPH of unit free radical scavenging activity (220 nm).
Embodiment
Utilize salt method, acid system or enzyme process to prepare aquatic collagen protein, or hot-water process prepare aquatic products gelatin.Simulated gastric fluid is prepared according to American Pharmacopeia (USP30-NF25).
In conjunction with following instance, the present invention is further elaborated:
embodiment 1:
Take 50 g collagen proteins in triangular flask, add 1000 mL simulated gastric fluids, after sealing, in the isothermal vibration water bath of 37 ° of C, 150 rpm, simulate peptic digestion 3 h, then regulate pH to 6.8 with 1.0 mol/L NaOH, add 10 g pancreatin to continue simulation small intestine and digest 5 h.After digestion finishes, boiling water bath 12 min termination reactions, 10000 g are centrifugal, and 15 min get supernatant.
Utilize Millipore Pellicon ultrafiltration system, be the regenerated cellulose ultra-filtration membrane surface of 1 kDa by above-mentioned digestion liquid pump to molecular weight cut-off, collect filtrate until it is close to 0 at 220 nm place light absorption values, then rotary evaporation is concentrated into 100 mL left and right, vacuum lyophilization, approximately 55 g.
Above-mentioned membrane sepn obtained component is dissolved in 0.05 mol/L HAc-NaAc damping fluid (pH=4.5) to 50 mg/mL, then get 8 mL and be splined on the Toyopearl SP-650M ion exchange column (2.5 × 30 cm) with this damping fluid balance, carry out linear elution (0-1.0 mol/L) by the same buffer that contains NaCl, flow velocity is 1.0 mL/min.Collect active elution fraction (70-140 min) taking the light absorption value DPPH of unit free radical scavenging activity (220 nm) as index, rotary evaporation is concentrated into after approximately 100 mg/mL, get again the upper Sephadex G10 molecular exclusion chromatography post (1.5 × 75 cm) of 2 ml, under the flow velocity of 0.5 mL/min, use distilled water wash-out, collect that active elution fraction (130-170 min) rotary evaporation is concentrated, gained odorless after vacuum lyophilization, tasteless, white powder, be target antioxidant collagen peptide, be about 1.6 g, IC
50value is 153.8 μ g/mL.
embodiment 2:
Take 50 g gelatin in digestion bottle, add 500 mL simulated gastric fluids, after sealing, in the isothermal vibration water bath of 37 ° of C, 180 rpm, simulate peptic digestion 2 h, then regulate pH to 6.8 with 1.0 mol/L NaOH, add 5 g pancreatin to continue simulation small intestine and digest 4 h.After digestion finishes, boiling water bath 10 min termination reactions, 10000 g are centrifugal, and 10 min get supernatant.
Utilize Millipore Pellicon ultrafiltration system, be the regenerated cellulose ultra-filtration membrane surface of 1 kDa by above-mentioned digestion liquid pump to molecular weight cut-off, collect filtrate until it is close to 0 at 220 nm place light absorption values, then rotary evaporation is concentrated into 100 mL left and right, vacuum lyophilization, approximately 52 g.
Above-mentioned membrane sepn obtained component is dissolved in 0.05 mol/L HAc-NaAc damping fluid (pH=4.5) to 100 mg/mL, then get 5 mL and be splined on the Toyopearl SP-650M ion exchange column (2.5 × 30 cm) with this damping fluid balance, carry out linear elution (0-1.0 mol/L) by the same buffer that contains NaCl, flow velocity is 1.0 mL/min.Collect active elution fraction (70-140 min) taking the light absorption value DPPH of unit free radical scavenging activity (220 nm) as index, rotary evaporation is concentrated into after approximately 150 mg/mL, get again the upper Sephadex G10 molecular exclusion chromatography post (1.5 × 75 cm) of 1.5 ml, under the flow velocity of 0.5 mL/min, use distilled water wash-out, collect that active elution fraction (130-170 min) rotary evaporation is concentrated, gained odorless after vacuum lyophilization, tasteless, white powder, be target antioxidant collagen peptide, be about 1.5 g, IC
50value is 145.2 μ g/mL.
embodiment 3:
Take 50 g gelatin in triangular flask, add 1250 mL simulated gastric fluids, after sealing, in the isothermal vibration water bath of 37 ° of C, 120 rpm, simulate peptic digestion 4 h, then regulate pH to 6.8 with 1.0 mol/L NaOH, add 12.5 g pancreatin to continue simulation small intestine and digest 6 h.After digestion finishes, boiling water bath 15 min termination reactions, 10000 g are centrifugal, and 20 min get supernatant.
Utilize Millipore Pellicon ultrafiltration system, be the regenerated cellulose ultra-filtration membrane surface of 1 kDa by above-mentioned digestion liquid pump to molecular weight cut-off, collect filtrate until it is close to 0 at 220 nm place light absorption values, then rotary evaporation is concentrated into 100 mL left and right, vacuum lyophilization, approximately 56 g.
Above-mentioned membrane sepn obtained component is dissolved in 0.05 mol/L HAc-NaAc damping fluid (pH=4.5) to 200 mg/mL, then get 3 mL and be splined on the Toyopearl SP-650M ion exchange column (2.5 × 30 cm) with this damping fluid balance, carry out linear elution (0-1.0 mol/L) by the same buffer that contains NaCl, flow velocity is 1.0 mL/min.Collect active elution fraction (70-140 min) taking the light absorption value DPPH of unit free radical scavenging activity (220 nm) as index, rotary evaporation is concentrated into after approximately 200 mg/mL, get again the upper Sephadex G10 molecular exclusion chromatography post (1.5 × 75 cm) of 1 ml, under the flow velocity of 0.5 mL/min, use distilled water wash-out, collect that active elution fraction (130-170 min) rotary evaporation is concentrated, gained odorless after vacuum lyophilization, tasteless, white powder, be target antioxidant collagen peptide, be about 1.6 g, IC
50value is 156.5 μ g/mL.
The prepared antioxidant collagen peptide of example 1,2,3 is through Agilent ZORBAX Eclipse XDB C18 post (4.6 × 150 mm, 5 μ are m) further after separation and purification, utilize UPLC-ESI-MS/MS technology to identify a kind of high reactivity anti-oxidation peptide Gly-Pro-Met(303.38 Da), this collagen peptide IC
50value is 25.64 μ g/mL.
Can find out from Fig. 1 a, collagen simulation digestion product ultrafiltration component obtains multiple components after Toyopearl SP-650M column chromatography, and wherein the elution fraction of 75-125 min has shown the highest anti-oxidant activity; By this component, again through Sephadex G10 column chromatography, (Fig. 1 b), wherein only has component III to have higher anti-oxidant activity to five components of I-V of getting back, and this component is the target product of this patented technology.
Claims (4)
1. a preparation method for antioxidant collagen peptide, is characterized in that, carries out according to following steps:
(1) the external film of collagen is intended pipe intestinal digesting:
According to American Pharmacopeia USP30-NF25 preparation simulated gastric fluid; Under 37 ° of C conditions, collagen and gastric juice are mixed, in the peptic digestion of oscillating condition Imitating, then regulate pH with NaOH, add pancreatin to mixed solution, continue the little intestinal digestion of simulation; After digestion finishes, carry out boiling water bath, centrifuging and taking supernatant after termination reaction;
(2) separation and purification of antioxidant collagen peptide:
1. membrane sepn: utilizing molecular weight cut-off is that the membrane ultrafiltration of 1 kDa separates above-mentioned Digestive system, collects filtrate until it is close to 0 at 220 nm place light absorption values, then rotary evaporation concentrate, lyophilize;
2. column chromatography: above-mentioned membrane sepn obtained component is splined on to the ion exchange column of HAc-NaAc damping fluid balance, uses the same buffer that contains NaCl to carry out linear elution; Collect active elution fraction taking DPPH free radical scavenging activity as index, be splined on again size-exclusion post after concentrated, distilled water wash-out, collect active elution fraction concentrated, dry after gained odorless, tasteless, white powder, be target antioxidant collagen peptide.
2. the preparation method of a kind of antioxidant collagen peptide according to claim 1, is characterized in that, described in step (1), collagen and gastric juice ratio are 1g:10-25mL; Described oscillation frequency is 120-180 rpm; The described simulation peptic digestion time is 2-4 h; Described pH is 6.8; The ratio of described pancreatin and mixed solution is 1g:100mL; Described simulation small intestine digestion time is 4-6 h; The described boiling water bath time is 10-15 min; Described centrifugal be the above centrifugal 10-20 min of 10000 g.
3. the preparation method of a kind of antioxidant collagen peptide according to claim 1, is characterized in that, wherein described in step (2), HAc-NaAc buffer concentration is 0.05 mol/L, and pH is 4.5; The described concentration that has NaCl in the damping fluid that carries out linear elution is 0-1.0 mol/L; Described ion exchange column is Toyopearl SP-650M ion exchange column; Described size-exclusion post is Sephadex G10 size-exclusion post.
4. the preparation method of a kind of antioxidant collagen peptide according to claim 1, it is characterized in that, collagen described in technique scheme comprises all taking processing of aquatic products waste: skin, squama, bone, fin are as raw material, with salt method, acid system, enzyme process, prepared collagen protein and the gelatin of hot-water process.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263786A (en) * | 2014-09-09 | 2015-01-07 | 江苏大学 | Method for preparing rapeseed dreg protein antioxidative peptide solution by gastrointestinal simulated digestion |
| CN104263789A (en) * | 2014-09-29 | 2015-01-07 | 江苏大学 | Method for preparing navodon septentrionalis skin anti-oxidative peptide liquid through in vitro simulation of gastrointestinal digestion |
| CN106434805A (en) * | 2016-09-30 | 2017-02-22 | 青岛琛蓝海洋生物工程有限公司 | Preparation method for medical ultrapure high-activity fish skin collagen |
| CN107338278A (en) * | 2017-08-10 | 2017-11-10 | 江苏大学 | The method that ultrasonic in combination simulation digestion prepares collagen gel antioxidation polypeptide liquid |
| CN107541538A (en) * | 2017-08-10 | 2018-01-05 | 江苏大学 | The method that in-vitro simulated gastro-intestinal digestion prepares collagen gel antioxidation polypeptide liquid |
| CN107604028A (en) * | 2017-08-10 | 2018-01-19 | 江苏大学 | The method that heating combined simulation digestion prepares collagen antioxidant polypeptide liquid |
| CN110713534A (en) * | 2019-11-29 | 2020-01-21 | 福建农林大学 | Collagen peptide with photoaging improvement effect and preparation method thereof |
| CN111635920A (en) * | 2020-05-21 | 2020-09-08 | 内蒙古元本生物医药科技有限公司 | Method for preparing donkey bone collagen peptide powder by two-stage bionic enzymatic hydrolysis technology |
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| CN1749275A (en) * | 2004-09-15 | 2006-03-22 | 天津科技大学 | Method for processing hydrolytic fish skin collagen |
| CN1858230A (en) * | 2006-03-03 | 2006-11-08 | 中国海洋大学 | Process for preparing oligomeric peptide capable of inhibiting angiotonase activity |
| CN103184261A (en) * | 2011-12-30 | 2013-07-03 | 刘若楼 | Co-production processing process of pharmaceutical gelatin and high-grade collagen |
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Patent Citations (3)
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| CN1749275A (en) * | 2004-09-15 | 2006-03-22 | 天津科技大学 | Method for processing hydrolytic fish skin collagen |
| CN1858230A (en) * | 2006-03-03 | 2006-11-08 | 中国海洋大学 | Process for preparing oligomeric peptide capable of inhibiting angiotonase activity |
| CN103184261A (en) * | 2011-12-30 | 2013-07-03 | 刘若楼 | Co-production processing process of pharmaceutical gelatin and high-grade collagen |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263786A (en) * | 2014-09-09 | 2015-01-07 | 江苏大学 | Method for preparing rapeseed dreg protein antioxidative peptide solution by gastrointestinal simulated digestion |
| CN104263786B (en) * | 2014-09-09 | 2017-06-06 | 江苏大学 | The method that stomach and intestine simulation digestion prepares rapeseed dregs protein antioxidant peptide liquid |
| CN104263789A (en) * | 2014-09-29 | 2015-01-07 | 江苏大学 | Method for preparing navodon septentrionalis skin anti-oxidative peptide liquid through in vitro simulation of gastrointestinal digestion |
| CN104263789B (en) * | 2014-09-29 | 2017-06-06 | 江苏大学 | The method that in-vitro simulated gastro-intestinal digestion prepares green fin black scraper Puffer fish-skin anti-oxidation peptide liquid |
| CN106434805A (en) * | 2016-09-30 | 2017-02-22 | 青岛琛蓝海洋生物工程有限公司 | Preparation method for medical ultrapure high-activity fish skin collagen |
| CN107338278A (en) * | 2017-08-10 | 2017-11-10 | 江苏大学 | The method that ultrasonic in combination simulation digestion prepares collagen gel antioxidation polypeptide liquid |
| CN107541538A (en) * | 2017-08-10 | 2018-01-05 | 江苏大学 | The method that in-vitro simulated gastro-intestinal digestion prepares collagen gel antioxidation polypeptide liquid |
| CN107604028A (en) * | 2017-08-10 | 2018-01-19 | 江苏大学 | The method that heating combined simulation digestion prepares collagen antioxidant polypeptide liquid |
| CN110713534A (en) * | 2019-11-29 | 2020-01-21 | 福建农林大学 | Collagen peptide with photoaging improvement effect and preparation method thereof |
| CN111635920A (en) * | 2020-05-21 | 2020-09-08 | 内蒙古元本生物医药科技有限公司 | Method for preparing donkey bone collagen peptide powder by two-stage bionic enzymatic hydrolysis technology |
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Effective date of registration: 20190104 Address after: Room 1001, 70 Fenghuang Street, Fangzi District, Weifang City, Shandong Province Patentee after: Shandong Aojing Biotechnology Co., Ltd. Address before: No. 301, Xuefu Road, Jingkou District, Zhenjiang, Jiangsu Province Patentee before: Jiangsu University |
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