CN103882083A

CN103882083A - Method for preparing antioxidant collagen peptide

Info

Publication number: CN103882083A
Application number: CN201410064576.4A
Authority: CN
Inventors: 王林; 梁秋芳; 徐军民; 王振斌; 马海乐
Original assignee: Jiangsu University
Current assignee: Shandong Aojing Biotechnology Co Ltd
Priority date: 2014-02-26
Filing date: 2014-02-26
Publication date: 2014-06-25
Anticipated expiration: 2034-02-26
Also published as: CN103882083B

Abstract

The invention belongs to the field of food biotechnology, and in particular relates to a preparation method of antioxidant collagen peptide. The main technical methods are as follows: at 37°C, mix collagen and artificial gastric juice (prepared according to the United States Pharmacopoeia), simulate gastric digestion under shaking conditions, then adjust the pH to 6.8 with NaOH, add trypsin to continue simulating small intestine digestion , the reaction was terminated after a boiling water bath, and the supernatant was collected by centrifugation. The digested solution was separated by membrane ultrafiltration with a molecular weight cut-off of 1 kDa, and the filtrate was collected until the absorbance value at 220 nm was close to 0, concentrated, dried, redissolved, and loaded on a Toyopearl SP-650M column. NaAc buffer eluted linearly. The eluted active components were collected, concentrated and then loaded on the SephadexG10 column, eluted with distilled water, and the collected active eluted components were concentrated and dried to obtain an odorless, tasteless, white powder, which was the target antioxidant collagen peptide.

Description

A kind of preparation method of antioxidant collagen peptide

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:

Figure 2014100645764100002DEST_PATH_IMAGE001

, 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

1. a preparation method of antioxidant collagen peptide, is characterized in that, carries out according to the following steps:

(1) The extracorporeal membrane of collagen simulates gastrointestinal digestion:

Prepare artificial gastric juice according to United States Pharmacopoeia USP30-NF25; mix collagen and gastric juice at 37°C, simulate gastric digestion under shaking conditions, then adjust pH with NaOH, add trypsin to the mixture, and continue to simulate small intestine digestion ; After the digestion is completed, carry out a boiling water bath, and centrifuge to obtain the supernatant after terminating the reaction;

(2) Separation and purification of antioxidant collagen peptides:

① Membrane separation: Utilize membrane ultrafiltration with a molecular weight cut-off of 1 kDa to separate the above digested liquid, collect the filtrate until its absorbance value at 220 nm is close to 0, then concentrate by rotary evaporation and freeze-dry;

② Column chromatography: The components obtained from the above membrane separation are loaded on an ion-exchange column equilibrated with HAc-NaAc buffer, and linearly eluted with the same buffer containing NaCl; the active elution is collected based on the DPPH free radical scavenging rate Components are concentrated and then loaded on a molecular exclusion column, eluted with distilled water, and the active eluted components are collected, concentrated and dried to obtain an odorless, tasteless, white powder, which is the target antioxidant collagen peptide.

2. The preparation method of an antioxidant collagen peptide according to claim 1, wherein the ratio of collagen to gastric juice in step (1) is 1g:10-25mL; the oscillation frequency is 120-180 rpm; the simulated gastric digestion time is 2-4 h; the pH is 6.8; the ratio of the trypsin and the mixed solution is 1g:100mL; the simulated small intestine digestion time is 4-6 h; the boiling water bath The time is 10-15 min; the centrifugation is above 10000 g for 10-20 min.

3. the preparation method of a kind of antioxidant collagen peptide according to claim 1, is characterized in that, wherein the concentration of HAc-NaAc buffer solution described in step (2) is 0.05 mol/L, and pH is 4.5; The concentration of NaCl in the buffer for linear elution is 0-1.0 mol/L; the ion exchange column is Toyopearl SP-650M ion exchange column; the molecular exclusion column is Sephadex G10 molecular exclusion column.

4. The preparation method of a kind of antioxidant collagen peptide according to claim 1, it is characterized in that, the collagen described in the above-mentioned technical scheme includes all with aquatic product processing waste: skin, scale, bone, fin as raw material, Collagen and gelatin prepared by salt method, acid method, enzyme method, hot water method.