CN102846553B - Preparation method for chlorella polypeptide-chitosan nanoparticle - Google Patents
Preparation method for chlorella polypeptide-chitosan nanoparticle Download PDFInfo
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Abstract
The invention provides a preparation method for a chlorella polypeptide-chitosan nanoparticle. The method is characterized by comprising the following steps: extracting chlorella protein by using a low temperature ultrahigh pressure continuous flow cell crusher; hydrolyzing the chlorella protein with papain; filtering the hydrolyzed chlorella protein with an ultrafiltration centrifuge tube; then carrying out separation and purification by using ion exchange chromatography DEAE-52 and gel chromatography dextrangel G-25 columns; and finally utilizing ionic cross-linking with sodium tripolyphosphate to prepare the chlorella polypeptide-chitosan nanoparticle. The chlorella polypeptide-chitosan nanoparticle prepared in the invention has antitumor activity, e.g., the chlorella polypeptide-chitosan nanoparticle has inhibitory effects on in vitro growth of human hepatoma carcinoma cells HepG2 and the inhibition rate of the chlorella polypeptide-chitosan nanoparticle reaches 32% when concentration is 400 mu g/mL. Therefore, the chlorella polypeptide-chitosan nanoparticle prepared in the invention is beneficial for development and utilization of antitumor health food and medicinal products.
Description
Technical field
The present invention relates to chlorella deep processing and biological technical field, be specifically related to the preparation method of chlorella polypeptide-chitosan nano.
Background technology
Chlorella (Chlorella pyenoidosa) is the general natural disposition monoplast green alga of a class, belong to Chlorophyta, Chlorella, fast growth, be easy to cultivate, using value is high. and chlorella contains abundant bioactive substance and medicinal ingredient, as a kind of novel healthy food and medicine, is containing great potential.Chlorella has anti-tumor activity, increases immunity, removing toxic substances protects the liver, hypotensive effect etc., its crude protein content high (50% left and right), and quality better, has become very active, the standby valued aspect of chlorella application.
Biologically active peptide refers to that those have the peptide class of special physiological activity or functional characteristic.Most protein is all the precursor substance with the biologically active peptide of certain function, in its peptide chain structure, exist and there is certain bioactive aminoacid sequence fragment (functional areas), under normal condition, its functional areas peptide section is hidden in peptide chain, once but discharge from protein peptide chain separately, under suitable environment, just can demonstrate unique biological activity, this functional peptide fragment is exactly biologically active peptide.Modern biological metabolism research is found: the protein of mankind's picked-up, after gastral plurality of enzymes hydrolysis, is more that the form with low peptide directly absorbs, and has higher nutritive value and biological value.Enzyme hydrolysis method obtains the Main Means that biologically active peptide has become suitability for industrialized production biologically active peptide.
Malignant tumor is one of disease of serious threat human health and life, though current existing antitumor drug has certain curative effect to most of tumors, but still exist that therapeutic efficiency is low, poor selectivity, toxicity greatly, easily produce the problems such as oncocyte drug resistance.Therefore, from different approaches, find the task of top priority that efficient, low toxicity, special strong antitumor drug are still Drug therapy tumor.In recent years, people more and more pay attention to the research and development of biologically active peptide, various biologically active peptides are constantly found and prepare, polypeptide drug because its molecular weight is little, non-immunogenicity, simple in structure, side effect is little, the research of its anti-tumor activity is causing the extensive concern of Chinese scholars.
Albumen and polypeptide drug thereof in drug delivery process, be very easily subject to complex physiologic environment particularly a large amount of enzyme materials effect and destroyed; its weak penetration capacity and natural fragility have determined the activity of easier forfeiture itself in addition; in order to improve the bioavailability of polypeptide; opposing gastric acid and pepsic Degradation; must carry out suitable protection to it, make its stable playing a role in small intestinal.Chitosan is nontoxic, has good biocompatibility, degradability, film property and plasticity. and chitosan also has certain antibacterial, antitumor, antiviral activity.Therefore, the application of chitosan is subject to people's attention day by day, at food and biomedicine field, has broad prospects.Especially for biologically active peptide, be chitosan imbeddedly expected to improve its stability and bioavailability, reach better trophic function and physiological function.
Summary of the invention
For expanding chlorella in the application of food and biomedical sector, the object of this invention is to provide the preparation method of chlorella polypeptide-chitosan nano.
For realizing the object of the invention, adopt following technical scheme:
1, the preparation method of chlorella polypeptide-chitosan nano, specifically comprises the steps:
(1) will after chlorella powder and pure water mixing, stir to obtain mixed solution, in described mixed solution, extract chlorella protein, the crude extract obtaining extracts as solution to be extracted next time again, by the solution centrifugal obtaining, remove precipitation and obtain albumen supernatant, vacuum lyophilization again, obtains chlorella protein powder.
(2) take the chlorella protein powder that step (1) obtains is basis, configuration concentration is 1 ~ 4%(w/v, g/mL) chlorella protein solution, adding hydrolytic enzyme is hydrolyzed, after hydrolysis, enzyme denaturing, be cooled to after room temperature hydrolyzed solution is centrifugal, get supernatant, then, the ultra-filtration centrifuge tube that is 10 KD through molecular cut off filters, and filtrate is filtered through the ultra-filtration centrifuge tube of 3 KD, 5 KD again, and can obtain molecular size range scope is the chlorella polypeptide solution of < 3 KD, 3-5 KD, 5-10 KD, >10 KD.
(3) take the 3-5 KD chlorella polypeptide solution that step (2) obtains is basis, use ion exchange chromatography DEAE-52 separation and purification, according to peak sequence, collect altogether four peak A1 ~ A4, the absorption peak A of collecting 2 is carried out to the separation and purification of gel chromatography sephadex G-25 again, according to peak sequence, collect altogether two peak A2-1 and A2-2.
(4) take the chlorella polypeptide solution A2-1 of the purification that step (3) obtains is basis, then adopts sodium tripolyphosphate (TPP) ionic cross-linking can obtain chlorella polypeptide-chitosan nano.
The preparation method of above-mentioned chlorella polypeptide-chitosan nano, the mass ratio of the described chlorella powder of step (1) and pure water is 1:10～1:40, described mixing time is 30～120 minutes.
The preparation method of above-mentioned chlorella polypeptide-chitosan nano, the described extraction chlorella of step (1) protein for to extract under the condition of 4 ℃～10 ℃ of temperature, pressure 50MPa～250MPa.
The preparation method of above-mentioned chlorella polypeptide-chitosan nano, the number of times extracting again described in step (1) is 1 time～8 times, each time of extracting is 10min～60min; Described centrifugal be 4 ~ 8 ℃ of temperature, rotating speed is centrifugal 10 ~ 60 min under 5000 ~ 10000 r/min.
The preparation method of above-mentioned chlorella polypeptide-chitosan nano, the described hydrolytic enzyme of step (2) is papain, hydrolysising condition is 30 ~ 80 ℃ of temperature, pH=5 ~ 10, the ratio of enzyme and chlorella protein aqueous solution is 1% ~ 5%(w/v, g/mL), hydrolysis time is 3 ~ 10h.
The preparation method of above-mentioned chlorella polypeptide-chitosan nano, the described enzyme denaturing of step (2) is enzyme denaturing 10 min in 100 ℃ of water; Described centrifugal be centrifugal 25 min under 8000 r/min.
The preparation method of above-mentioned chlorella polypeptide-chitosan nano, is filtered under 8000 g, 4 ℃ of conditions described in step (2) and filters through ultra-filtration centrifuge tube.
The preparation method of above-mentioned chlorella polypeptide-chitosan nano, the concrete separation condition of the described use ion exchange chromatography of step (3) DEAE-52 separation and purification is: loading volume is 3 ~ 15 mL, elution speed is 0.2 ~ 2 mL/min, and eluent is distilled water, and detecting wavelength is 280 nm; The concrete separation condition of described gel chromatography sephadex G-25 separation and purification is: loading volume is 3 ~ 15 mL, and elution speed is 0.2 ~ 2 mL/min, and eluent is distilled water, and detecting wavelength is 280 nm.
The preparation method of above-mentioned chlorella polypeptide-chitosan nano, the chitosan-acetic acid solution that the chlorella polypeptide solution A2-1 that the described sodium tripolyphosphate of step (4) (TPP) ionic cross-linking is is 0.2 ~ 2 mg/mL 1 ~ 5mL concentration is 1 ~ 3 mg/mL with 3 ~ 10 mL concentration is mixed homogeneously, the TPP aqueous solution that by concentration is again 0.1 ~ 2 mg/mL is added drop-wise in A2-1 and chitosan-acetic acid solution system gradually, stirring reaction 30 min, high speed centrifugation 30 min under 4 ℃, 10000 r/min, collecting precipitation thing, it is dry in 45 ℃ of baking ovens.
Compared with prior art, tool of the present invention has the following advantages and technique effect: chlorella polypeptide-chitosan nano that the present invention obtains has following anti-tumor activity: human liver cancer cell HepG2 growth in vitro is had to certain inhibitory action, when concentration is 400 μ g/mL, suppression ratio can reach 32%. thereby chlorella polypeptide-chitosan nano of obtaining of the present invention be conducive to the exploitation of antineoplastic health product and medical product.
Accompanying drawing explanation
Fig. 1 is chlorella papain 3-5 KD hydrolyzed solution (active component A) ion exchange chromatography DEAE-52 column chromatography elution curve in embodiment 1;
Fig. 2 is chlorella active component A 2 gel chromatography sephadex G-25 column chromatography elution curves in embodiment 1;
Fig. 3 is the size distribution curve of the prepared chlorella polypeptide-chitosan nano of embodiment 1;
Fig. 4 is scanning electron microscope (SEM) figure of the prepared chlorella polypeptide-chitosan nano surface topography of embodiment 1.
The specific embodiment
Below in conjunction with accompanying drawing and example, specific embodiment of the invention is described further, but enforcement of the present invention and protection domain are not limited to this.
The preparation method of chlorella polypeptide-chitosan nano, step is as follows:
(1) adopt low-temperature ultrahigh-pressure continuous flow cell disintegrating machine to extract chlorella protein: chlorella powder and pure water are stirred 50 minutes to obtain to mixed solution after the mass ratio mixing with 1:20.By above-mentioned mixed solution, in temperature, be to extract chlorella protein under 6 ℃, the pressure condition that is 100MPa, obtain crude extract and extract as solution to be extracted next time again, the number of times of extraction is 3 times, and each time of extracting is 20min.Finally, by the solution obtaining, in 4 ℃ of temperature, rotating speed is centrifugal 30 min under 6000 r/min, remove precipitation and obtain albumen supernatant, then vacuum lyophilization rapidly, obtain chlorella protein powder.
(2) take the chlorella protein powder that step (1) obtains is basis, configuration concentration is 2% chlorella protein solution, add papain to be hydrolyzed. experiment condition is 40 ℃ of temperature, pH=5, after ratio 3%. hydrolysis 5 h of enzyme-to-substrate, enzyme denaturing 10 min in 100 ℃ of water, by hydrolyzed solution centrifugal 25 min under 8000 r/min, get supernatant after room temperature is cooling.Then, the ultra-filtration centrifuge tube that is 10 KD through molecular cut off under 8000 g, 4 ℃ of conditions filters.Filtrate is filtered through the ultra-filtration centrifuge tube of 3 KD, 5 KD again.Can obtain molecular size range scope is the chlorella polypeptide solution of 0-3 KD, 3-5 KD, 5-10 KD, >10 KD.
(3) take the 3-5 KD papain hydrolysis liquid (active component A) that step (2) obtains is basis, use ion exchange chromatography DEAE-52 separation and purification. concrete separation condition is: loading volume is 10 mL, elution speed is 0.3 mL/min, eluent is distilled water, and detecting wavelength is 280 nm.And collect altogether four peak A1 ~ A4 (Fig. 1) according to peak sequence. the absorption peak A of collecting 2 is carried out to the separation and purification of gel chromatography sephadex G-25 again.Concrete separation condition is: loading volume is 5 mL, and elution speed is 0.2 mL/min, and eluent is distilled water, and detecting wavelength is 280 nm.And collect altogether two peak A2-1 and A2-2 (Fig. 2) according to peak sequence.
(4) take the chlorella polypeptide solution A2-1 of the purification that step (3) obtains is basis, then adopts sodium tripolyphosphate (TPP) ionic cross-linking to prepare chitin nanometer.Specific procedure is: 1mL concentration is that the chitosan-acetic acid solution that the component A2-1 of 0.4 mg/mL is 1 mg/mL with 5 mL concentration is mixed homogeneously.The TPP aqueous solution that again concentration is respectively to 0.3 mg/mL is added drop-wise in A2-1 and chitosan-acetic acid solution system gradually.Stirring reaction 30 min, by the colloid solution making high speed centrifugation 30 min under 4 ℃, 10000 r/min, collecting precipitation thing, dryly in 45 ℃ of baking ovens can obtain chlorella polypeptide-chitosan nano by it.
Adopt laser particle analyzer to carry out particle diameter detection to the chlorella polypeptide-chitosan nano of step (1) ~ (4) gained.As can be seen from Figure 3, chlorella polypeptide-chitosan nano mean diameter is 129.2nm.
Chlorella polypeptide-the chitosan nano of step (1) ~ (4) gained is carried out to scanning electron microscope (SEM) analysis.As can be seen from Figure 4, the form of the spherical in shape or almost spherical of chlorella polypeptide-chitosan nano complex, size is homogeneous relatively.Size is 170 nm, substantially identical to the granularmetric analysis result of chitin nanometer complex with above-mentioned laser particle size scatterometer.
Chlorella polypeptide-the chitosan nano of step (1) ~ (4) gained is carried out to anti-tumor activity detection (using MTT detection method), result shows, chlorella polypeptide-chitosan nano has certain inhibitory action to human liver cancer cell HepG2 growth in vitro, when concentration is 400 μ g/mL, suppression ratio can reach 32%.
The preparation method of chlorella polypeptide-chitosan nano, step is as follows:
(1) adopt low-temperature ultrahigh-pressure continuous flow cell disintegrating machine to extract chlorella protein: chlorella powder and pure water are stirred 80 minutes to obtain to mixed solution after the mass ratio mixing with 1:30.By above-mentioned mixed solution, in temperature, be to extract chlorella protein under 8 ℃, the pressure condition that is 150MPa, obtain crude extract and extract as solution to be extracted next time again, the number of times of extraction is 6 times, and each time of extracting is 50min.Finally, by the solution obtaining, in 4 ℃ of temperature, rotating speed is centrifugal 20 min under 5000 r/min, remove precipitation and obtain albumen supernatant, then vacuum lyophilization rapidly, obtain chlorella protein powder.
(2) take the chlorella protein powder that step (1) obtains is basis, configuration concentration is 2% chlorella protein solution, add papain to be hydrolyzed. experiment condition is 70 ℃ of temperature, pH=9, after ratio 4%. hydrolysis 6 h of enzyme-to-substrate, enzyme denaturing 10 min in 100 ℃ of water, by hydrolyzed solution centrifugal 25 min under 8000 r/min, get supernatant after room temperature is cooling.Then, the ultra-filtration centrifuge tube that is 10 KD through molecular cut off under 8000 g, 4 ℃ of conditions filters.Filtrate is filtered through the ultra-filtration centrifuge tube of 3 KD, 5 KD again.Can obtain molecular size range scope is the chlorella polypeptide solution of 0-3 KD, 3-5 KD, 5-10 KD, >10 KD.
(3) take the 3-5 KD papain hydrolysis liquid (active component A) that step (2) obtains is basis, use ion exchange chromatography DEAE-52 separation and purification. concrete separation condition is: loading volume is 10 mL, elution speed is 0.5 mL/min, eluent is distilled water, and detecting wavelength is 280 nm.And according to peak sequence, collect altogether four peak A1 ~ A4. the absorption peak A of collecting 2 is carried out to the separation and purification of gel chromatography sephadex G-25 again.Concrete separation condition is: loading volume is 5 mL, and elution speed is 1.2 mL/min, and eluent is distilled water, and detecting wavelength is 280 nm.And collect altogether two peak A2-1 and A2-2 according to peak sequence.
(4) take the chlorella polypeptide solution A2-1 of the purification that step (3) obtains is basis, then adopts sodium tripolyphosphate (TPP) ionic cross-linking to prepare chitin nanometer.Specific procedure is: 1mL concentration is that the chitosan-acetic acid solution that the component A2-1 of 0.7 mg/mL is 1.5 mg/mL with 5 mL concentration is mixed homogeneously.The TPP aqueous solution that again concentration is respectively to 0.15 mg/mL is added drop-wise in A2-1 and chitosan-acetic acid solution system gradually.Stirring reaction 30 min, by the colloid solution making high speed centrifugation 30 min under 4 ℃, 10000 r/min, collecting precipitation thing, dryly in 45 ℃ of baking ovens can obtain chlorella polypeptide-chitosan nano by it.Detection method and result are with embodiment 1.
The preparation method of chlorella polypeptide-chitosan nano, step is as follows:
(1) adopt low-temperature ultrahigh-pressure continuous flow cell disintegrating machine to extract chlorella protein: chlorella powder and pure water are stirred 100 minutes to obtain to mixed solution after the mass ratio mixing with 1:35.By above-mentioned mixed solution, in temperature, be to extract chlorella protein under 9 ℃, the pressure condition that is 200MPa, obtain crude extract and extract as solution to be extracted next time again, the number of times of extraction is 2 times, and each time of extracting is 10min.Finally, by the solution obtaining, in 4 ℃ of temperature, rotating speed is centrifugal 30 min under 8000 r/min, remove precipitation and obtain albumen supernatant, then vacuum lyophilization rapidly, obtain chlorella protein powder.
(2) take the chlorella protein powder that step (1) obtains is basis, configuration concentration is 2% chlorella protein solution, add papain to be hydrolyzed. experiment condition is 40 ℃ of temperature, pH=5, after ratio 4%. hydrolysis 4 h of enzyme-to-substrate, enzyme denaturing 10 min in 100 ℃ of water, by hydrolyzed solution centrifugal 25 min under 8000 r/min, get supernatant after room temperature is cooling.Then, the ultra-filtration centrifuge tube that is 10 KD through molecular cut off under 8000 g, 4 ℃ of conditions filters.Filtrate is filtered through the ultra-filtration centrifuge tube of 3 KD, 5 KD again.Can obtain molecular size range scope is the chlorella polypeptide solution of 0-3 KD, 3-5 KD, 5-10 KD, >10 KD.
(3) take the 3-5 KD papain hydrolysis liquid (active component A) that step (2) obtains is basis, use ion exchange chromatography DEAE-52 separation and purification. concrete separation condition is: loading volume is 10 mL, elution speed is 1 mL/min, eluent is distilled water, and detecting wavelength is 280 nm.And according to peak sequence, collect altogether four peak A1 ~ A4. the absorption peak A of collecting 2 is carried out to the separation and purification of gel chromatography sephadex G-25 again.Concrete separation condition is: loading volume is 5 mL, and elution speed is 1.5 mL/min, and eluent is distilled water, and detecting wavelength is 280 nm.And collect altogether two peak A2-1 and A2-2 according to peak sequence.
(4) take the chlorella polypeptide solution A2-1 of the purification that step (3) obtains is basis, then adopts sodium tripolyphosphate (TPP) ionic cross-linking to prepare chitin nanometer.Specific procedure is: 1mL concentration is that the chitosan-acetic acid solution that the component A2-1 of 1.2 mg/mL is 2 mg/mL with 5 mL concentration is mixed homogeneously.The TPP aqueous solution that again concentration is respectively to 1 mg/mL is added drop-wise in A2-1 and chitosan-acetic acid solution system gradually.Stirring reaction 30 min, by the colloid solution making high speed centrifugation 30 min under 4 ℃, 10000 r/min, collecting precipitation thing, dryly in 45 ℃ of baking ovens can obtain chlorella polypeptide-chitosan nano by it.Detection method and result are with embodiment 1.
embodiment 4
The preparation method of chlorella polypeptide-chitosan nano, step is as follows:
(1) adopt low-temperature ultrahigh-pressure continuous flow cell disintegrating machine to extract chlorella protein: chlorella powder and pure water are stirred 90 minutes to obtain to mixed solution after the mass ratio mixing with 1:10.By above-mentioned mixed solution, in temperature, be to extract chlorella protein under 9 ℃, the pressure condition that is 250MPa, obtain crude extract and extract as solution to be extracted next time again, the number of times of extraction is 4 times, and each time of extracting is 40minn.Finally, by the solution obtaining, in 4 ℃ of temperature, rotating speed is centrifugal 60 min under 10000 r/min, remove precipitation and obtain albumen supernatant, then vacuum lyophilization rapidly, obtain chlorella protein powder.
(2) take the chlorella protein powder that step (1) obtains is basis, configuration concentration is 2% chlorella protein solution, add papain to be hydrolyzed. experiment condition is temperature 60 C, pH=7, after ratio 5%. hydrolysis 10 h of enzyme-to-substrate, enzyme denaturing 10 min in 100 ℃ of water, by hydrolyzed solution centrifugal 25 min under 8000 r/min, get supernatant after room temperature is cooling.Then, the ultra-filtration centrifuge tube that is 10 KD through molecular cut off under 8000 g, 4 ℃ of conditions filters.Filtrate is filtered through the ultra-filtration centrifuge tube of 3 KD, 5 KD again.Can obtain molecular size range scope is the chlorella polypeptide solution of 0-3 KD, 3-5 KD, 5-10 KD, >10 KD.
(3) take the 3-5 KD papain hydrolysis liquid (active component A) that step (2) obtains is basis, use ion exchange chromatography DEAE-52 separation and purification. concrete separation condition is: loading volume is 10 mL, elution speed is 1.5 mL/min, eluent is distilled water, and detecting wavelength is 280 nm.And according to peak sequence, collect altogether four peak A1 ~ A4. the absorption peak A of collecting 2 is carried out to the separation and purification of gel chromatography sephadex G-25 again.Concrete separation condition is: loading volume is 5 mL, and elution speed is 2 mL/min, and eluent is distilled water, and detecting wavelength is 280 nm.And collect altogether two peak A2-1 and A2-2. according to peak sequence
(4) take the chlorella polypeptide solution A2-1 of the purification that step (3) obtains is basis, then adopts sodium tripolyphosphate (TPP) ionic cross-linking to prepare chitin nanometer.Specific procedure is: 1mL concentration is that the chitosan-acetic acid solution that the component A2-1 of 0.2 mg/mL is 3 mg/mL with 5 mL concentration is mixed homogeneously.The TPP aqueous solution that again concentration is respectively to 0.1 mg/mL is added drop-wise in A2-1 and chitosan-acetic acid solution system gradually.Stirring reaction 30 min, by the colloid solution making high speed centrifugation 30 min under 4 ℃, 10000 r/min, collecting precipitation thing, dryly in 45 ℃ of baking ovens can obtain chlorella polypeptide-chitosan nano by it.Detection method and result are with embodiment 1.
Claims (4)
1. the preparation method of chlorella polypeptide-chitosan nano, is characterized in that specifically comprising the steps:
(1) will after chlorella powder and pure water mixing, stir to obtain mixed solution, in described mixed solution, extract chlorella protein, the crude extract obtaining extracts as solution to be extracted next time again, by the solution centrifugal obtaining, remove precipitation and obtain albumen supernatant, vacuum lyophilization again, obtains chlorella protein powder; Described extraction chlorella protein for to extract under the condition of 4 ℃～10 ℃ of temperature, pressure 50MPa～250MPa; The described number of times extracting is again 1 time～8 times, and each time of extracting is 10min～60min; Described centrifugal be 4 ~ 8 ℃ of temperature, rotating speed is centrifugal 10 ~ 60 min under 5000 ~ 10000 r/min;
(2) take the chlorella protein powder that step (1) obtains is basis, configuration concentration is 1 ~ 4%(w/v) chlorella protein solution, adding hydrolytic enzyme is hydrolyzed, after hydrolysis, enzyme denaturing, be cooled to after room temperature hydrolyzed solution is centrifugal, get supernatant, then, the ultra-filtration centrifuge tube that is 10 KD through molecular cut off filters, filtrate is filtered through the ultra-filtration centrifuge tube of 3 KD, 5 KD again, and can obtain molecular size range scope is the chlorella polypeptide solution of < 3 KD, 3-5 KD, 5-10 KD, >10 KD; Described hydrolytic enzyme is papain, and hydrolysising condition is 30 ~ 80 ℃ of temperature, and pH=5 ~ 10, and enzyme is 1% ~ 5%(w/v with the ratio of chlorella protein aqueous solution), hydrolysis time is 3 ~ 10h;
(3) take the 3-5 KD chlorella polypeptide solution that step (2) obtains is basis, use ion exchange chromatography DEAE-52 separation and purification, according to peak sequence, collect altogether four peak A1 ~ A4, the absorption peak A of collecting 2 is carried out to the separation and purification of gel chromatography sephadex G-25 again, according to peak sequence, collect altogether two peak A2-1 and A2-2; The concrete separation condition of described use ion exchange chromatography DEAE-52 separation and purification is: loading volume is 3 ~ 15 mL, and elution speed is 0.2 ~ 2 mL/min, and eluent is distilled water, and detecting wavelength is 280 nm; The concrete separation condition of described gel chromatography sephadex G-25 separation and purification is: loading volume is 3 ~ 15 mL, and elution speed is 0.2 ~ 2 mL/min, and eluent is distilled water, and detecting wavelength is 280 nm;
(4) take the chlorella polypeptide solution A2-1 of the purification that step (3) obtains is basis, then adopts sodium tripolyphosphate (TPP) ionic cross-linking can obtain chlorella polypeptide-chitosan nano; The chitosan-acetic acid solution that the chlorella polypeptide solution A2-1 that described sodium tripolyphosphate (TPP) ionic cross-linking is is 0.2 ~ 2 mg/mL 1 ~ 5mL concentration is 1 ~ 3 mg/mL with 3 ~ 10 mL concentration is mixed homogeneously, the TPP aqueous solution that by concentration is again 0.1 ~ 2 mg/mL is added drop-wise in A2-1 and chitosan-acetic acid solution system gradually, stirring reaction 30 min, high speed centrifugation 30 min under 4 ℃, 10000 r/min, collecting precipitation thing, it is dry in 45 ℃ of baking ovens.
2. the preparation method of chlorella polypeptide-chitosan nano according to claim 1, the mass ratio that it is characterized in that the described chlorella powder of step (1) and pure water is 1:10～1:40, described mixing time is 30～120 minutes.
3. the preparation method of chlorella polypeptide-chitosan nano according to claim 1, is characterized in that the described enzyme denaturing of step (2) is for enzyme denaturing 10 min in 100 ℃ of water; Described centrifugal be centrifugal 25 min under 8000 r/min.
4. the preparation method of chlorella polypeptide-chitosan nano according to claim 1, is characterized in that being filtered under 8000 g, 4 ℃ of conditions and filtering through ultra-filtration centrifuge tube described in step (2).
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| CN102304168A (en) * | 2011-08-17 | 2012-01-04 | 华南理工大学 | Low-temperature ultrahigh-pressure preparation method of chlorella protein |
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| CN102284063A (en) * | 2011-08-17 | 2011-12-21 | 华南理工大学 | Application of carbon nanotube-chitosan-phycocyanin compound in preparing antineoplastic drugs |
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