CN106501191A - The high throughput screening system of functional components of chlorella vulgaris CPE, screening technique and preparation method thereof - Google Patents
The high throughput screening system of functional components of chlorella vulgaris CPE, screening technique and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of high throughput screening system of functional components of chlorella vulgaris CPE, screening technique and preparation method thereof.Screening Platform includes：1) antioxidation test：2) Culture in vitro test：3) redox metal ion chelating test：4) advanced glycation end products AGEs tests are suppressed.Preparation method includes：Chlorella pyrenoidesa algae powder is added in distilled water, under high pressure, 110-130 DEG C of heating 30-60min is extracted；Sample is taken out, after cooling down under room temperature, 10000rpm is centrifuged 30min, and after supernatant is through the sucking filtration that reduces pressure, 4 DEG C save backup；Algae mud after by centrifugation, adds equivalent distilled water resuspended, repeats aforesaid operations, merges filtrate twice, and lyophilization is obtained chlorella CPE extract powders.The functional components of chlorella vulgaris CPE yield that extracts through the inventive method is high, and the Protein and nucleic acid in extract is abundant, is suitable for industrialized production, to reduce the development cost of the microalgae energy, improves the overall availability of the microalgae energy.
Description
Technical field
The invention belongs to functional components of chlorella vulgaris CPE screenings, preparation extractive technique field, more particularly to a kind of high throughput screening system of functional components of chlorella vulgaris CPE, screening technique, and the method for preparing functional components of chlorella vulgaris CPE based on high pressure breaking cellular wall method.
Background technology
Microalgae due to growth cycle short, biomass yield is high, carbon dioxide fixation ability is strong, unit soil production efficiency is high and do not strive with grain etc. advantage, just gradually beyond tradition energy crop becomes third generation a new generation bioenergy.Although the study on the industrialization of the microalgae energy comes into the pilot scale stage at present, but still difficult, wherein topmost problem is high cost.
One of chlorella (Chlorella), is commonly called as chlorella, be the microalgae energy greatly developing both at home and abroad and cultivate with the main algae kind of microalgae carbon sequestration this emerging strategic industries.Chlorella is a kind of nutritious and safe food in itself, and there is effect at many aspects such as immunomodulating.Therefore, if the high value added product with multiple efficacies such as immunomodulating can be extracted from the algae-residue after extraction oils and fatss, can be greatly reduced the production cost with chlorella as the microalgae energy of algae kind, further promote the process of microalgae energy industry.
Contain various active material, such as polysaccharide, glycoprotein, aminoacid and polypeptide etc. in chlorella, with extensive biological activity, such as antitumor, the beneficial bacteria growing of promotion, enhancing immunity, removing toxic substances, blood pressure lowering etc..More with regard to the applied research of chlorella hot water extract both at home and abroad, research is concentrated mainly on immunocompetent research and the anti-tumor activity mediated by immunocompetence.Chlorella pyrenoidesa hot water extract (Hot water extract of Chlorella pyrenoidosa, CPE) is mainly the mixture of water-soluble substanceses, including nucleic acid, polysaccharide, polypeptide, albumen, water soluble vitamins etc..Due to CPE in chlorella rich content, for the immune class health product of exploitation more valuable.
Hidaka has found that CPE can improve the immunocompetence of malnutrition mice, it is proposed that using chlorella as functional food.Yamagishi etc. has found that chlorella can treat the disease of carbohydrate metabolism imbalance, the potential value with human diseasess such as treatment diabetes.But which composition remains a blank in terms of there is the research for promoting growth of animal and enhance immunity function so far in the numerous compositions of CPE actually, therefore further determine the effective ingredient composition in CPE, structurally and functionally mechanism there is basic research meaning not only, and be with a wide range of applications.
At present, the exploitation of the microalgae such as chlorella and spirulina, salt alga constitute an important branch of China's field of food, have broad prospects.Chlorella powder protein content is very high, is excellent single cell protein sources, and the bead algae glycoproteins for wherein containing are present among the waste water of algae amyloid proteins production in a large number, are isolated extraction according to appropriate means, will produce significant economic benefit and social benefit.
CPE extracting method species is various at present, causes its lytic activity difference big, it is difficult to situations such as determining major constituent.Therefore before exploitation chlorella high value added product, it is necessary to numerous CPE extracting method are compared, a kind of method for being suitable to industrialized production is therefrom filtered out.For example, due to the presence of tough and tensile cell wall, from chlorella, effective component extracting has whether breaking cellular wall.
Content of the invention
It is an object of the invention to overcoming the deficiencies in the prior art, there is provided a kind of high throughput screening system of functional components of chlorella vulgaris CPE, screening technique and preparation method thereof.
The purpose of the present invention is achieved through the following technical solutions：
The first object of the present invention is to provide a kind of high throughput screening system of functional components of chlorella vulgaris CPE, including：
1) antioxidation test：Tested using Griess reagents, the total antioxidant capacity by spectrophotometry sample and the scavenging action to free radical；
2) Culture in vitro test：
A. impact of the four tetrazolium bromide mtt assay detected components to mouse macrophage Ana-1 survival rates；
B. impact of the dimethyl diaminophenazine chloride NR staining detected components to Ana-1 phagocytic functions；
C. the change of fluorescence probe method detection sample NO secretions intracellular to Ana-1；
3) redox metal ion chelating test：
A. using luxuriant and rich with fragrance Lip river piperazine as Fe2+Chelating reagent, by the Fe of spectrophotometry sample2+Sequestering power；
B. using pyrocatechol violet as Cu2+Chelating reagent, by the Cu of spectrophotometry sample2+Sequestering power；
4) advanced glycation end products AGEs tests are suppressed：
Non-enzymatic glycation system：The glucose of the bovine serum albumin BSA and 0.5mol/L of 10mg/mL is dissolved in the phosphate buffer PBS of 0.02mol/L, pH 7.4, non-enzymatic glycation system is obtained；
By fluorescent spectrometry, impact of different phase of the sample in Maillard reaction to AGEs and pentosidine is analyzed.
The high throughput screening system of the functional components of chlorella vulgaris CPE of the present invention, by improvement and combination to existing Screening Platform, propose a kind of high-throughput screening method of the physiological functions such as antioxidation, immunomodulating and diabetes, active spectral limit is wide, functional activity is high, is easy to the separation and Extraction of CPE and the industrialized production of chlorella functional product.
The second object of the present invention is to provide a kind of high-throughput screening method of functional components of chlorella vulgaris CPE, including：
1) antioxidation test：Concentration is taken for 0,100,200,500,1000 μ g/mL samples, add Griess reagents, terminating reaction after 37 DEG C of reaction 1min, with 630nm as reference wavelength, determine OD value of the reactant at 550nm, according to the radical scavenging activity that equation below calculates sample, the percentage clearance rate of OH is represented with sample；
2) Culture in vitro test：
A. impact of the four tetrazolium bromide mtt assay detected components to mouse macrophage Ana-1 survival rates；
Concrete operations are as follows：Take the logarithm the cell of trophophase, after trypsinization, cell suspension is made with the RPMI-1640 culture medium containing 10% hyclone, with 2 × 104The even density in individual/hole is added in 96 porocyte culture plates, put 37 DEG C, saturated humidity, containing 5%CO2Cell culture incubator in quiescent culture.After cell attachment and after growing to suitable density (about 24h), the medicines such as CPE of variable concentrations are added per hole.After continuing culture certain time, the MTT solution of 20 μ L0.5mg/mLs, 37 DEG C incubation 4hs are added per hole.After the completion of incubation, 100 μ L, tri- liquid (10%SDS, 5% isobutanol, 12mmol/L HCl), 37 DEG C of overnight incubations is added to react formed purple formazan crystallizations to dissolve cell with MTT per hole.Then concussion mixes 5min, with 630nm wavelength as reference, detects OD value of each hole at 492nm wavelength, and calculates cell survival rate according to formula.
B. impact of the dimethyl diaminophenazine chloride NR staining detected components to Ana-1 phagocytic functions；
Concrete operations are as follows：After Ana-1 cells are through the sample treatment certain time such as CPE, add 20 μ L of freshly prepared 0.1%NR suspensions per hole, after gently mixing, continue culture 3h.Careful suction abandons the culture fluid containing NR afterwards, softly washes away the NR not swallowed with physiology PBS.Add 150 μ L cell pyrolysis liquid (dehydrated alcohol per hole:Acetic acid=1:1 (v/v)), 2h is stood, dissolves cell, concussion determines OD values at 540nm after mixing.The change that cell is secreted with above-mentioned formula c. fluorescence probe method detection sample NO intracellular to Ana-1 to the phagocytic rate calculation of NR；
Concrete operations are as follows：Fluorescent probe DAF-FM DA are diluted to 5 μm of ol/L of final concentration with DAF-FM DA diluents.The cell of the amount of taking fully exponential phase, digests and dispels, and washed once with the physiology PBS of pH 7.4 after being collected by centrifugation.With the DAF-FM DA working solution re-suspended cells for having diluted, cell concentration is adjusted to 1 × 106Individual/mL, 37 DEG C of incubation 20min.Period is mixed reverse for mixture every 3-5min, is fully contacted probe and cell.PBS washed cells three times, are introduced into intracellular DAF-FM DA with abundant removal.The cell for having loaded uniformly is spread in 96 hole black cell culture plates, and adds the CPE samples of variable concentrations.After 37 DEG C of temperature bath certain times, fluorescent value of each hole under 495/515nm excitation/emission wavelength is detected, and calculates the change of the lower Ana-1 cell NO levels of each sample effect.
3) redox metal ion chelating test：
a.Fe2+Chelating test
The respective metal solion of 5mmol/L is added in the HAc-NaAc buffer of pH 4.9 so as to final concentration of 1.25mmol/L；
With concentration as 2.5,5.0, and the EDTA solution of 7.5mmol/L is used as positive control, remaining CPE sample for variable concentrations；
The interior distilled water of each reaction tube is supplied, and makes each system volume reach 240 μ L, room temperature reaction 30min；
The luxuriant and rich with fragrance Lip river piperazine solution colour developing 30min of 50mmol/L after reaction terminates, is added, and is detected its OD value at 535nm or 612nm, sample is calculated to Fe according to following formula2+Chelation percent；
b.Cu2+Chelating test
The respective metal solion of 5mmol/L is added in the HAc-NaAc buffer of pH 6.0 so as to final concentration of 1.25mmol/L；
With concentration as 2.5,5.0, and the EDTA solution of 7.5mmol/L is used as positive control, remaining CPE sample for variable concentrations；
The interior distilled water of each reaction tube is supplied, and makes each system volume reach 240 μ L, room temperature reaction 30min；
The pyrocatechol violet solution colour developing 30min of 20mmol/L after reaction terminates, is added, and is detected its OD value at 535nm or 612nm, sample is calculated to Cu according to following formula2+Chelation percent；
4) advanced glycation end products AGEs tests are suppressed：
The glucose of the bovine serum albumin BSA and 0.5mol/L of 10mg/mL is dissolved in the phosphate buffer PBS of 0.02mol/L, pH 7.4, non-enzymatic glycation system；
Sample is added in above-mentioned reaction system and is incubated so as to which final concentration is respectively 10,100, and 1000 μ g/mL, while with the 10mg/mL BSA that are individually incubated in PBS as blank；
Microporous filter membrane sucking filtration sterilizing of all samples through 0.22 μm of diameter, after 37 DEG C of lucifuges incubations 1,3,7,14,28,42d, will incubation product dilution to 1mg/mL, detection incubation fluorescent value of the product at 370/440nm and 335/385nm respectively；
With the fluorescent value of 1mg/mL BSA as a flat fluorescent, according to fluorescence intensity FI that following formula calculate the lower incubation product of CPE effects；
The high-throughput screening method of the functional components of chlorella vulgaris CPE of the present invention, by improvement and combination to existing Screening Platform, propose a kind of high-throughput screening method of the physiological functions such as antioxidation, immunomodulating and diabetes, active spectral limit is wide, functional activity is high, is easy to the separation and Extraction of CPE and the industrialized production of chlorella functional product.
The third object of the present invention is to provide a kind of preparation method of functional components of chlorella vulgaris CPE, including：
1) Chlorella pyrenoidesa algae powder is added in distilled water, at pressure 0.05-0.25MPa, temperature 110-130 DEG C, heats 30-60min, extracted；
2) sample is taken out, after cooling down under room temperature, 10000rpm is centrifuged 30min, and after supernatant is through the sucking filtration that reduces pressure, 4 DEG C save backup；
3) by step 2) centrifugation after algae mud, add resuspended with 1) equal-volume distilled water, at pressure 0.1MPa, temperature 110-130 DEG C, heating 30-60min, extract again；
4) sample is taken out, after cooling down under room temperature, 10000rpm is centrifuged 30min, takes supernatant, reduce pressure sucking filtration, merges filtrate twice, lyophilization is obtained chlorella CPE extract powders.
Preferably, the step 1) in pressure be 0.05-0.25MPa.
Preferably, the step 1) in temperature be 110-130 DEG C.
Preferably, the step 1) in heat time heating time be 30-60min.
The step 1) in Chlorella pyrenoidesa algae powder even suspension in distilled water, the proportioning of Chlorella pyrenoidesa algae powder and distilled water is 1g:10ml.
Preferably, the step 3) in pressure be 0.05-0.25MPa.
Preferably, the step 3) in temperature be 110-130 DEG C.
Preferably, the step 3) in heat time heating time be 30-60min.
Described extraction is extracted with hot high pressure water law.
The method that chlorella CPE is prepared based on high pressure breaking cellular wall method of the present invention, the method only need water and common device autoclave, breaking cellular wall and extraction synchronously to carry out, breaking cellular wall is abundant, operating procedure is few, and required time significantly shortens, with low cost, many advantages such as pollution-free and fast and convenient.
Compared with prior art, the positive effect of the present invention is as follows：
The present invention is by introducing high pressure wall breaking technology, it is achieved that the efficient preparation of chlorella CPE.Through the chlorella CPE that the inventive method is extracted, yield is up to 251.67mg/g algae powder, and the Protein and nucleic acid in extract enriches, CPE activity is high, it is extremely suitable for industrialized production, research and development for chlorella high added value bioactive substance lays the foundation, and to reduce the development cost of the microalgae energy, improves the overall availability of the microalgae energy.
Description of the drawings
Fig. 1 is clearance rate of two kinds of CPE to OH；
Fig. 2 is impacts of two kinds of CPE to Ana-1 survival rates；
Fig. 3 is impacts of the CPE1 to Ana-1 survival rates and Ana-1 to the cell phagocytic rate of NR；
Fig. 4 be CPE1 to Fe2+Chelation；
Fig. 5 be CPE1 to Cu2+Chelation；
Fig. 6 is the impact that CPE1 is formed to total AGEs and penosidine；
CPE yield prepared by Fig. 7 Different Extraction Methods；
Fig. 8 is clearance rate of four kinds of CPE crude products to OH；
Fig. 9 is impacts of four kinds of CPE to macrophage Ana-1 survival rates；
Figure 10 be four kinds of CPE crude extracts to Fe2+And Cu2+Chelation percent.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.In addition, it is to be understood that after the content for having read instruction of the present invention, those skilled in the art can be made various changes or modifications to the present invention, these equivalent form of values equally fall within the application appended claims limited range.
Embodiment 1
The high flux screening active ingredients system of functional components of chlorella vulgaris CPE
1) antioxidation test：Tested using Griess reagents, the total antioxidant capacity by spectrophotometry sample and the scavenging action to free radical；
2) Culture in vitro test：
A. impact of the four tetrazolium bromide mtt assay detected components to mouse macrophage Ana-1 survival rates；
B. impact of the dimethyl diaminophenazine chloride NR staining detected components to Ana-1 phagocytic functions；
C. the change of fluorescence probe method detection sample NO secretions intracellular to Ana-1；
3) redox metal ion chelating test：
A. using luxuriant and rich with fragrance Lip river piperazine as Fe2+Chelating reagent, by the Fe of spectrophotometry sample2+Sequestering power；
B. using pyrocatechol violet as Cu2+Chelating reagent, by the Cu of spectrophotometry sample2+Sequestering power；
4) advanced glycation end products AGEs tests are suppressed：
Non-enzymatic glycation system：The glucose of the bovine serum albumin BSA and 0.5mol/L of 10mg/mL is dissolved in the phosphate buffer PBS of 0.02mol/L, pH 7.4, non-enzymatic glycation system is obtained；
By fluorescent spectrometry, impact of different phase of the sample in Maillard reaction to AGEs and pentosidine is analyzed.
Embodiment 2
What in the present embodiment, CPE1 and CPE2 chose is obtained by ultrasonic enzymatic isolation method, and concrete grammar is：10g Chlorella pyrenoidesa powder even suspensions are in 100mL distilled water.After ultrasonication 30min, 30min is extracted in 95 DEG C of water-baths.After liquid cooling to be extracted, cellulase (80mg/g algae powder), pectase (10mg/g algae powder) is added.50 DEG C of water-bath 2h, degrade to polysaccharide.Boiling water bath 30min makes enzyme-deactivating, 10000rpm be centrifuged 30min.Supernatant is collected, by 4 times of volume concentrations, and lyophilization, CPE ultrasound enzymatic hydrolysate is obtained, wherein CPE1 has digested 4h, and CPE2 has then digested 16h.
The high flux method for screening active ingredients of functional components of chlorella vulgaris CPE
1) oxidation resistance test, i.e. scavenging hydroxyl (OH) aptitude tests
Method of testing：0,100,200,500,1000 μ g/mL concentration samples are taken, adds Griess reagents, terminating reaction after 37 DEG C of accurate response 1min with 630nm as reference wavelength, to determine OD value of the reactant at 550nm.
The radical scavenging activity of sample, is calculated according to formula, represents that formula is as follows with sample to the percentage clearance rate of OH：
Interpretation of result：(clearance rate of two kinds of CPE to OH, n=6 as shown in Figure 1；Compared with matched group, * p<0.05, * * p<0.01) OH during, two kinds of CPE are to system has certain Scavenging activity, and this Scavenging activity and concentration positive correlation, and with the raising of concentration of participating in the experiment, clearance rate increases.Especially when concentration reaches 1000 μ g/mL, CPE1 reaches 29.46% (p to the clearance rate of OH<0.01), and CPE2 has higher Scavenging activity to OH, its clearance rate reaches 34.05% (p<0.01).
2) Culture in vitro functional test
The cell survival rate test of a.Ana-1
Concrete operations are as follows：Take the logarithm the cell of trophophase, after trypsinization, cell suspension is made with the RPMI-1640 culture medium containing 10% hyclone, with 2 × 104The even density in individual/hole is added in 96 porocyte culture plates, put 37 DEG C, saturated humidity, containing 5%CO2Cell culture incubator in quiescent culture.After cell attachment and after growing to suitable density (about 24h), the medicines such as CPE of variable concentrations are added per hole.After continuing culture certain time, the MTT solution of 20 μ L0.5mg/mLs, 37 DEG C incubation 4hs are added per hole.After the completion of incubation, 100 μ L, tri- liquid (10%SDS, 5% isobutanol, 12mmol/L HCl), 37 DEG C of overnight incubations is added to react formed purple formazan crystallizations to dissolve cell with MTT per hole.Then concussion mixes 5min, with 630nm wavelength as reference, detects OD value of each hole at 492nm wavelength, and calculates cell survival rate according to formula.
Interpretation of result：(the impact n=5 of two kinds of CPE to Ana-1 survival rates as shown in Figure 2；Compared with matched group, * p<0.05, * * p<0.01), in the concentration range of 0-500 μ g/mL, two kinds of CPE without obvious cytotoxic effect, promote cell propagation on the contrary, but the rate of increase of cell are substantially below 10% to Ana-1.
(impacts of the CPE1 to Ana-1 survival rates, n=6 as shown in Figure 3；Compared with matched group, * p<0.05, * * p<0.01) by the highest of CPE1 participate in the experiment concentration bring up to after 1000 μ g/mL find, though CPE1 does not make significant difference to the growth of Ana-1 cells in low concentration, but when its concentration of participating in the experiment is higher than 600 μ g/mL, the survival rate of macrophage can be made to improve significantly to 120% of matched group or so.The studies above result shows that CPE is conducive to the propagation of macrophage on the contrary without obvious cytotoxicity.
B.Ana-1 cytophagies are tested
Concrete operations are as follows：After Ana-1 cells are through the sample treatment certain time such as CPE, add 20 μ L of freshly prepared 0.1%NR suspensions per hole, after gently mixing, continue culture 3h.Careful suction abandons the culture fluid containing NR afterwards, softly washes away the NR not swallowed with physiology PBS.Add 150 μ L cell pyrolysis liquid (dehydrated alcohol per hole:Acetic acid=1:1 (v/v)), 2h is stood, dissolves cell, concussion determines OD values at 540nm after mixing.Cell is to the same above-mentioned formula of the phagocytic rate calculation of NR.
Interpretation of result：(impacts of the Ana-1 to the cell phagocytic rate of NR, n=6 as shown in Figure 3；Compared with matched group, * p<0.05, * * p<0.01) under CPE1 effects, cell is significantly increased to the phagocytic activity of NR.When CPE1 concentration is 200 μ g/mL, Ana-1 increases to 156.00% (p of matched group to the cell phagocytic rate of NR<0.05)；When CPE1 concentration brings up to 600 μ g/mL, cell phagocytic rate can reach 211.72% (Fig. 3, p of matched group<0.01).With the cell survival rate under the same terms incremental compared with, the cell phagocytic rate increasing degree of Ana-1 is apparently higher than the former.
3) redox metal ion chelating test
The Fe of a.CPE2+Sequestering power is tested
The FeSO of 10 μ L 5mmol/L is added in the HAc-NaAc buffer of 200 μ L pH 4.94(final concentration of 1.25mmol/L).With 20 μ L concentration as 2.5,5.0, and the EDTA solution of 7.5mmol/L is used as positive control, remaining 20 μ L of CPE samples for variable concentrations.The interior distilled water of each reaction tube is supplied, and makes each system volume reach 240 μ L.Room temperature reaction 30min.FZ (the luxuriant and rich with fragrance Lip river piperazine) solution of 20 μ L 50mmol/L after reaction terminates, is added, develop the color 30min, detects its OD value at 535nm or 612nm, sample is calculated to Fe according to following formula2+Chelation percent：
Interpretation of result：(CPE1 is to Fe as shown in Figure 42+Chelation), metal ion-chelant positive control EDTA, can be in concentration dependent ground chelating systems in Fe2+；When its concentration reaches 7.5mmol/L, EDTA is to Fe2+Chelation percent reach 99.72%.But CPE1 is to Fe2+Substantially without chelation.
The Cu of b.CPE2+Sequestering power is tested
The CuSO of 10 μ L 5mmol/L is added in the HAc-NaAc buffer of 200 μ L pH 6.04(final concentration of 1.25mmol/L).With 20 μ L concentration as 2.5,5.0, and the EDTA solution of 7.5mmol/L is used as positive control, remaining 20 μ L of CPE samples for variable concentrations.The interior distilled water of each reaction tube is supplied, and makes each system volume reach 240 μ L.Room temperature reaction 30min.PV (pyrocatechol violet) solution of 5 μ L 20mmol/L after reaction terminates, is added, develop the color 30min, detects its OD value at 535nm or 612nm, sample is calculated to Cu according to following formula2+Chelation percent：
Interpretation of result：(CPE1 is to Cu as shown in Figure 52+Chelation), positive control EDTA in 5mmol/L, to Cu2+Sequestering power reach 83.80%, and CPE1 is to Cu2+Also chelation, and the increase with CPE1 concentration, chelation strengthen.
4) advanced glycation end products (AGEs) aptitude tests are suppressed
By the glucose of the bovine serum albumin (BSA) and 0.5mol/L of 10mg/mL be dissolved in 0.02mol/L phosphate buffer (PBS, pH 7.4) in, become the vitro reactions system of protein non-enzyme glycosylation.CPE is added in nonenzymatic glycosylation incubation system so as to final concentration of 10,100, and 1000 μ g/mL, while with the 10mg/mL BSA that are individually incubated in PBS as blank.Microporous filter membrane sucking filtration sterilizing of all samples through 0.22 μm of diameter, after 37 DEG C of lucifuges incubations 1,3,7,14,28,42d, will incubation product dilution to 1mg/mL, detection incubation fluorescent value of the product at 370/440nm and 335/385nm respectively.With the fluorescent value of 1mg/mL BSA as a flat fluorescent, according to the fluorescence intensity (FI) that following formula calculate the lower incubation product of CPE effects.
Interpretation of result：335/385nm and 370/440nm are the distinctive fluorescence excitation of the pentosidine of one of AGEs components and total AGEs and launch wavelength respectively.By the change of FI at periodic detection 335/385nm and 370/440nm, impact of different phases of the CPE in Maillard reaction to AGEs and pentosidine levels is studied.Maillard reaction has persistently been carried out 4 weeks altogether, the dynamic effect that CPE is formed to AGEs (impact that CPE1 is formed to total AGEs and penosidine, n=4 as shown in Figure 6；A is the impact that CPE1 is formed to total AGEs, and B is the impact that CPE1 is formed to AGEs precursors penosidine；Compared with AGEs groups, #p<0.05, ##p<0.01), early stages of two kinds of CPE in Maillard reaction, especially the 1st day and the 3rd day, are respectively provided with facilitation, and the concentration of thing of participating in the experiment are higher to the formation of total AGEs and penosidine, the bigger (p of its facilitation<0.05)；But the continuous prolongation with incubation time, especially from the beginning of the 3rd week, CPE fades away to the facilitation that AGEs is formed, and suppresses the formation of total AGEs, and the increase of penosidine levels is also eased.
The above results are pointed out, and CPE has certain potentiality in terms of preventing and treating chronic complicating diseases of diabetes.
4 kinds of different preparation methoies of functional components of chlorella vulgaris CPE are following example illustrated, and is tested using above-mentioned screening system to extracting product, be described as follows：
Embodiment 3
Based on the method that high pressure breaking cellular wall method prepares functional components of chlorella vulgaris CPE
10g Chlorella pyrenoidesa algae powder is taken, even suspension is in 100mL distilled water.It is placed in pressure cooker, in 0.15MPa, at 120 DEG C, heats 40min.Sample is taken out, is cooled down at room temperature.10000rpm is centrifuged 30min, after supernatant is through the sucking filtration that reduces pressure, saves backup in 4 DEG C.Algae mud after centrifugation, adds 100mL distilled water resuspended, is placed in pressure cooker, in 0.15MPa, heats 40min, extract at 120 DEG C again.Sample is taken out, is cooled down at room temperature.10000rpm is centrifuged 30min, takes supernatant, and reduce pressure sucking filtration.Merge filtrate, lyophilization twice, obtain CPE extract powders, be labeled as CPE-a.
Embodiment 4
Based on the method that high pressure breaking cellular wall method prepares chlorella CPE
10g Chlorella pyrenoidesa algae powder is taken, even suspension is in 100mL distilled water.It is placed in pressure cooker, in 0.05MPa, at 110 DEG C, heats 60min.Sample is taken out, is cooled down at room temperature.10000rpm is centrifuged 30min, after supernatant is through the sucking filtration that reduces pressure, saves backup in 4 DEG C.Algae mud after centrifugation, adds 100mL distilled water resuspended, is placed in pressure cooker, in 0.05MPa, heats 60min, extract at 110 DEG C again.Sample is taken out, is cooled down at room temperature.10000rpm is centrifuged 30min, takes supernatant, and reduce pressure sucking filtration.Merge filtrate, lyophilization twice, obtain CPE extract powders.
Embodiment 5
Based on the method that high pressure breaking cellular wall method prepares chlorella CPE
10g Chlorella pyrenoidesa algae powder is taken, even suspension is in 100mL distilled water.It is placed in pressure cooker, in 0.25MPa, at 130 DEG C, heats 30min.Sample is taken out, is cooled down at room temperature.10000rpm is centrifuged 30min, after supernatant is through the sucking filtration that reduces pressure, saves backup in 4 DEG C.Algae mud after centrifugation, adds 100mL distilled water resuspended, is placed in pressure cooker, in 0.25MPa, heats 30min, extract at 130 DEG C again.Sample is taken out, is cooled down at room temperature.10000rpm is centrifuged 30min, takes supernatant, and reduce pressure sucking filtration.Merge filtrate, lyophilization twice, obtain CPE extract powders.
Comparative example 1
Ultrasonication method is extracted
10g Chlorella pyrenoidesa powder even suspensions are in 100mL distilled water.After ultrasonication 30min, 30min is extracted in 95 DEG C of water-baths.After liquid cooling to be extracted, suspension is centrifuged 30min in 10000rpm, supernatant is saved backup by 4 DEG C after 4 times of volume concentrations.Algae mud is resuspended in equivalent distilled water, and supersound extraction and centrifugation again.Merge supernatant twice, by 4 times of volume concentrations, and lyophilization, CPE crude extracts are obtained, CPE-b is labeled as.
Comparative example 2
Ultrasonic enzymatic isolation method is extracted
10g Chlorella pyrenoidesa powder even suspensions are in 100mL distilled water.After ultrasonication 30min, 30min is extracted in 95 DEG C of water-baths.After liquid cooling to be extracted, cellulase (80mg/g algae powder), pectase (10mg/g algae powder) is added.50 DEG C of water-bath 2h, degrade to polysaccharide.Boiling water bath 30min makes enzyme-deactivating, 10000rpm be centrifuged 30min.Supernatant is collected, by 4 times of volume concentrations, and lyophilization, CPE ultrasound enzymatic hydrolysate is obtained, CPE-c is labeled as.
Comparative example 3
Enzymatic isolation method is extracted
10g Chlorella pyrenoidesa algae powder even suspensions are in 100mL distilled water.Algae powder suspension is placed in immersion 10h in 70 DEG C of water-baths.In chlorella powder suspension after immersion, in 80mg/g algaes powder and the ratio of 10mg/g algae powder, pectase and cellulase is added, in 50 DEG C of water enzyme digestion 12h, form breaking cellular wall algae mud.After pectase and cellulase degradation terminate, E.C. 3.4.21.64 and ribonuclease A (RNase A), 50 DEG C of water enzyme digestion 2h are added with the concentration of 100mg/L and 30mg/L.After the enzymolysis such as RNase terminates, chlorella enzymatic hydrolysate is in 100 DEG C of water-bath enzyme denaturing work 30min.10000rpm is centrifuged 30min, takes supernatant.Supernatant is obtained CPE extract powders, is labeled as CPE-d by lyophilization after 4 times of volume concentrations.
Extract evaluation test：
Extraction product by the various embodiments described above：High pressure breaking cellular wall method (product is CPE-a), ultrasonication method (product is CPE-b), ultrasonication-enzymatic isolation method (product is CPE-c), and enzymatic isolation method (product is CPE-d), carry out yield, hot water extract desired value (OD respectively260), protein and sugared content, and the evaluation test such as biological activity.
1st, yield and component analyses
As can be seen from Figure 1 (CPE-e is the CPE extracts that classical warm method is obtained), in the present invention, significantly lower than other three kinds of products, the yield of CPE-b shows that the efficiency that ultrasonication and conventional hot water extract is very low.After ultrasonication obtains digesting breaking cellular wall auxiliary, efficiency of pcr product is significantly improved, from the 279.25mg/g algae powder that the 148.36mg/g algae powder of CPE-b rises very rapidly up to CPE-c.The yield of tri- kinds of products of CPE-a, CPE-c, and CPE-d is higher, and respectively 251.67,279.25, and 258.85mg/g algae powder (Fig. 7).
The yield of CPE-a is high, is the product of 251.67mg/g algae powder.The above results show, in high pressure extraction method, proper extension extraction time, increase extraction time contribute to the lifting of extraction efficiency.
As enzyme itself is expensive, thoroughly removal is difficult to after reaction, and enzyme digestion reaction operating procedure is complicated, time-consuming long, with enzyme solution preparation CPE and inadvisable.
And high pressure breaking cellular wall method only needs water and common device autoclave, breaking cellular wall and extraction synchronously to carry out, operating procedure is few, and required time also significantly shortens compared with enzymatic isolation method, with many advantages such as inexpensive, pollution-free and fast and convenient.Therefore, consider that high pressure breaking cellular wall method is to prepare CPE high efficiency methods from efficiency of pcr product.
2nd, protein and sugar content analysis
By uv absorption analysis being carried out to four kinds of products, as a result show that four kinds of products are respectively provided with rich in protein and nucleic acid, wherein CPE-a is the most abundant material of four kinds of extract amplifying nucleic acids and protein content.
By phend-sulphuric acid, later stage detects that total sugar content, DNS methods detect that content of reducing sugar and Coomassie Brilliant Blue detect protein content, as a result as shown in table 1.
The protein of 1 CPE products of table and sugared content analysis (n=3)
Note：OD1200, OD5000 are commercially available " CGF " commodity
Compared with OD1200, * * p<0.01；Compared with OD5000, ##p<0.01；
Compared with CPE-a ,+p<0.05, ++ p<0.01.N is not detected
Similar to uv absorption comparative result, the protein content highest of CPE-a in four kinds of products.Using Coomassie Brilliant Blue, the protein content of CPE-a is all remarkably higher than OD1200 (p<0.01), and the protein content of CPE-c and CPE-d is substantially less than OD5000.
Sugared content testing result shows that the total sugar content of four kinds of CPE compares (p less than two kinds " CGF "<0.01), but wherein CPE-c and CPE-d abundant compared with the CPE-a (p of sugared content<0.05).Additionally, except CPE-c, can't detect the presence of reducing sugar in remaining product.
3rd, activity analysiss
1) the OH Scavenging activities of four kinds of CPE
Method of testing：Above-mentioned 4 kinds of samples that concentration is 0,100,200,500,1000 μ g/mL are taken, adds Griess reagents, terminating reaction after 37 DEG C of accurate response 1min with 630nm as reference wavelength, to determine OD value of the reactant at 550nm.The radical scavenging activity of sample, is calculated according to formula, the percentage clearance rate of OH is represented with sample.
Four kinds of CPE are analyzed from Fig. 2 (n=6 is compared to the clearance rate of OH；Compared with blank, * p<0.05, * * p<0.01；Compared with CPE-a ,+p<0.05, ++ p<0.01), above-mentioned four kinds of CPE are respectively provided with the ability for removing OH, and the raising with concentration of participating in the experiment, and clearance rate is gradually increasing.The clearance rate of four kinds of CPE is compared in same concentration of participating in the experiment, it is found that CPE-a is optimal to the elimination effect of OH, in the range of 40-400 μ g/mL, the OH clearance rate of its excess-three kind CPE is substantially less than CPE-a (p<0.05)；When concentration brings up to 1000 μ g/mL, CPE-a can reach 74.31% to the clearance rate of OH.
2) impact of four kinds of CPE to macrophage survival rate
Impact (n=6s of four kinds of CPE to macrophage Ana-1 survival rates is analyzed from Fig. 3；Compared with matched group, * p<0.05, * * p<0.01；Compared with CPE-a ,+p<0.05, ++ p<0.01), four kinds of CPE can remarkably promote macrophage Ana-1 propagation.In the concentration range of 25-200 μ g/mL, four kinds of CPE can promote Ana-1 cells to breed in the way of concentration dependant, and wherein optimal with the effect of CPE-a, in 100 μ g/mL, CPE-a is significantly higher than other three kinds of CPE (p to the rush rate of increase of Ana-1<0.05)；During 200 μ g/mL, CPE-a makes the survival rate of Ana-1 bring up to 162.98%, and the proliferation pole of other three kinds of CPE is substantially less than CPE-a (p<0.01).
3) metal ion chelating capacity of four kinds of CPE
Method of testing：In 200 μ L pH, 4.9 (Fe2+Chelating test) or 6.0 (Cu of pH2+Chelating test) HAc-NaAc buffer in each respective metal solion (final concentration of 1.25mmol/L) for adding 10 μ L 5mmol/L.With 20 μ L concentration as 2.5,5.0, and the EDTA solution of 7.5mmol/L is used as positive control, remaining 20 μ L of CPE samples for variable concentrations.The interior distilled water of each reaction tube is supplied, and makes each system volume reach 240 μ L.Room temperature reaction 30min.After reaction terminates, FZ (the luxuriant and rich with fragrance Lip river piperazine) solution (Fe of 20 μ L 50mmol/L is separately added into2+Chelating test) or 5 μ L 20mM PV (pyrocatechol violet) solution (Cu2+Chelating test) colour developing 30min, detects its OD value at 535nm or 612nm, calculates sample to Cu according to above-mentioned formula2+And Fe2+Chelation percent.
Four kinds of CPE crude extracts are analyzed to Fe from Fig. 42+And Cu2+Chelation percent (n=3；Compared with matched group, * p<0.05, * * p<0.01；Compared with CPE-a ,+p<0.05), when concentration is less than 1mg/mL, four kinds of CPE are to Fe2+Chelation is there is no, but when concentration reaches 10mg/mL, four kinds of CPE show certain Fe2+Sequestering power (p<0.01).
Four kinds of CPE are to Cu2+There is certain chelation, and as CPE participates in the experiment the raising of concentration, chelation percent also accordingly increases.In 1mg/mL, each group is to Cu2+Chelation percent be below 10% (p<0.05) without significant difference, and between each group；When concentration reaches 10mg/mL, four kinds of CPE are to Cu2+Sequestering power accordingly improve.
4) impact that four kinds of CPE are formed to AGEs
Method of testing：By the glucose of the bovine serum albumin (BSA) and 0.5mol/L of 10mg/mL be dissolved in 0.02mol/L phosphate buffer (PBS, pH 7.4) in, become the vitro reactions system of protein non-enzyme glycosylation.CPE is added in nonenzymatic glycosylation incubation system so as to final concentration of 10,100, and 1000 μ g/mL, while with the 10mg/mL BSA that are individually incubated in PBS as blank.Microporous filter membrane sucking filtration sterilizing of all samples through 0.22 μm of diameter, after 37 DEG C of lucifuges incubations 1,3,7,14,28,42d, will incubation product dilution to 1mg/mL, detection incubation fluorescent value of the product at 370/440nm and 335/385nm respectively.With the fluorescent value of 1mg/mL BSA as a flat fluorescent, according to the fluorescence intensity (FI) that above-mentioned formula calculates the lower incubation product of CPE effects.
Using the screening system and screening technique of above-mentioned functional components of chlorella vulgaris CPE, screening and assessment is carried out to the functional components of chlorella vulgaris CPE that 4 kinds of different preparation methoies are obtained, as a result find that the functional components of chlorella vulgaris CPE overall merits prepared by high pressure breaking cellular wall method are best, and the preparation method is simple, low cost, is especially suitable for industrialized production.
Ultimate principle, principal character and the advantages of the present invention of the present invention has been shown and described above.It should be understood by those skilled in the art that; the present invention is not restricted to the described embodiments; merely illustrating the principles of the invention described in above-described embodiment and description; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements are both fallen within scope of the claimed invention.The claimed scope of the invention is defined by appending claims and its equivalent.
Claims (8)
1. a kind of high throughput screening system of functional components of chlorella vulgaris CPE, including：
1) antioxidation test：
Tested using Griess reagents, by the total antioxidant capacity of spectrophotometry sample and to the clear of free radical
Except effect；
2) Culture in vitro test：
A. impact of the four tetrazolium bromide mtt assay detected components to mouse macrophage Ana-1 survival rates；
B. impact of the dimethyl diaminophenazine chloride NR staining detected components to Ana-1 phagocytic functions；
C. the change of fluorescence probe method detection sample NO secretions intracellular to Ana-1；
3) redox metal ion chelating test：
A. using luxuriant and rich with fragrance Lip river piperazine as Fe2+Chelating reagent, by the Fe of spectrophotometry sample2+Sequestering power；
B. using pyrocatechol violet as Cu2+Chelating reagent, by the Cu of spectrophotometry sample2+Sequestering power；
4) advanced glycation end products AGEs tests are suppressed：
Non-enzymatic glycation system：The glucose of the bovine serum albumin BSA and 0.5mol/L of 10mg/mL is dissolved
In the phosphate buffer PBS of 0.02mol/L, pH 7.4, non-enzymatic glycation system is obtained；By fluorescent spectrometry,
Analyze impact of different phase of the sample in Maillard reaction to AGEs and pentosidine.
2. a kind of high-throughput screening method of functional components of chlorella vulgaris CPE, including：
1) antioxidation test：Concentration is taken for 0,100,200,500,1000 μ g/mL samples, addition Griess reagents, 37 DEG C
Terminating reaction after reaction 1min, with 630nm as reference wavelength, determines OD value of the reactant at 550nm, according to
Equation below calculates the radical scavenging activity of sample, the percentage clearance rate of OH is represented with sample；
2) Culture in vitro test：
A. impact of the four tetrazolium bromide mtt assay detected components to mouse macrophage Ana-1 survival rates；
Concrete operations are as follows：Take the logarithm the cell of trophophase, after trypsinization, with the RPMI-1640 containing 10% hyclone
Culture medium makes cell suspension, with 2 × 104The even density in individual/hole is added in 96 porocyte culture plates, put 37 DEG C,
Saturated humidity, contain 5%CO2Cell culture incubator in quiescent culture.Treat cell attachment and grow to suitable density (about 24h)
Afterwards, the medicines such as the CPE of variable concentrations are added per hole.After continuing culture certain time, 20 μ L 0.5mg/mL are added per hole
MTT solution, 37 DEG C incubation 4h.After the completion of incubation, 100 μ L, tri- liquid (10%SDS, 5% isobutyl is added per hole
Alcohol, 12mmol/L HCl), 37 DEG C of overnight incubations react formed purple formazan to dissolve cell with MTT
Crystallization.Then concussion mixes 5min, with 630nm wavelength as reference, detects OD value of each hole at 492nm wavelength,
And cell survival rate is calculated according to formula.
B. impact of the dimethyl diaminophenazine chloride NR staining detected components to Ana-1 phagocytic functions；
Concrete operations are as follows：After Ana-1 cells are through the sample treatment certain time such as CPE, add freshly prepared 0.1%NR suspended per hole
20 μ L of liquid, continue culture 3h after gently mixing.Careful suction abandons the culture fluid containing NR afterwards, is softly washed away with physiology PBS
The NR not swallowed.Add 150 μ L cell pyrolysis liquid (dehydrated alcohol per hole:Acetic acid=1:1 (v/v)), 2h is stood, is made
Cell dissolves, and concussion determines OD values at 540nm after mixing.Cell is to the same above-mentioned formula of the phagocytic rate calculation of NR.
C. the change of fluorescence probe method detection sample NO secretions intracellular to Ana-1；
Concrete operations are as follows：Fluorescent probe DAF-FM DA are diluted to 5 μm of ol/L of final concentration with DAF-FM DA diluents.
The cell of the amount of taking fully exponential phase, digests and dispels, and washed once with the physiology PBS of pH 7.4 after being collected by centrifugation.With
The DAF-FM DA working solution re-suspended cells for having diluted, adjustment cell concentration to 1 × 106Individual/mL, 37 DEG C of incubations
20min.Period is mixed reverse for mixture every 3-5min, is fully contacted probe and cell.PBS washed cells three
Secondary, intracellular DAF-FM DA are introduced into abundant removal.The cell for having loaded uniformly is spread into 96 hole black cells
In culture plate, and add the CPE samples of variable concentrations.After 37 DEG C of temperature bath certain times, detect each hole in 495/515nm
Fluorescent value under excitation/emission wavelength, and calculate the change of the lower Ana-1 cell NO levels of each sample effect.
3) redox metal ion chelating test：
a.Fe2+Chelating test
The respective metal solion of 5mmol/L is added in the HAc-NaAc buffer of pH 4.9 so as to final concentration of
1.25mmol/L；
With concentration as 2.5,5.0, and the EDTA solution of 7.5mmol/L, used as positive control, remaining is variable concentrations
CPE samples；
The interior distilled water of each reaction tube is supplied, and makes each system volume reach 240 μ L, room temperature reaction 30min；
The luxuriant and rich with fragrance Lip river piperazine solution colour developing 30min of 50mmol/L after reaction terminates, is added, detects which in 535nm or 612nm
The OD values at place, calculate sample to Fe according to following formula2+Chelation percent；
b.Cu2+Chelating test
The respective metal solion of 5mmol/L is added in the HAc-NaAc buffer of pH 6.0 so as to final concentration of
1.25mmol/L；
With concentration as 2.5,5.0, and the EDTA solution of 7.5mmol/L, used as positive control, remaining is variable concentrations
CPE samples；
The interior distilled water of each reaction tube is supplied, and makes each system volume reach 240 μ L, room temperature reaction 30min；
After reaction terminates, add the pyrocatechol violet solution colour developing 30min of 20mmol/L, detect which in 535nm or
OD values at 612nm, calculate sample to Cu according to following formula2+Chelation percent；
4) advanced glycation end products AGEs tests are suppressed：
The glucose of the bovine serum albumin BSA and 0.5mol/L of 10mg/mL is dissolved in 0.02mol/L, pH 7.4
Phosphate buffer PBS in, non-enzymatic glycation system；
Sample is added in above-mentioned reaction system and is incubated so as to which final concentration is respectively 10,100, and 1000 μ g/mL, with
When with the 10mg/mL BSA that are individually incubated in PBS as blank；
Microporous filter membrane sucking filtration sterilizing of all samples through 0.22 μm of diameter, 37 DEG C of lucifuges incubations 1,3,7,14,28,
After 42d, product dilution will be incubated to 1mg/mL, detection incubation product is at 370/440nm and 335/385nm respectively
Fluorescent value；
With the fluorescent value of 1mg/mL BSA as a flat fluorescent, the lower incubation product of CPE effects is calculated according to following formula
Fluorescence intensity FI；
3. a kind of preparation method of functional components of chlorella vulgaris CPE, including：
1) Chlorella pyrenoidesa algae powder is added in distilled water, at pressure 0.05-0.25MPa, temperature 110-130 DEG C, plus
Hot 30-60min, is extracted；
2) sample is taken out, after cooling down under room temperature, 10000rpm is centrifuged 30min, after supernatant is through the sucking filtration that reduces pressure, 4 DEG C of guarantors
Deposit standby；
3) by step 2) centrifugation after algae mud, add resuspended with 1) equal-volume distilled water, in pressure 0.1MPa, temperature
At 110-130 DEG C, 30-60min is heated, is extracted again；
4) sample is taken out, after cooling down under room temperature, 10000rpm is centrifuged 30min, takes supernatant, reduce pressure sucking filtration, merges two
Secondary filtrate, lyophilization are obtained chlorella CPE extract powders.
4. a kind of preparation method of functional components of chlorella vulgaris CPE according to claim 3, it is characterised in that：The step 1)
It is 0.05-0.25MPa with the pressure in 3).
5. a kind of preparation method of functional components of chlorella vulgaris CPE according to claim 3, it is characterised in that：The step 1)
It it is 110-130 DEG C with the temperature in 3).
6. a kind of preparation method of functional components of chlorella vulgaris CPE according to claim 3, it is characterised in that：The step 1)
It is 30-60min with the heat time heating time in 3).
7. a kind of preparation method of functional components of chlorella vulgaris CPE according to claim 3, it is characterised in that：The step 1)
In Chlorella pyrenoidesa algae powder even suspension in distilled water, Chlorella pyrenoidesa algae powder with the proportioning of distilled water is
1g:10ml.
8. a kind of preparation method of functional components of chlorella vulgaris CPE according to claim 3, it is characterised in that：Described extraction
It is to be extracted with hot high pressure water law.
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| CN107125288A (en) * | 2017-05-18 | 2017-09-05 | 湖北工业大学 | The method that Cyanidin suppresses AGEs in chiffon cake |
| EP3403662A1 (en) * | 2017-05-02 | 2018-11-21 | National Institute of Ocean Technology | Pharmacoactive nutrient and a process of production thereof from marine algae |
| CN111718856A (en) * | 2020-06-29 | 2020-09-29 | 重庆工商大学 | Method for preparing biodiesel by screening large amount of oil-containing microalgae of cyanophyta |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP3403662A1 (en) * | 2017-05-02 | 2018-11-21 | National Institute of Ocean Technology | Pharmacoactive nutrient and a process of production thereof from marine algae |
| CN107125288A (en) * | 2017-05-18 | 2017-09-05 | 湖北工业大学 | The method that Cyanidin suppresses AGEs in chiffon cake |
| CN111718856A (en) * | 2020-06-29 | 2020-09-29 | 重庆工商大学 | Method for preparing biodiesel by screening large amount of oil-containing microalgae of cyanophyta |
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