Method for biological wall breaking of haematococcus pluvialis

Abstract

The invention discloses a method for biological wall breaking of haematococcus pluvialis, which comprises the following steps: co-culturing Trametes trogii S0301 and haematococcus pluvialis, cracking the haematococcus pluvialis by the Trametes trogii S0301, promoting the release of astaxanthin in the haematococcus pluvialis, and realizing the extraction of the astaxanthin in the haematococcus pluvialis; the method is easy to operate, low in cost and safe, and compared with a grinding method, the method hardly affects the antioxidant activity of astaxanthin, and the method is suitable for industrial production and market popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial treatment, and in particular relates to a method for breaking cell walls of Haematococcus pluvialis through microbial treatment to promote the extraction of astaxanthin from Haematococcus pluvialis. Background Art

[0002] Haematococcus pluvialis is the species with the highest known accumulation of astaxanthin. Under adverse conditions, the accumulation of astaxanthin can reach more than 4% of the cell dry weight, and it is widely used in the production of astaxanthin. While accumulating astaxanthin, Haematococcus pluvialis forms a thick and dense three-layer cell wall structure (~2.2μm), which is mainly composed of algin biopolymers and cellulose polysaccharides; this cell wall structure protects the Haematococcus pluvialis cells, but also hinders the subsequent extraction of astaxanthin.

[0003] At present, methods such as bead milling, high pressure homogenization (HPH), ultrasonic treatment, microwave radiation, pulsed electric field (PEF) application, acid treatment, ionic liquid (IL) treatment and enzyme lysis have been used to break the cell wall of Haematococcus pluvialis to assist in the extraction of astaxanthin. However, the existing processing technology has problems such as high facility costs and the impact of the processing process on the activity, safety and stability of astaxanthin. Therefore, it is still necessary to develop efficient, safe and low-cost extraction technology to meet market demand and promote its widespread application. Summary of the invention

[0004] The present invention provides a method for breaking the wall of Haematococcus pluvialis organisms, wherein the method breaks the wall of the thermotolerant Trametes Trametes trogii ) S0301 is co-cultured with Haematococcus pluvialis, and the heat-resistant Trichoderma S0301 lyses the Haematococcus pluvialis, promotes the release of astaxanthin in the Haematococcus pluvialis, and realizes the extraction of astaxanthin from the Haematococcus pluvialis. The method of the invention solves the problems of high cost and safety in the development and utilization of Haematococcus pluvialis.

[0005] The method of the present invention specifically comprises the following steps: culturing the thermotolerant tricholoma S0301 in a solid culture medium for 3 days, laying Haematococcus pluvialis on the thermotolerant tricholoma S0301, culturing at 28°C in the dark for 2 days, collecting Haematococcus pluvialis and the thermotolerant tricholoma S0301 cells, extracting with anhydrous ethanol, and obtaining astaxanthin; wherein the solid culture medium comprises 4-6 g malt extract powder, 18-22 g algae powder shell, Mg 2 SO 4 •7H 2 O 1-2g, agar 20g, water 1000mL.

[0006] The thermotolerant trichomoniasis fungus ( Trametes trogii) S0301 has been disclosed in non-patent literature before the filing date of this invention, and has been disclosed in "Gao Sheng. Identification of homokaryons of thermotolerant Trametes S0301 and preparation of polyclonal antibodies against heat shock transcription factor HSF2 [D]. Kunming University of Science and Technology, 2018." The applicant guarantees that the biological material will be released to the public within twenty years from the filing date.

[0007] Compared with the prior art, the present invention has the following advantages: The thermotolerant Trametes S0301 is co-cultured with Haematococcus pluvialis, and the thermotolerant Trametes S0301 breaks the wall of Haematococcus pluvialis, thereby achieving the purpose of extracting astaxanthin from Haematococcus pluvialis; using thermotolerant Trametes S0301 to extract astaxanthin from Haematococcus pluvialis is simple to operate, low in cost, and safe, and is suitable for industrial production. In large-scale production, this method shows higher economic benefits than traditional extraction methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 is the standard curve of astaxanthin; Figure 2 The results of the effects of culturing S0301 with different C sources on the algae powder wall breaking; Figure 3 The results of the effects of culturing S0301 with different N sources on the algae powder wall breaking; Figure 4 Figure 3 is a cell morphology diagram of Haematococcus pluvialis under an optical microscope, wherein Figure A shows the cells of Haematococcus pluvialis before control ethanol extraction, Figure B shows the cells of Haematococcus pluvialis after control ethanol extraction, Figure C shows the cells of the experimental group co-cultured with S0301 and Haematococcus pluvialis before ethanol extraction of astaxanthin, and Figure D shows the cells of the experimental group co-cultured with S0301 and Haematococcus pluvialis after ethanol extraction of astaxanthin; Figure 5 The cell morphology of Haematococcus pluvialis under a scanning electron microscope. The left picture shows the cells of the control group before ethanol extraction, and the right picture shows the cells of the experimental group co-cultured with S0301 and Haematococcus pluvialis before ethanol extraction. Figure 6 The results of the scavenging activity of the thermotolerant Trichoderma S0301 biologically broken extract on ABTS free radicals, where MCK is the grinding extract, TR is the biologically broken extract, and VC is ascorbic acid; Figure 7 The scavenging activity of the thermotolerant Trichoderma S0301 biological wall-broken extract on DPPH free radicals, wherein MCK is the grinding extract, TR is the biological wall-broken extract, and VC is ascorbic acid. DETAILED DESCRIPTION

[0009] The present invention is further explained by examples below, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified; GYP medium contains 20 g glucose, 5 g peptone, 5 g yeast extract, Mg 2 SO 4 •7H 2 O 1g, agar 20g, water 1000mL; Standard curve for astaxanthin determination by spectrophotometer: accurately weigh 1 mg of free astaxanthin standard, add 20 mL of anhydrous ethanol, dilute to 20 mL, and prepare a mother solution with a concentration of 50 µg / mL. After serial dilution, a series of standard solutions with concentrations of 2 µg / mL, 4 µg / mL, 6 µg / mL, 8 µg / mL, 10 µg / mL, and 12 µg / mL were obtained. With mass concentration as the abscissa and absorbance as the ordinate, draw the astaxanthin standard curve, see Figure 1 , the linear equation is y=0.1143x+0.0075; The astaxanthin in the sample to be tested was obtained by substituting the obtained absorbance value into the above equation to obtain the astaxanthin concentration; At the same time, the astaxanthin concentration obtained by anhydrous ethanol extraction of completely ground Haematococcus pluvialis of the same mass is the complete astaxanthin content in the treated sample, and the extraction rate of the treated sample is calculated by the following formula: Astaxanthin relative extraction rate (%) = (astaxanthin concentration in the treated sample extract × the volume of the solvent used for extraction) / (astaxanthin concentration in the ground sample extract × the volume of the solvent used for extraction); Example 1: Method for breaking the cell wall of Haematococcus pluvialis on GYP medium After the thermotolerant Trichoderma S0301 was activated, it was inoculated on the GYP medium and cultured at 28°C for 3 days. Then, 0.15 g of Haematococcus pluvialis algae powder was spread on the thermotolerant Trichoderma and cultured at 28°C in the dark for 2 days. The mixture of algae powder and bacteria was scraped and placed in a 50 mL centrifuge tube. 10 mL of anhydrous ethanol was added to extract in the dark for 1 hour. The mixture was centrifuged at 8000 rpm for 5 minutes, the supernatant was aspirated, 10 mL of anhydrous ethanol was added to the precipitate to extract in the dark for 1 hour, and the precipitate was extracted again. The extraction was repeated once. The three extracts were collected and combined, and the mixture was mixed at OD 475 The absorbance value was measured at 40°C and substituted into the above linear equation to obtain the astaxanthin concentration; at the same time, a control without S0301 was set (Haematococcus pluvialis powder was directly extracted with anhydrous ethanol, PCK); At the same time, 0.15 g of Haematococcus pluvialis powder was taken and completely ground. It was extracted three times with anhydrous ethanol according to the above method. 475The absorbance value was measured and substituted into the above linear equation to obtain the astaxanthin concentration; the extraction rate of biological wall breaking was calculated according to the above extraction rate formula to be 34%; the extraction rate of Haematococcus pluvialis powder in the control group was 0. The results showed that the heat-resistant Trichoderma S0301 could lyse Haematococcus pluvialis on GYP culture medium.

[0010] Example 2: Optimization of carbon source in culture medium for biological cell wall breaking The culture medium used in this example is the same as that in Example 1, except that sodium carboxymethyl cellulose, pectin, starch, xylan, algae powder shell, xylose, and glucose are used as carbon sources, respectively. After the thermotolerant Trachoma spp. S0301 is activated, it is inoculated on the GYP culture medium, and after culturing at 28°C for 3 days, 0.15 g of Haematococcus pluvialis algae powder is spread on the thermotolerant Trachoma spp., and cultured at 28°C in the dark for 2 days; the mixture of algae powder and bacteria is scraped and put into a 50 mL centrifuge tube, 10 mL of anhydrous ethanol is added to extract in the dark for 1 hour, centrifuged at 8000 rpm for 5 minutes, the supernatant is aspirated, 10 mL of anhydrous ethanol is added to the precipitate to extract in the dark for 1 hour, and the precipitate is extracted once more; the three extracts are collected and combined, mixed, and the mixture is stirred at OD 475 The absorbance value was measured at 40 °C, and the astaxanthin concentration was obtained by substituting it into the above linear equation to calculate the extraction rate. Meanwhile, the extraction of Haematococcus pluvialis directly with anhydrous ethanol was set as a control (PCK). The results are shown in Figure 2, from which it can be seen that when the carbon source is algae powder shell, the astaxanthin extraction rate is the highest.

[0011] Example 3: Optimization of nitrogen source in culture medium for biological cell wall breaking The culture medium used in this embodiment is the same as that in Example 1, except that peptone, ammonium nitrate, yeast extract powder, malt extract powder, corn flour, yeast extract powder + malt extract powder are used as nitrogen sources, and algae powder shell is used as carbon source. The thermotolerant Trichoderma S0301 is activated and inoculated on the GYP culture medium. After culturing at 28°C for 3 days, 0.15g of Haematococcus pluvialis algae powder is spread on the thermotolerant Trichoderma, and the culture is continued at 28°C in the dark for 2 days; the mixture of algae powder and bacteria is scraped and put into a 50mL centrifuge tube, 10mL of anhydrous ethanol is added to extract in the dark for 1h, centrifuged at 8000rpm for 5 minutes, the supernatant is aspirated, 10mL of anhydrous ethanol is added to the precipitate to extract in the dark for 1h, and the precipitate is extracted once more; the three extracts are collected and combined, mixed, and the mixture is stirred at OD 475 The absorbance value was measured at 40 °C, and the astaxanthin concentration was obtained by substituting it into the above linear equation to calculate the extraction rate. At the same time, the extraction of Haematococcus pluvialis directly with anhydrous ethanol was set as a control (PCK). The results are shown in Figure 3 ,It can be seen from the figure that when the nitrogen source is malt extract powder, the astaxanthin extraction rate is the highest.

[0012] Example 4: Optical microscope and scanning electron microscope observation The culture medium used in this example is 5g of malt extract powder, 20g of algae shell powder, Mg 2 SO 4 •7H 2 O 1g, agar 20g, water 1000mL; using the culture medium to culture the thermotolerant Trichoderma S0301 and lyse Haematococcus pluvialis, the method is the same as that in Example 1, and setting the Haematococcus pluvialis directly extracted with anhydrous ethanol as a control group (PCK); The morphology of Haematococcus pluvialis cells before and after astaxanthin extraction with anhydrous ethanol was observed under an optical microscope. Figure 4 From the figure, it can be seen that in the control group, there was no change in the Haematococcus pluvialis cells before and after ethanol extraction; after S0301 was co-cultured with Haematococcus pluvialis, the Haematococcus pluvialis cells were obviously broken, and after astaxanthin was extracted with anhydrous ethanol, a large number of empty shell cells appeared, indicating that S0301 can lyse Haematococcus pluvialis cells.

[0013] The cells of the experimental group co-cultured with S0301 and Haematococcus pluvialis before ethanol extraction and the cells of the control group before ethanol extraction were placed in a 55°C oven for 4 hours and then observed under a scanning electron microscope. The results are shown in Figure 5. Compared with the control group, the algal cells in the experimental group co-cultured with S0301 and Haematococcus pluvialis obviously had many cell fragments, indicating that S0301 can lyse Haematococcus pluvialis cells.

[0014] Example 5: Experiment on the effect of thermotolerant Trichoderma S0301 biological cell wall breaking on astaxanthin 1. Use culture medium (5g malt extract powder, 20g algae powder shell, Mg 2 SO 4 •7H 2 O 1g, agar 20g, water 1000mL) culture thermotolerant Trichoderma S0301 and lyse Haematococcus pluvialis, the method is the same as Example 1, to obtain biological wall-breaking extract (TR); At the same time, grinding was performed by grinding method and extraction with anhydrous ethanol, and the grinding extract (MCK) was obtained in the same manner as above; 2. Determination of ABTS free radical scavenging activity Accurately weigh 0.192g of ABTS free radical, dissolve it in methanol and dilute it to 50 mL to obtain 7mmol / L ABTS free radical solution; accurately weigh 0.033g of potassium persulfate, dissolve it in distilled water and dilute it to 50 mL to obtain 2.45mmol / L potassium persulfate solution; mix 7mmol / L ABTS free radical solution and 2.45mmol / L potassium persulfate solution in a brown volumetric flask at a volume ratio of 1:1 to obtain ABTS working solution, and store it in a refrigerator at 4°C; 180 µL of ABTS working solution was mixed with 20 µL of different concentrations of biological wall-broken extract, grinding extract, and ascorbic acid (4 µg / mL, 8 µg / mL, 16 µg / mL, 32 µg / mL, 64 µg / mL, 128 µg / mL) respectively; after being sealed and kept away from light at room temperature for 30 minutes, the absorbance was recorded at 734 nm on a spectrophotometer. All measurements were performed three times; the scavenging rate Q of the extract and ascorbic acid on ABTS was calculated using the following formula: ABTS free radical scavenging rate Q (%) = [A0-(A1-A2)] / A0×100% Wherein, A0: absorbance value of 180 μL ABTS and 20 μL anhydrous ethanol; A1: absorbance of 180 µL ABTS and 20 µL extract or ascorbic acid; A2: Absorbance of 180 µL of extract or ascorbic acid and 20 µL of absolute ethanol.

[0015] Calculation of IC values ​​of biological cell wall-broken extract, ground extract and ascorbic acid for ABTS free radical scavenging 50 (half-efficiency clearance concentration) were 16.00 µg / mL, 14.40 µg / mL, and 25.17 µg / mL ( Figure 6 ), the results showed that S0301 can lyse Haematococcus pluvialis cells and will not affect the quality of astaxanthin compared with the grinding method.

[0016] 3. DPPH free radical scavenging activity assay Accurately weigh 0.0012 g of DPPH free radical and dissolve it in anhydrous ethanol to 50 mL to obtain a 100 µmol / L DPPH working solution, which was then stored in a 4°C refrigerator away from light. 100 µL of DPPH solution was mixed with 100 µL of different concentrations of biological wall-broken extract, grinding extract, and ascorbic acid (4 µg / mL, 8 µg / mL, 16 µg / mL, 32 µg / mL, 64 µg / mL, 128 µg / mL) respectively; the absorbance was recorded at 517 nm on a spectrophotometer after being sealed and kept away from light at room temperature for 30 minutes. All measurements were performed three times; the scavenging rate K of the sample for DPPH was calculated using the following formula: DPPH free radical scavenging rate K (%) = [A0-(A1-A2)] / A0×100% Wherein, A0: absorbance of 100 µL DPPH and 100 µL anhydrous ethanol; A1: absorbance of 100 µL DPPH and 100 µL sample reaction solution; A2: absorbance of 100 µL extract or ascorbic acid + 100 µL anhydrous ethanol.

[0017] Calculation of IC values ​​of biological cell wall-broken extract, ground extract and ascorbic acid for scavenging DPPH free radicals 50 (half-efficiency clearance concentration) were 19.00 µg / mL, 15.00 µg / mL, 9.00 µg / mL ( Figure 7 ), the results showed that S0301 can lyse Haematococcus pluvialis cells and will not affect the quality of astaxanthin compared with the grinding method.

Claims

1. A method for breaking the wall of Haematococcus pluvialis, characterized in that: Thermotolerant Trametes Trametes trogii ) S0301 is co-cultured with Haematococcus pluvialis. The thermotolerant Trichoderma S0301 lyses the Haematococcus pluvialis, promotes the release of astaxanthin in the Haematococcus pluvialis, and realizes the extraction of astaxanthin from the Haematococcus pluvialis.

2. The method for biological cell wall breaking of Haematococcus pluvialis according to claim 1, characterized in that: After culturing the thermotolerant Trametes S0301 in a solid culture medium at 28°C for 3 days, Haematococcus pluvialis was spread on the thermotolerant Trametes, and after culturing at 28°C in the dark for 2 days, the Haematococcus pluvialis and the thermotolerant Trametes S0301 cells were collected and extracted with anhydrous ethanol to obtain astaxanthin; the solid culture medium formula was 4-6g malt extract powder, 18-22g algae powder shell, 1-2g Mg2SO4•7H2O, 20g agar, and 1000mL water.

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