Application of pseudoarthrobacter in preparation of astaxanthin

Abstract

The invention discloses new application of Pseudomonas sp. F13, namely application of the Pseudomonas sp. F13 in splitting haematococcus pluvialis to extract astaxanthin, the Pseudomonas sp. F13 and the haematococcus pluvialis are jointly fermented and cultured, the Pseudomonas sp. F13 can split the haematococcus pluvialis, so that the astaxanthin in the haematococcus pluvialis is fully released, and the extraction rate reaches 76%. The method is simple and easy to operate, low in cost and suitable for industrial production. Solvents and wall breaking equipment are not needed in extraction, so that the negative influence on the environment is reduced, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and biocatalysis, and specifically relates to application of a strain of Pseudoarthrobacter F13 in the preparation of astaxanthin. Background Art

[0002] Astaxanthin (3,3′-dihydroxy-4,4′-diketo-β,β′-carotene) is a red keto-carotenoid with a strong ability to scavenge oxygen free radicals. Its antioxidant capacity is 550 times that of vitamin E and 10 times that of β-carotene, and it is known as a "super antioxidant". In addition, astaxanthin also has anti-cancer, anti-radiation, and immune-enhancing effects, as well as good coloring properties. It has broad application potential in aquaculture, feed additives, food, medicine, cosmetics and other fields (J. Tissue. Eng, 2024, 15, 20417314241287487).

[0003] Haematococcus pluvialis Haematococcus pluvialis ) is one of the best sources of natural astaxanthin, and its extraction method has always been the focus of research. Common methods include acid-assisted extraction, mechanical crushing, chemical solvent extraction, and the application of genetic engineering and random mutation technologies (Bioresource. Technol, 2022, 343, 126124). Acid-assisted extraction is a commonly used extraction method. Acid treatment can destroy the cell wall of Haematococcus pluvialis, thereby effectively improving the extraction efficiency of astaxanthin. The mechanical crushing method is to use physical means (such as ultrasound, high-pressure homogenization, etc.) to destroy the cell wall to extract astaxanthin. However, these methods have high requirements for experimental conditions and equipment and are only suitable for laboratories and small-scale production. The chemical solvent extraction method uses organic solvents (such as ethanol, acetone, etc.) to dissolve astaxanthin in cells. This method is simple to operate, but there may be problems such as solvent residues and environmental pollution. The yield of astaxanthin in Haematococcus pluvialis can be increased through genetic engineering and random mutation. Due to its technical complexity and high cost, it has certain limitations in practical applications.

[0004] The cell wall of Haematococcus pluvialis has a high degree of physical and chemical resistance, which makes the extraction process of astaxanthin complicated and inefficient. Especially in large-scale production, the extraction efficiency of astaxanthin is still low. Since astaxanthin is unstable, factors such as light, temperature and oxidation during the extraction process will affect astaxanthin, thereby affecting the quality of the final product. Although there are many methods for the extraction of astaxanthin from Haematococcus pluvialis, they all have certain limitations and challenges, so there is still a need to develop efficient, safe and low-cost extraction technologies to meet market demand and promote its widespread application. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a strain of Pseudomonas aeruginosa ( Pseudarthrobacter sp.) F13, namely its application in extracting astaxanthin by lysing Haematococcus pluvialis, wherein Pseudomonas aeruginosa ( Pseudarthrobacter sp.) F13 was deposited in the China Center for Type Culture Collection on August 28, 2024, with the deposit number CCTCC NO: M 20241866, and the deposit address is Wuhan University, Wuhan, China.

[0006] The purpose of the present invention is achieved through the following technical solutions: 1. After the pseudoarthrobacter F13 stored on the slant was activated by inoculation on LB solid medium, it was inoculated into LB liquid medium, cultured at 37°C and 150rpm, the cells were collected by centrifugation, washed, and resuspended to obtain pseudoarthrobacter suspension; 2. Inoculate the Pseudomonas aeruginosa suspension into the YEP liquid fermentation medium, mix well and add to the Haematococcus pluvialis, culture at 37°C and 150rpm for 1-3 days, collect the culture supernatant by centrifugation and detect the astaxanthin content.

[0007] Compared with the prior art, the present invention has the following advantages: 1) Pseudoarthrobacter F13 is co-cultured with Haematococcus pluvialis. Pseudoarthrobacter F13 can lyse Haematococcus pluvialis, allowing the astaxanthin in the Haematococcus pluvialis to be fully released; 2) The present invention uses Pseudoarthrobacter F13 to extract astaxanthin from Haematococcus pluvialis, which is simple to operate and low in cost. No solvent or wall-breaking equipment is required during the extraction, which reduces the negative impact on the environment. It 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 The results show the effects of different carbon sources on the production of pectinase by Pseudomonas aeruginosa F13; Figure 2 The results show the effects of different nitrogen sources on the pectinase production of Pseudomonas aeruginosa F13; Figure 3 The results of the effects of different pH on the production of pectinase by Pseudomonas aeruginosa F13; Figure 4 The results of the effects of different lysis time on the lysis of Haematococcus pluvialis by Pseudomonas aeruginosa F13. The upper figure is the result of optical microscope observation, and the lower figure is the statistical result. Figure 5 The scanning electron micrographs of algal cells after fermentation with the addition of Pseudomonas aeruginosa F13 (Fig. b) and without the addition of F13 (Fig. a); Figure 6 The algal cells after fermentation before extraction with Pseudoarthrobacter F13 (Figure A) and after extraction with Pseudoarthrobacter F13 (Figure B); Figure 7 The state diagram of the supernatant after fermentation centrifugation (Figure A) and the precipitate after adding ethanol extraction and centrifugation (Figure B), where the left test tube is the control without adding F13, and the right test tube is the one with adding F13; Figure 8 The results of astaxanthin extraction rate after fermentation with and without adding Pseudomonas aeruginosa F13. 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; The pectinase activity determination method in the following examples is as follows: Take two 1.5mL centrifuge tubes, add 0.2mL 100mmol / L Tris-HCl buffer (pH 8) and 0.1mL 1% (w / v) pectin solution respectively; add 0.1mL of the enzyme solution to be tested to one centrifuge tube, and add 0.1mL of the enzyme solution to be tested that has been inactivated in boiling water for 5min to the other centrifuge tube as a control, mix well and incubate in a 37℃ water bath for 1h; The activity of pectinase was calculated by the amount of D-galacturonic acid produced. 0.3 mL of 3.5-dinitrosalicylic acid was added to the incubated solution, heated in a boiling water bath for 5 min, and quickly cooled to room temperature. The absorbance at 540 nm was measured. A standard curve was prepared with different concentrations of D-galacturonic acid. One unit of enzyme activity (U) was defined as 1 μg of D-galacturonic acid produced by 1 mL of crude enzyme solution hydrolyzing pectin per minute.

[0010] The enzyme activity calculation formula is: U=[(A×N×1000) / T]×V, where A: D-galacturonic acid content, μg / mL; N: enzyme solution dilution multiple; T: reaction time, min; V: enzyme solution volume, mL.

[0011] Example 1: Preparation of Pseudomonas aeruginosa F13 bacterial suspension Pseudoarthrobacter F13 isolated from the soil of a vegetable greenhouse in a farmland in Yuxi County, Yunnan Province, where the pesticide procymidone was used all year round, was activated by streaking on LB solid medium and cultured at 37°C. When a large single colony grew on the LB solid medium, a single colony was inoculated into 30 mL of sterilized LB liquid medium and cultured at 37°C and 150 rpm overnight. 600 When the pH value is 1.2-1.4, the culture solution is centrifuged at 8000 rpm for 5 min, the supernatant is discarded, the bacteria are washed twice with 0.9% NaCl solution, and then the bacteria are resuspended in 3 mL of sterile saline to prepare Pseudomonas aeruginosa F13 bacterial suspension for later use.

[0012] Example 2: Effect of carbon source on pectinase production by Pseudomonas aeruginosa F13 YEP fermentation medium (3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L potassium dihydrogen phosphate, pH 6) was prepared with 10 g / L lactose, pectin, glucose, mannitol, fructose, sorbitol, and xylose as carbon sources, respectively. 50 μL of the suspension of Pseudomonas aeruginosa F13 in Example 1 was inoculated into 5 mL of YEP fermentation medium. After culturing at 37°C for 2 days, the mixture was centrifuged at 8000 rpm for 10 min, the supernatant was collected, and the pectinase activity of the supernatant was determined. The detection method was the same as in Example 1. The results are shown in Table 1. Figure 1 ,From the figure, we can see that when pectin is used as the carbon source, the ,enzyme activity of pectinase in the supernatant is the highest, which is 5 U / mL.

[0013] Example 3: Effect of nitrogen source on pectinase production by Pseudomonas aeruginosa F13 YEP fermentation medium (10 g / L pectin, 2 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, pH 6) was prepared with 3 g / L methionine, casein, cysteine, yeast extract, phenylalanine, and ammonium sulfate as nitrogen sources, respectively. 50 μL of the suspension of Pseudomonas aeruginosa F13 in Example 1 was inoculated into 5 mL of YEP fermentation medium. After culturing at 37°C for 2 days, the mixture was centrifuged at 8000 rpm for 10 min, the supernatant was collected, and the pectinase activity of the supernatant was determined. The detection method was the same as in Example 1. The results are shown in Table 1. Figure 2 ,It can be seen from the figure that when yeast extract powder is used as the nitrogen source, the pectinase activity in the supernatant is the highest, which is 16 U / mL.

[0014] Example 4: Effect of pH on Pectinase Production by Pseudomonas aeruginosa F13 YEP fermentation medium (10 g / L pectin, 3 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 2 g / L potassium dihydrogen phosphate) with pH values ​​of 5, 6, 7, 8, and 9 were prepared, 50 μL of the suspension of Pseudomonas aeruginosa F13 in Example 1 was inoculated into 5 mL of YEP fermentation medium, cultured at 37°C for 2 days, centrifuged at 8000 rpm for 10 min, the supernatant was collected, and the pectinase activity of the supernatant was determined by the same detection method as in Example 1. The results are shown in Table 1. Figure 3 ,It can be seen from the figure that when the pH is 8, the pectinase activity in the supernatant is the highest, which is 32U / mL.

[0015] Example 5: Effect of treatment time on lysis of Haematococcus pluvialis by Pseudomonas aeruginosa F13 50 μL of the suspension of Pseudomonas aeruginosa F13 in Example 1 was inoculated into 5 mL of YEP fermentation medium (10 g / L pectin, 3 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, pH 8). At the same time, YEP fermentation medium without inoculation of Pseudomonas aeruginosa F13 was used as a control. After culturing at 37°C for 2 days, 0.5% (w / v) Haematococcus pluvialis was added to the above experimental group, and the culture was continued at 37°C. Samples were taken on the 1st, 2nd and 3rd days for optical microscopy observation. The results are shown in FIG. Figure 4 ,It can be seen from the figure that the amount of astaxanthin released from Haematococcus pluvialis increases with time, indicating that Pseudomonas aeruginosa F13 can lyse Haematococcus pluvialis.

[0016] Example 6: Experiment on extracting astaxanthin from Haematococcus pluvialis by lysing Haematococcus pluvialis with Pseudoarthrobacter F13 50 μL of the suspension of Pseudoarthrobacter F13 in Example 1 was inoculated into 5 mL of YEP fermentation medium (10 g / L pectin, 3 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, pH 8). At the same time, the YEP fermentation medium without inoculation of Pseudoarthrobacter F13 was used as a control (CK). After culturing at 37°C for 2 days, 0.025 g of Haematococcus pluvialis was added to the above experimental group, and the culture was continued at 37°C for 2 days; 1 mL was taken and centrifuged at 8000 rpm for 10 min, and the supernatant was collected ( Figure 7 A), the astaxanthin content in the supernatant was determined at 475 nm; 1 mL of anhydrous ethanol was added to the precipitate after centrifugation, mixed and allowed to stand for 1 h, and centrifuged at 8000 rpm for 10 min ( Figure 7 B), collect the supernatant as the astaxanthin extract, and determine the astaxanthin content in the supernatant at 475 nm; add the astaxanthin content detected in the supernatant and the astaxanthin content extracted from the precipitate to obtain the total astaxanthin extraction amount A; The above precipitation was observed by scanning electron microscopy for algal cell lysis. Figure 5 From the figure, it can be seen that after adding Pseudomonas aeruginosa F13, the Haematococcus pluvialis was deformed and cracked. Without adding Pseudomonas aeruginosa F13, the Haematococcus pluvialis was full in shape and had no obvious deformation. The results of optical microscope observation are shown in Figure 6 It can be clearly seen from the figure that after adding Pseudomonas aeruginosa F13, the astaxanthin in the Haematococcus pluvialis was released to the outside world, and the inside of the Haematococcus pluvialis became lighter; before adding Pseudomonas aeruginosa F13, the Haematococcus pluvialis did not release astaxanthin; from Figure 7 It can be seen that no red astaxanthin is produced in the control precipitate ( Figure 7 B left), while adding the precipitate separated by F13 fermentation ( Figure 7 B (right) shows astaxanthin, indicating that F13 lysed the algal cells; At the same time, 0.025g of Haematococcus pluvialis was added with 0.05g of silicon dioxide for grinding for 30min, 5mL of anhydrous ethanol was added for static extraction for 1h, centrifuged at 8000 rpm for 10min, the supernatant was collected, and the extraction process was repeated until the supernatant was colorless, the supernatants were combined and the astaxanthin content was determined at 475 nm, and the astaxanthin content was taken as the completely extracted astaxanthin amount B; the astaxanthin extraction rate after adding F13 fermentation (100%) = total astaxanthin extraction amount A / completely extracted astaxanthin amount B; The extraction rate results are shown in Figure 8 From the figure, it can be seen that no astaxanthin was extracted from the control group without adding F13, while after adding F13, the algal cells were lysed to produce astaxanthin, and the extraction rate was 76%.

Claims

1. A strain of Arthrobacter pseudoarthritis ( Pseudarthrobacter sp.) F13 in the lysis of Haematococcus pluvialis to extract astaxanthin, the pseudoarthrobacter ( Pseudarthrobacter sp.) F13 is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20241866.

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