New strain of Paracoccus producing astaxanthin and its use

Abstract

The present invention belongs to the field of microbial fermentation, and specifically relates to a new strain of Paracoccus that produces astaxanthin and its use. The existing strains for producing astaxanthin have defects such as long culture cycle, complex culture conditions, great difficulty in extraction and separation, and low yield, and there are few types of functional microorganisms that produce astaxanthin, which need to be developed. The present invention provides a new strain of Paracoccus that produces astaxanthin. The new strain is deposited with CGMCC NO.22042. The temperature tolerance range is 20-40°C, the ethanol tolerance concentration is ≤6%, the salinity tolerance is ≤6%, and the pH tolerance range is 5-10. In addition, the new strain can produce astaxanthin in high yield, can provide new functional microbial strains for astaxanthin production, enrich the microbial resources for astaxanthin production, and has good application value in the fields of medicine, food, cosmetics, etc.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and in particular relates to a new strain of Paracoccus producing astaxanthin and a use thereof. Background Art

[0002] The percentage of culturable microorganisms in the natural environment is very low, such as less than 0.1% in seawater and about 0.3% in soil. The culturable microorganisms in traditional solid liquor are no exception. Through high-throughput sequencing, we found that most of the microorganisms are unculturable. However, using traditional bacterial culture methods, only a small part of the bacteria in the natural environment can be cultured. The microorganisms cultured using traditional methods are all known microorganisms, and no new microbial species have been discovered.

[0003] In addition, astaxanthin is the most powerful antioxidant ketocarotenoid in nature. It can effectively remove oxygen free radicals in cells and has strong antioxidant capacity. It can remove nitrogen dioxide, sulfide, etc. It can also reduce lipid peroxidation and effectively inhibit lipid peroxidation caused by free radicals. At the same time, it has many physiological effects such as inhibiting tumor occurrence, enhancing immunity, and removing free radicals in the body. It has a good therapeutic effect on skin cancer caused by ultraviolet rays and prevents and treats eye diseases caused by diabetes. It has broad application prospects in health products, medicine, cosmetics, food additives and aquaculture. In addition to being obtained by biological extraction, astaxanthin can also be obtained by chemical synthesis, and can also be produced by algae, bacteria, yeast, etc. Chemically synthesized astaxanthin is significantly different from natural astaxanthin in terms of structure, function, application and safety. Its stability, antioxidant activity and coloring are also significantly lower than those of natural astaxanthin. Therefore, large-scale production generally tends to extract natural astaxanthin. Currently, the main biological sources of astaxanthin are Phaffia rhodozyma and Haematococcus pluvialis, but the production of astaxanthin from these sources has defects such as long culture cycle, complex culture conditions, difficulty in extraction and separation, and low yield, and the production process does not meet industrial production requirements.

[0004] Currently, there are relatively few types of functional microorganisms that can produce astaxanthin, and further development is needed. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the existing strains for producing astaxanthin have defects such as long culture cycle, complex culture conditions, great difficulty in extraction and separation, and low yield, and most new microbial species have not yet been cultured and need to be developed; at the same time, the existing functional microbial species that produce astaxanthin are relatively few and need to be developed.

[0006] The technical solution of the present invention to solve the above technical problems is: providing a new strain of Paracoccus that produces astaxanthin. The new strain of Paracoccus described in the present invention has a preservation number of CGMCC NO.22042. The preservation time is March 22, 2021, and the preservation unit is the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC), and the address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0007] The nucleotide sequence of 16S rDNA of the new strain of Paracoccus is shown in SEQ ID NO:1.

[0008] SEQ ID NO:1 16S rDNA nucleotide sequence of a new strain of Paracoccus

[0009]

[0010] The temperature tolerance range of the new strain of the genus Paracoccus is 20-40°C, the ethanol tolerance concentration is ≤6%, the salinity tolerance is ≤6%, and the pH tolerance range is 5-10.

[0011] The novel strain of the genus Paracoccus can produce astaxanthin, and furthermore, the astaxanthin content is 5.04 μg / g of bacterial body.

[0012] The cytological characteristics of the new strain of Paracoccus are as follows: the colonies are yellow, the surface is moist and smooth, and the edges are irregular; the cell morphology is spherical.

[0013] Among them, the biological characteristics of the above-mentioned new strain of Paracoccus are:

[0014] The test results of API50CH showed that it could utilize D-ribose, erythritol, L-arabinose, D-galactose, esculin (ferric citrate), D-cellobiose, D-maltose, D-lactose, D-melbiose, D-gentiobiose, D-lyxose, and D-fucose; it could not utilize glycerol, D-arabinose, D-xylose, L-xylose, adonol, β-methyl-xyloside, D-glucose, D-fructose, D-mannose, L-sorbose, and L-rhamnose. , dulcitol, inositol, D-mannitol, sorbitol, α-methyl-D-mannoside, α-methyl-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-sucrose, D-trehalose, inulin, D-melezitose, D-raffinose, starch, glycogen, xylitol, D-toulose, D-tagatose, L-fucose, D-arabinol, L-arabinol, potassium gluconate, 2-keto-gluconate, 5-keto-gluconate.

[0015] API ZYM showed that alkaline phosphatase, esterase, lipidase, leucine arylamine, cystine arylamine, acid phosphatase, naphthol-AS-BI-phosphohydrolase and β-glucosidase were all positive; esterase, valine arylamine, trypsin, chymotrypsin, α-galactosidase, β-galactosidase, β-uronidase, α-glucosidase, N-acetylglucosaminidase, α-mannosidase and β-fucosidase were all negative. Catalase was positive.

[0016] The present invention also provides use of the novel strain of Paracoccus in microbial fermentation.

[0017] The present invention also provides use of the novel strain of the genus Paracoccus in fermentation to produce astaxanthin or in the preparation of a microbial agent for producing astaxanthin.

[0018] Beneficial effects:

[0019] The present invention screened a new strain of Paracoccus from the fermentation environment of Luzhou-flavor liquor, with a preservation number of CGMCC NO.22042. The temperature tolerance range of the new strain of Paracoccus of the present invention is 20-40°C, the ethanol tolerance concentration is ≤6%, the salinity tolerance is ≤6%, and the pH tolerance range is 5-10; it can produce astaxanthin. It can provide a new functional microbial strain for astaxanthin production, enrich the microbial resources for astaxanthin production, and has good application value in the fields of medicine, food, cosmetics, etc.

[0020] The new strain of the genus Paracoccus of the present invention was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on March 22, 2021, with the deposit number: CGMCC NO.22042. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. The classification name is Paracoccus sp. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The morphological diagram of the new strain of Paracoccus of the present invention, wherein A: colony diagram; B: cell diagram;

[0022] Figure 2 16S rDNA phylogenetic tree diagram of the new strain of Paracoccus of the present invention;

[0023] Figure 3 is a temperature tolerance diagram of the new strain of Paracoccus of the present invention;

[0024] Figure 4 is a graph showing the ethanol resistance of the new strain of the genus Paracoccus of the present invention;

[0025] Figure 5 is a diagram showing the salinity tolerance of the novel strain of the genus Paracoccus according to the present invention;

[0026] Figure 6 This is a pH tolerance diagram of the new strain of the genus Paracoccus according to the present invention. DETAILED DESCRIPTION

[0027] The present invention provides a new strain of Paracoccus, which was screened in the fermentation environment of Luzhou-flavor liquor of Wuliangye Yibin Co., Ltd. and named WLY-B-L5. The new strain of the present invention was deposited on March 22, 2021, with the deposit number CGMCC NO.22042, and the deposit unit is the General Microbiological Center (CGMCC) of China General Microbiological Culture Collection Committee, and the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0028] The temperature tolerance range of the new Paracoccus strain WLY-B-L5 of the present invention is 20-40°C, the ethanol tolerance concentration is ≤6%, the salinity tolerance is ≤6%, and the pH tolerance range is 5-10.

[0029] The new Paracoccus strain WLY-B-L5 of the present invention produces astaxanthin.

[0030] The Paracoccus WLY-B-L5 of the present invention is a new strain, which supplements new and valuable bacterial species resources and has high application value.

[0031] The specific implementation modes of the present invention will be further explained below through examples, but it is not intended to limit the protection scope of the present invention to the scope described in the examples.

[0032] Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0033] The separation medium PCA used in the example comprises: 5 parts of tryptone, 2.5 parts of yeast extract powder, 1 part of glucose, 2 parts of natamycin, dissolved in 1000 parts of distilled water, and then mixed and adjusted to pH 7.2 with NaOH.

[0034] The composition of the culture medium NA used in the example includes: 3 parts of beef extract, 5 parts of peptone, 2.5 parts of glucose, 1000 ml of distilled water, and NaOH to adjust the pH to 7.0.

[0035] The slant and plate used in the examples were prepared by adding 2% agar powder to the corresponding culture medium.

[0036] The ethanol concentrations described in the examples are volume concentrations.

[0037] Example 1 Isolation, Screening and Identification of a New Paracoccus Strain WLY-B-L5

[0038] (I) Separation and screening

[0039] Samples were taken from the brewing environment of Wuliangye Yibin Co., Ltd., and PCA culture medium was used to screen bacteria. Samples were collected for one minute (100L of air) using a planktonic bacteria sampler (Zhejiang Sujing Purification Equipment Co., Ltd., FKC-I model), and the sampling was repeated three times. The samples were cultured at 37°C for 3 days, and colonies on the plates were selected for streaking and culture, and the process was repeated three times until a purified strain was isolated and stored on a slant. The strain was named WLY-B-L5.

[0040] (II) Classification and identification

[0041] 1. Morphological characteristics of strains

[0042] The isolated WLY-B-L5 strain is Gram-negative, with yellow colonies, moist and smooth surfaces, and irregular edges; the cell morphology is spherical.

[0043] 2. Physiological and biochemical characteristics

[0044] The biological characteristics of the new strain of Paracoccus are as follows:

[0045] Can utilize D-ribose, erythritol, L-arabinose, D-galactose, esculin (ferric citrate), D-cellobiose, D-maltose, D-lactose, D-melbiose, D-gentiobiose, D-lyxose, D-fucose; cannot utilize glycerol, D-arabinose, D-xylose, L-xylose, adonol, β-methyl-xyloside, D-glucose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol , D-mannitol, sorbitol, α-methyl-D-mannoside, α-methyl-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-sucrose, D-trehalose, inulin, D-melezitose, D-raffinose, starch, glycogen, xylitol, D-toulose, D-tagatose, L-fucose, D-arabinol, L-arabinol, potassium gluconate, 2-keto-gluconate, 5-keto-gluconate.

[0046] Alkaline phosphatase, esterase, lipidase, leucine arylamine, cystine arylamine, acid phosphatase, naphthol-AS-BI-phosphohydrolase and β-glucosidase were all positive; esterase, valine arylamine, trypsin, chymotrypsin, α-galactosidase, β-galactosidase, β-uronidase, α-glucosidase, N-acetylglucosaminidase, α-mannosidase and β-fucosidase were all negative. Catalase was positive.

[0047] 3. 16S rDNA identification

[0048] The strain WLY-B-L5 was inoculated into NA medium and cultured at 37°C for 3 days. 1 mL of bacterial solution was collected by centrifugation, and genomic DNA was extracted using a bacterial genome extraction kit (Shanghai Sangon Biotechnology, SK8255) and stored at -20°C for later use.

[0049] The bacterial genome 16S rDNA was amplified using primers 27F and 1492R, 27F: AGTTTGATCMTGGCTCAG (SEQ ID NO: 2), 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO: 3).

[0050] Amplification system (total 25 μL): 10×PCR buffer 2.5 μL, dNTP (2.5 mmol / L each) 1 μL, DNA 10 ng, primer F (10 μmol / L) 0.5 μL, primer R (10 μmol / L) 0.5 μL, add double distilled water to 25 μL. Amplification program: 94℃ 4 min; 94℃ 45 s, 55℃ 45 s, 72℃ 60 s, 30 cycles in total; 72℃ 10 min, 4℃ termination reaction.

[0051] 16S rDNA sequence determination: The PCR amplification product was purified using a gel recovery kit (NO.B110092, Shanghai Sangon Biotechnology Co., Ltd.); the sequencing of the purified product was completed by the China Industrial Microbiological Culture Collection Administration Center. The 16S rDNA sequence of strain WLY-B-L9 was obtained as shown in SEQ ID NO: 1.

[0052] Phylogenetic tree construction: The bacterial 16S rDNA gene sequences were analyzed by BLASTn similarity analysis with the GenBank database, and the model strain Paracoccus acridae SCU-M53, which was the closest relative to the experimental strain, was identified. T The similarity of (KT634253) was 98.48%, which was less than the critical value of 98.65%. The standard strains with relatively close relationship with the experimental strains were selected for sequence alignment using Clustalw software, and phylogenetic analysis was performed using MEGA7 software to construct a phylogenetic tree, such as Figure 2 shown.

[0053] 4. Whole-genome dDDH analysis and ANI

[0054] The paracoccus WLY-B-L5 was sent to the China Industrial Microbiology Collection Center (CICC) for whole genome dDDH and ANI analysis. The results showed that it was similar to the model strain Paracoccus acridae SCU-M53. T The dDDH value of (KT634253) was 26.1%, which was far below the critical value of 70%. The results showed that it was similar to the model strain Paracoccus acridae CGMCC1.15419 T The ANI value of (GCA-014642735) was 91.19%, which was lower than the critical value of 95%.

[0055] Based on the bacterial morphology, physiological and biochemical characteristics, 16S rDNA gene sequence, whole-genome dDDH analysis and ANI analysis, strain WLY-B-L5 was identified as a new strain of Paracoccus sp. The strain WLY-B-L5 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on March 22, 2021, with the deposit number CGMCC NO.22042.

[0056] Example 2 Temperature resistance test of the new strain WLY-B-L5 of the genus Paracoccus

[0057] The strain WLY-B-L5 was inoculated into liquid NA medium for activation and cultured until the OD value of the logarithmic growth phase was reached. 600 =1.8, and 5% of the inoculum volume ratio was inoculated into 50 mL of liquid NA medium, and placed in an incubator at different temperatures (15, 20, 25, 30, 35, 40, and 45°C) for 3 days. The absorbance OD was measured using a UV / visible spectrophotometer (Eppendorf BioSpectrometer) 600 Calculate the OD before and after culture 600 The difference, the result is Figure 3 shown.

[0058] Depend on Figure 3 It can be seen that the OD of strain WLY-B-L5 after being cultured at 20℃ and 40℃ 600 The differences were 0.88 and 0.68 respectively, and when the temperature was 15℃ and 45℃, the OD 600 The difference is almost 0, indicating that there is basically no growth. Therefore, the temperature tolerance of strain WLY-B-L5 is 20-40°C.

[0059] Example 3 Ethanol tolerance test of the new strain WLY-B-L5 of the genus Paracoccus

[0060] The strain WLY-B-L5 was inoculated into liquid NA medium for activation and cultured until the OD value of the logarithmic growth phase was reached. 600 =1.8, and the inoculum volume ratio was 5% and inoculated into 100 mL of liquid NA medium containing different concentrations of ethanol (2%, 4%, 6%, 8% and 10%), and cultured at 37°C for 3 days. The absorbance OD was measured using a UV / visible spectrophotometer (Eppendorf Bio Spectrometer) 600 Calculate the OD before and after culture 600 The difference, the result is Figure 4 shown.

[0061] Depend on Figure 4 It can be seen that the OD of strain WLY-B-L5 after being cultured in NA medium with an ethanol concentration of 6% 600 The difference is 0.91, and when the ethanol concentration is 8%, the OD 600 The difference is almost 0, indicating that there is essentially no growth. Therefore, the ethanol tolerance of strain WLY-B-L5 is 6%.

[0062] Example 4 Salt tolerance of the new Paracoccus strain WLY-B-L5

[0063] The strain WLY-B-L5 was inoculated into liquid NA medium for activation and cultured until the OD value of the logarithmic growth phase was reached. 600 =1.8, and the inoculum volume ratio was 5% and inoculated into 100 mL of liquid NA medium containing different NaCl (0%, 2%, 4%, 6% and 8%), and cultured at 37°C for 3 days. The absorbance OD was measured using a UV / visible spectrophotometer (Eppendorf Bio Spectrometer) 600 Calculate the OD before and after culture 600 The difference, the result is Figure 5 shown.

[0064] Depend on Figure 5 It can be seen that the OD of strain WLY-B-L5 after being cultured in NA medium with 6% NaCl 600 The difference is 0.33, and when the NaCl concentration is 8%, the OD 600 The difference is almost 0, indicating no growth. Therefore, the NaCl tolerance of strain WLY-B-L5 is 6%.

[0065] Example 5 Acid resistance of the new Paracoccus strain WLY-B-L5

[0066] The strain WLY-B-L5 was inoculated into liquid NA medium for activation and cultured until the OD value of the logarithmic growth phase was reached. 600 =1.8, and the inoculum volume ratio was 5% and inoculated into 100 mL of liquid NA medium containing different pH values ​​(4, 5, 6, 7, 8, 9, 10 and 11), and cultured at 37°C for 3 days. The absorbance OD was measured using an Eppendorf Bio Spectrometer. 600 Calculate the OD before and after culture 600 The difference, the result is Figure 6 shown.

[0067] Depend on Figure 6 It can be seen that the OD of strain WLY-B-L5 after being cultured in NA medium with pH 5 and 10 600 The difference was 0.56 and 0.48, and when the pH was 4 and 11, the OD600 The difference is almost 0, indicating no growth. Therefore, the pH tolerance range of strain WLY-B-L5 is 5-10.

[0068] Example 6 Determination of fatty acids in the new strain of Paracoccus WLY-B-L5

[0069] The strain WLY-B-L5 was inoculated into liquid NA medium for activation and cultured until the OD value of the logarithmic growth phase was reached. 600 =1.8, inoculated into 250mL liquid NA medium at a volume ratio of 5%, cultured at 37°C for 3 days, the culture solution was dispensed into 50mL centrifuge tubes, centrifuged at 5000r / min for 10min, the supernatant was discarded, and the cells were collected and sent to China Industrial Microbiological Culture Collection (CICC) for fatty acid composition determination. Gas chromatography was used for detection. The results showed that the main fatty acid components were: C 18:1 ω7c or C 18:1 The ω6c content is 80.54%.

[0070] Example 7 Determination of polar lipids of new strain WLY-B-L5 of Paracoccus

[0071] The strain WLY-B-L5 was inoculated into liquid NA medium for activation and cultured until the OD value of the logarithmic growth phase was reached. 600 =1.8, inoculated into 250mL liquid NA medium at a volume ratio of 5%, cultured at 37°C for 3 days, the culture solution was dispensed into 50mL centrifuge tubes, centrifuged at 5000r / min for 10min, the supernatant was discarded, and the cells were collected and sent to the China Industrial Microbiology Collection Center (CICC) for polar lipid component determination. Thin layer chromatography was used for detection. The results showed that the main polar lipid components were: cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, amino lipids (UAL 1-2), unidentified aminoglycolipid (UAGL), unidentified phospholipid (UPL), glycolipid (UL 1-5), glycolipid (GL).

[0072] Example 8 Astaxanthin production test of new strain WLY-B-L5 of Paracoccus

[0073] 1. Astaxanthin extraction

[0074] The strain WLY-B-L5 was inoculated into liquid NA medium for activation and cultured until the OD value of the logarithmic growth phase was reached. 600=1.8, inoculated into 250mL NA medium at a volume ratio of 5%, cultured at 37℃ shaker for 3 days at 180rpm. After the culture is completed, the fermentation liquid is divided into 50mL centrifuge tubes, centrifuged at 10000rpm for 5min, the supernatant is discarded, the cells are collected, washed twice with sterile water, frozen overnight, and then vacuum freeze-dried. Accurately weigh 0.1g of dried bacteria, add 2.5ml methanol solution to dissolve the bacteria, shake at room temperature in the dark for 30min, centrifuge at 10000rpm for 5min, collect the liquid, repeat the extraction twice until the bacteria are colorless, and store the extract at -20℃.

[0075] 2. Determination of astaxanthin content

[0076] According to the astaxanthin enzyme-linked immunosorbent assay kit of Shanghai ELISA Biotechnology Co., Ltd., the astaxanthin extract was diluted to 10 -3 , follow the instructions, and finally use a Thermoscientific microplate reader to measure the absorbance (OD value) of the standard wells and sample wells at a wavelength of 450nm.

[0077] The standard curve equation was y = -0.2211x + 0.2693, R 2 =0.9988, x represents the logarithmic value of astaxanthin concentration, y represents the percentage absorbance value, the average value of the percentage absorbance value of the sample test is 0.2686, and the astaxanthin content of the astaxanthin stock solution is calculated to be 0.1008ppm. The final astaxanthin content of the new strain Paracoccus WLY-B-L5 is calculated to be 5.04μg / g bacteria.

Claims

1. A novel strain of Paracoccus, characterized in that: The classification name is Paracoccus sp., and the preservation number is CGMCCNO.22042.

2. Use of the novel strain of the genus Paracoccus according to claim 1 in fermentation production of astaxanthin or preparation of a microbial agent for producing astaxanthin.

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