A marine bacterium for producing carotenoids and its isolation and culture method

Abstract

The present invention relates to the field of marine bacteria, and discloses a marine bacterium for producing carotenoids and its isolation and culture method. This strain is isolated from seawater and named Roseovarius huanghaiensis NDS ( R.marisflavi NDS ), which is deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit number of CGMCC NO. 32287 and the deposit date of October 21, 2024. This strain is extracted from seawater, has good ecological friendliness, and also has the characteristics of oligotrophy. The raw material cost of the culture medium is low and easy to obtain. At the same time, this strain has excellent growth rate, can reach the logarithmic growth phase within a few hours, and complete the accumulation of carotenoids in 20 h. The carotenoids produced account for 1.764% of the cell dry weight, and the production efficiency is high. This marine bacterium can efficiently produce carotenoids, is easy to mass-produce, and has less impact on the marine ecosystem.

Description

Technical Field

[0001] The present invention relates to the field of marine bacteria, and particularly to a marine bacterium for producing carotenoids and its isolation and culture method. Background Art

[0002] Carotenoids are a class of bioactive secondary metabolites, which have functions such as antioxidant, anti-inflammatory, and immune enhancement. Research shows that providing feed containing carotenoids during the breeding period of fish can significantly improve the quality of eggs and the survival rate of juvenile fish. In addition, for brightly colored aquatic products such as rainbow trout and shrimp, carotenoids also play a crucial role in maintaining body color and muscle health. However, natural carotenoids mainly exist in photosynthetic plants, bacteria, and microalgae. For most marine organisms, the sources of natural carotenoids are limited, and the concentration is often not sufficient to meet the needs of growth and reproduction. Therefore, an appropriate amount of carotenoids needs to be supplemented in aquaculture feed.

[0003] In industrial production, there are mainly two production routes for carotenoids: extraction from natural sources such as plants, algae, and microorganisms, and chemical synthesis. The most typical plants for extracting carotenoids are: extracting β-carotene from carrots and pumpkins; extracting lycopene from tomatoes; extracting zeaxanthin from corn and red peppers, etc. Algae include varieties such as Haematococcus pluvialis, Dunaliella salina, and Chlorella vulgaris. Under high light, nitrogen deficiency, or high salt stress conditions, the astaxanthin yield of Haematococcus pluvialis can reach 1.5 - 3% (dry weight) per liter of culture medium; in a large-scale culture system, the β-carotene yield of Dunaliella salina can reach 3 - 5% (dry weight) per liter of culture medium; in commercial culture, the carotenoid yield (total amount of lutein and β-carotene) of Chlorella vulgaris is about 0.5 - 2% (dry weight) per liter of culture medium. The main bacteria for producing carotenoids are photosynthetic bacteria: purple sulfur bacteria, green sulfur bacteria, Rhodospirillum; non-photosynthetic bacteria include Flavobacterium, Pseudomonas, and Lactobacillus delbrueckii. However, the carotenoid yields of these bacteria are all relatively low, and the carotenoid content per liter of culture medium is only 0.1 - 1% (dry weight). And the types of bacteria that are easy to mass-produce are also relatively limited.

[0004] However, compared with plants and algae, bacteria have the advantages of faster reproduction speed and lower cultivation cost. Therefore, finding a bacterium that can efficiently produce carotenoids and is easy to mass-produce is crucial for saving cultivation time and cost. At the same time, since the bacterium finally needs to be added to aquaculture feed to increase the carotenoid content, it is also necessary to make the impact of the bacterium on the marine ecosystem smaller when added to seawater with aquaculture feed. Summary of the Invention

[0005] The first object of the present invention is to provide a marine bacterium that can efficiently produce carotenoids, is easy to mass-produce, and has a relatively small impact on the marine ecosystem after being added to seawater, aiming at the disadvantages of low yield and difficulty in mass production of bacteria that can produce carotenoids in the prior art.

[0006] To solve the above technical problems, the present invention is solved by the following technical solutions:

[0007] A marine bacterium for producing carotenoids, named Rossellomorea huanghaiensis NDS ( R. marisflavi NDS ), is preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number CGMCC NO. 32287 and the preservation date of October 21, 2024.

[0008] Adopting the above scheme, this strain of bacterium was isolated from seawater obtained at a water depth of 0.5 m in the central sea area of Xiangshan Bay, Ningbo, Zhejiang (121°43'06''E, 29°34'44''N). Through 16S rRNA gene sequence analysis, it was found that this bacterium belongs to the genus Rossellomorea and has the highest homology with the strain Rossellomorea marisflavi A, and they both belong to Rossellomorea marisflavi. Therefore, we named it Rossellomorea huanghaiensis NDS ( R. marisflavi NDS ). This strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number CGMCC NO. 32287 and the preservation date of October 21, 2024.

[0009] This strain is extracted from seawater, has excellent growth rate, the carotenoids produced account for 1.764% of the cell dry weight, and the endotoxin meets the standard, making this bacterium capable of efficiently producing carotenoids, being easy to mass-produce, being able to coexist with marine organisms and having ecological friendliness.

[0010] Preferably, when this bacterium grows to 20 h, the accumulation amount of carotenoids reaches the maximum.

[0011] The second object of the present invention is to provide a separation and cultivation method for separating, purifying and culturing the above marine bacterium from seawater.

[0012] A separation and cultivation method for marine bacteria:

[0013] A1. Separation of marine bacteria from seawater

[0014] Collect seawater samples, dilute them and inoculate them into liquid culture medium, and culture them for 12 to 16 hours at a temperature of 36 to 38 °C and a shaking speed of 100 to 150 r / min to form a bacterial culture solution;

[0015] Take the bacterial culture solution and spread it on the plate culture medium. After culturing for 10-14 hours, pick the mixed bacterial colonies and streak them on the plate culture medium to obtain the mixed bacterial culture.

[0016] Take the mixed bacterial culture, dilute it with sterile water to form a bacterial solution, aspirate the bacterial solution and evenly spread it on the liquid culture medium, culture it at a temperature of 36-38 °C and a shaking speed of 100-150 r / min for 10-12 hours, pick out the yellow colonies separately, and perform zone streak purification culture on the plate culture medium to form purified bacteria;

[0017] A2. Cultivation of marine bacteria

[0018] The purified bacteria were inoculated into a liquid culture medium with an initial pH of 7-9 at an inoculation size of 0.1-0.15% and cultured at a temperature of 36-38°C and a shaking speed of 100-150 r / min for 8-16 hours.

[0019] The above scheme is adopted to inoculate the seawater sample into the liquid culture medium to form mixed bacterial colonies, streak the mixed bacterial colonies on the plate culture medium to obtain mixed bacterial culture, spread the mixed bacterial culture on the liquid culture medium, and pick out yellow colonies after they are cultured, and then streak them on the plate culture medium for purification and culture, and then preserve the purified bacteria for subsequent experiments. The purified bacteria are R. marisflavi NDS .

[0020] In subsequent experiments, the purified R. marisflavi NDS Cultivate and observe R. marisflavi NDS of growth, and R. marisflavi NDS Endotoxin content determination and carotenoid content determination were performed.

[0021] The results show that R. marisflavi NDS The endotoxin content is below the prescribed limit, and it is extracted from seawater, so it can coexist with marine life and is eco-friendly. R. marisflavi NDS The growth rate is excellent. And by comparison R. marisflavi NDS The growth and intracellular pigment accumulation of R. marisflavi NDS When the growth reaches the plateau stage for 20 hours, the pigment accumulation reaches the maximum. Samples are taken at the corresponding time point and the measured R. marisflavi NDSThe carotenoids account for 1.764% of the cell dry weight, which is significantly better than other bacteria.

[0022] Preferably, the liquid medium is a liquid beef extract peptone medium, which contains the following components: 0.3% beef extract, 1% peptone, 2% sodium chloride, and the rest is water.

[0023] Preferably, the plate medium is a solid beef extract peptone medium, which contains the following components: 0.3% beef extract, 1% peptone, 2% sodium chloride, 2% agar, and the rest is water.

[0024] Adopting the above scheme, the isolated R. marisflavi NDS has oligotrophic characteristics. The medium used is beef extract peptone medium, without the need to additionally add other carbon sources or nitrogen sources. The raw material cost is low and it is easy to obtain.

[0025] Due to the adoption of the above technical scheme, the present invention has remarkable technical effects: the R. marisflavi NDS is isolated from seawater and cultured through beef extract peptone medium. It has excellent growth rate, can reach the logarithmic growth phase within a few hours, and quickly completes the accumulation of carotenoids in the plateau phase. The produced carotenoids account for 1.764% of the cell dry weight, and the endotoxin meets the standard, making this bacterium capable of efficiently producing carotenoids, being easy to mass-produce, being able to coexist with marine organisms and having ecological friendliness. Description of the Drawings

[0026] Figure 1 is a schematic diagram in the plate medium in the embodiment; R. marisflavi NDS in the plate medium in the embodiment;

[0027] Figure 2 is a TEM image in the embodiment; R. marisflavi NDS in the embodiment;

[0028] Figure 3 is a 16s rRNA phylogenetic tree in the embodiment; R. marisflavi NDS in the embodiment;

[0029] Figure 4 is a growth curve graph in the embodiment; R. marisflavi NDS in the embodiment;

[0030] Figure 5 is a comparison graph of the growth situation and pigment accumulation situation in the embodiment; R. marisflavi NDS in the embodiment. Detailed Embodiments

[0031] The present invention will be further described in detail below in conjunction with the drawings and embodiments. Embodiment

[0032] A marine bacterium for producing carotenoids and a method for separating and culturing the same, comprising the marine bacterium and a method for separating, purifying and culturing the marine bacterium from seawater.

[0033] 1. Isolation of bacteria:

[0034] Seawater samples were collected from the middle waters of Xiangshan Port, Ningbo City, Zhejiang Province (121°43'06''E, 29°34'44''N) at a water depth of 0.5 m.

[0035] Take 50 mL of seawater sample and dilute it 8 to 12 times and inoculate it in liquid culture medium. After culturing for 12 to 16 hours at a temperature of 36 to 38 ° C and a shaking speed of 100 to 150 r / min, a bacterial culture solution is formed. In this embodiment, the seawater sample is diluted 10 times, the temperature is 37 ° C, the shaking speed is 120 r / min, and the culture is 14 hours. The culture medium is liquid beef extract peptone medium. The liquid beef extract peptone medium contains the following components: 0.3% beef extract, 1% peptone, 2% sodium chloride, and the rest is water.

[0036] Take 100 mL of bacterial culture solution, spread it on a plate culture medium, and after culturing for 10 to 14 hours, pick mixed bacterial colonies with good growth and different morphologies for streaking to obtain a mixed bacterial culture. In this embodiment, the culture time is 12 hours, and the plate culture medium is a solid beef extract peptone medium. The solid beef extract peptone medium contains the following components: 0.3% beef extract, 1% peptone, 2% sodium chloride, 2% agar, and the rest is water.

[0037] Glycerol magnetic beads are used to adsorb the mixed bacterial culture to form a bacterial glycerol storage solution, which is stored in a -80°C refrigerator. Glycerol magnetic beads and the adsorption method of glycerol magnetic beads on mixed bacterial cultures are both existing technologies and will not be described in detail here. In addition, glycerol magnetic beads only have the effect of extending the storage time of mixed bacterial cultures, and do not affect the subsequent purification, cultivation and subsequent experiments of bacteria.

[0038] Take 10 mL of bacterial glycerol storage solution and dilute it 10 to 20 times with sterile water to form a bacterial solution. In this example, the sterile water was diluted 12 times.

[0039] Pipette 100 mL of bacterial solution and spread it evenly on the liquid culture medium. After incubation at 36-38 °C and a shaking speed of 100-150 r / min for 10-12 h, pick out the yellow colonies and perform zone streak purification on the plate culture medium to form purified bacteria. For details, refer to Figure 1 In this embodiment, the temperature is 37°C, the shaking speed is 120 r / min, and the culture time is 11 h.

[0040] Store the purified bacteria in a refrigerator at -80 °C.

[0041] 2. Cultivation of bacteria:

[0042] Inoculate the purified bacteria into a liquid medium with an initial pH of 7 - 9, at an inoculation amount of 0.1 - 0.15%, and culture for 8 - 16 h under the conditions of a temperature of 36 - 38 °C and a shaker speed of 100 - 150 r / min. In this example, the initial pH of the liquid medium is 7.5, the inoculation amount is 0.12%, the cultivation temperature is 37 °C, and the shaker speed is 120 r / min.

[0043] When the bacteria are activated to the logarithmic growth phase and the OD 600 value reaches 0.4 - 0.8, place the pure strain under a transmission electron microscope to observe the morphology of the bacteria, specifically referring to Figure 2 . The transmission electron microscope image shows that the bacterial cells are surrounded by a dense outer shell, and the internal contents are separated by an inner membrane. The cells are long rod-shaped, with a length of 3.07 mm and a width of 431 nm.

[0044] 3. Identification and naming of bacteria

[0045] The phylogenetic position of this bacterium is determined by 16S rRNA gene sequence analysis. Isolate the DNA of this strain of bacteria, and amplify the 16S rRNA gene by PCR using the universal bacterial primers 27F and 1492R. Use the BLASTN program to align the 16S rRNA sequence of this bacterium with the GeneBank online database, and use the Mega 7.0 software to construct a phylogenetic tree, specifically referring to Figure 3 .

[0046] Rossellomorea marisflavi 16s rRNA sequence:

[0047]

[0048] The strain was identified as belonging to the genus Rossellomorea by morphological characteristics, 16S rDNA and whole genome sequencing analysis, and had the highest homology with the strain Rossellomorea marisflavi A, which we named R. marisflavi NDS .

[0049] 4. Determination of bacterial growth curve

[0050] Prepare liquid culture medium and sterilize at 121℃ for 20 min. Dispense the liquid culture medium into 50 mL cell culture bottles, 25 mL per bottle. Pick a single colony and culture it at 37℃ for 10 h with an oscillation speed of 120 r / min. Measure the OD 600 Then, 10 mL of the primary bacterial solution was transferred to a culture bottle and cultured under the same conditions (i.e., the secondary bacterial solution), and the OD was measured every 4 h. 600 Value, result reference Figure 4 It can be seen that R. marisflavi NDS The concentration began to increase significantly after 6 h of culture; during the period of 6 to 14 h, the bacteria grew rapidly and proliferated massively, entering the logarithmic growth phase; after 14 h of culture, the concentration of the bacterial solution did not change much, indicating that the cell growth had entered a plateau phase.

[0051] 5. Determination of bacterial endotoxicity

[0052] Bacterial endotoxins are a group of lipopolysaccharide (LPS) complexes that are widely present inside and outside cells. During the growth and reproduction of bacteria, they can have certain toxic (sensitizing / pathogenic) effects on the normal physiological activities of surrounding organisms. In order to determine whether the endotoxin content of the isolated strain is within the allowable range, an endotoxin detection reagent (Limulus amebocyte lysate colorimetric method) is used to quantitatively detect the endotoxin content in the bacteria. The calculation formula is: L = MVD × λ / c, L represents the bacterial endotoxin limit of the test sample, MVD is the maximum effective dilution multiple, and λ is the minimum endotoxin concentration in the standard curve. The method for determining cellular endotoxins is an existing technology and will not be described in detail here.

[0053] Experimental calculations show R. marisflavi NDS The endotoxin content of the sample was 0.085 EU / mL, which is lower than the prescribed limit of 0.1 EU / mL (Chinese Pharmacopoeia). R. marisflavi NDS The endotoxin content is at a low level, which means that this strain of bacteria is less likely to cause pathogenicity and allergy to other organisms during its growth and has a higher biosafety.

[0054] 6. Determination of carotenoid production content of bacteria

[0055] According to the comparison of bacterial growth (OD 600 ) and pigment accumulation in cells (OD 480 ) relationship, found in R. marisflavi NDS When the growth reaches 20 hours, the pigment accumulation is the largest. Figure 5 In view of this, bacterial cells cultured for 20 h were collected and the yellow pigment in the bacterial cells was extracted using a mixed organic solvent, wherein the mixed organic solvent included methanol and chloroform, and the ratio of methanol to chloroform was 1:2.

[0056] Bacterial cells were added to the mixed organic solvent, ultrasonically disrupted at 200-250W for 25 minutes at room temperature and in the dark, and then centrifuged at 8000×g for 10 minutes. The extraction was repeated several times until the cell fragments appeared white and free of pigment.

[0057] Collect the supernatant and test the OD 480 The precipitate was resuspended in 1× PBS buffer, and the resuspension was placed in an oven at 60°C and dried to constant weight.

[0058] The total carotenoid content (TCC, mg / g) was determined using the following formula: TCC = A × D × V / (E × W), where A: absorbance of total extracted carotenoids at 480 nm; D: sample dilution ratio; V: volume of extraction solvent (mL); E: extinction coefficient of carotenoids (0.16); and W: dry weight of bacterial cells (g).

[0059] R. marisflavi NDS It only takes 20 hours to accumulate 1.764 g / 100 g of carotenoids, accounting for 1.764% of the cell dry weight. The production efficiency is high.

[0060] In summary, R. marisflavi NDS It is extracted from seawater, and the endotoxin content meets the standard. Therefore, the bacteria is added to the ocean as a carotenoid supplement during the production of feed, which is friendly to the marine ecology. R. marisflavi NDS It can be cultured in beef extract peptone medium without the need to add other carbon or nitrogen sources. The raw material cost is low and easy to obtain. R. marisflavi NDS The growth rate is excellent, and it can reach the logarithmic growth phase within a few hours. The culture is simple, fast and easy to operate. It only takes 20 hours to accumulate 1.764 g / 100 g of carotenoids, accounting for 1.764% of the cell dry weight, with high production efficiency.

[0061] therefore, R. marisflavi NDS It is a marine bacterium that can efficiently produce carotenoids, is easy to mass-produce, and has little impact on the marine ecology.

[0062] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A marine bacterium for producing carotenoids, characterized in that: The strain was named Rosella yellowensis ( Rossellomorea marisflavi )NDS, deposited in the China General Microbiological Culture Collection Center (CGMCC), its deposit number is CGMCC NO. 32287, and the deposit date is October 21, 2024.

2. A marine bacterium for producing carotenoids according to claim 1, characterized in that: The carotenoid accumulation of the bacteria reached the maximum when the bacteria grew for 20 hours.

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