Marine bacterium for producing carotenoid and isolated culture mode thereof

Abstract

The invention relates to the field of marine bacteria, and discloses a marine bacterium for producing carotenoid and a separation culture mode thereof, the strain is separated from seawater, is named as Rosella Yellow Sea NDS (R. marissflavii NDS), is preserved in the China General Microbiological Culture Collection Center (CGMCC), has the preservation number of CGMCC NO.32287 and the preservation date of October 21, 2024, is extracted from seawater, and has the preservation number of CGMCC NO.32287, the preservation number of CGMCC NO.32287 and the preservation number of CGMCC NO.32287 and the preservation number of CGMCC NO.32287. The strain has good eco-friendliness and oligotrophic characteristics, the raw materials of the culture medium are low in cost and easy to obtain, meanwhile, the strain is excellent in growth speed and can reach the logarithmic phase within several hours, accumulation of carotenoid is completed within 20 hours, the produced carotenoid accounts for 1.764% of the dry cell weight, the production efficiency is high, and the production cost is low. The marine bacterium can efficiently produce carotenoid, is easy for mass production, and has small influence on marine ecology.

Description

Technical Field

[0001] The invention relates to the field of marine bacteria, in particular to marine bacteria for producing carotenoids and a separation and cultivation method thereof. Background Art

[0002] Carotenoids are a class of biologically active secondary metabolites that have antioxidant, anti-inflammatory and immune-enhancing effects. Studies have shown that providing feed containing carotenoids during the breeding period of fish can significantly improve the quality of eggs and the survival rate of young fish. In addition, for brightly colored aquatic products such as rainbow trout and shrimp, carotenoids also play a vital role in maintaining body color and muscle health. However, natural carotenoids are mainly found in photosynthetic plants, bacteria and microalgae. For most marine organisms, the source of natural carotenoids is limited, and the concentration is often not enough 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 two main ways to produce carotenoids: extraction from natural sources such as plants, algae and microorganisms, and chemical synthesis. The most typical carotenoids that can be extracted from plants are: β-carotene from carrots and pumpkins; lycopene from tomatoes, zeaxanthin from corn and red peppers, etc. Algae include varieties such as Haematococcus pluvialis, Dunaliella salina, and Chlorella. Under high light, nitrogen deficiency or high salt stress conditions, the astaxanthin production per liter of culture medium of Haematococcus pluvialis can reach 1.5~3% (dry weight); in large-scale culture systems, the β-carotene production of Dunaliella salina per liter of culture medium can reach 3~5% (dry weight); in commercial culture, the carotenoid production (total amount of lutein and β-carotene) of Chlorella per liter of culture medium is about 0.5~2% (dry weight). The main bacteria that produce carotenoids are photosynthetic bacteria: purple sulfur bacteria, green sulfur bacteria, and Rhodospirilla; non-photosynthetic bacteria include Flavobacterium, Pseudomonas, and Lactobacillus delbrueckii. However, the carotenoid production of these bacteria is low, with the carotenoid content in each liter of culture medium being only 0.1-1% (dry weight). In addition, the types of bacteria that are easy to mass produce are relatively limited.

[0004] However, compared with plants and algae, bacteria have the advantages of faster reproduction and lower cultivation costs. Therefore, it is crucial to find a bacterium that can efficiently produce carotenoids and is easy to mass produce in order to save cultivation time and cost. At the same time, since the bacteria need to be added to aquaculture feed to increase the carotenoid content, it is also necessary to make the bacteria added to seawater with aquaculture feed have less impact on the marine ecology. Summary of the invention

[0005] The first purpose of the present invention is to provide a marine bacterium that can efficiently produce carotenoids, is easy to mass produce, and has little impact on the marine ecology after being added to seawater, in view of the shortcomings of the bacteria that can produce carotenoids in the prior art, which have low yields and are difficult to mass produce.

[0006] In order to solve the above technical problems, the present invention is solved by the following technical solutions: A marine bacterium that produces carotenoids, the strain named Rosella yellowsea NDS ( R. marisflavi NDS ), deposited in the China General Microbiological Culture Collection Center (CGMCC), its deposit number is CGMCCNO. 32287, and the deposit date is October 21, 2024.

[0007] Using the above protocol, this bacterium was isolated from seawater obtained at a depth of 0.5 m in the middle waters of Xiangshan Port, Ningbo, Zhejiang (121°43'06''E, 29°34'44''N). Through 16S rRNA gene sequence analysis, it was found that the bacterium belongs to the genus Rossellomorea, and has the highest homology with the strain Rossellomorea marisflavi A, which belongs to the Yellow Sea Rossellomorea marisflavi. Therefore, we named it Yellow Sea Rossellomorea NDS ( R. marisflavi NDS ). The strain is deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC NO. 32287 and the deposit date is October 21, 2024.

[0008] The strain is extracted from seawater, has an excellent growth rate, produces carotenoids that account for 1.764% of the cell dry weight, and its endotoxicity meets the standards, making the bacteria efficient in producing carotenoids, easy to mass-produce, able to coexist with marine organisms, and eco-friendly.

[0009] Preferably, the carotenoid accumulation of the bacteria reaches a maximum when the bacteria grows for 20 hours.

[0010] The second object of the present invention is to provide a separation and cultivation method for separating, purifying and culturing the above-mentioned marine bacteria from seawater.

[0011] A method for separating and cultivating marine bacteria: A1. Isolation of marine bacteria from seawater 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; 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. 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; A2. Cultivation of marine bacteria 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.

[0012] 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 .

[0013] 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.

[0014] 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 NDS Carotenoids accounted for 1.764% of the cell dry weight, which was significantly higher than that of other bacteria.

[0015] Preferably, the liquid culture medium is a liquid beef extract peptone culture medium, comprising the following components: 0.3% beef extract, 1% peptone, 2% sodium chloride, and the remainder water.

[0016] Preferably, the plate culture medium is a solid beef extract peptone culture medium, comprising the following components: 0.3% beef extract, 1% peptone, 2% sodium chloride, 2% agar, and the remainder water.

[0017] Using the above scheme, the separated R. marisflavi NDS It has oligotrophic characteristics, and the culture medium used is beef extract peptone culture medium, which does not require the addition of other carbon sources or nitrogen sources. The raw material cost is low and easy to obtain.

[0018] The present invention has significant technical effects due to the adoption of the above technical solution: R. marisflavi NDS It is isolated from seawater and cultured in beef extract peptone medium. It has an 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 carotenoids produced account for 1.764% of the cell dry weight, and the endotoxicity meets the standards, making the bacteria efficient in producing carotenoids, easy to mass produce, able to coexist with marine organisms, and eco-friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 In the embodiment R. marisflavi NDS Schematic diagram on plate culture medium; Figure 2 In the embodiment R. marisflavi NDS Transmission electron microscopy images of Figure 3 In the embodiment R. marisflavi NDS 16s rRNA phylogenetic tree; Figure 4 In the embodiment R. marisflavi NDS Growth curve chart; Figure 5 In the embodiment R. marisflavi NDS Comparison of growth and pigment accumulation. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Example

[0021] 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.

[0022] 1. Isolation of bacteria: 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The purified bacteria were stored in a refrigerator at -80°C.

[0029] 2. Cultivation of bacteria: The purified bacteria were inoculated into a liquid culture medium with an initial pH of 7 to 9, with an inoculation amount of 0.1 to 0.15%, and cultured for 8 to 16 hours at a temperature of 36 to 38 °C and a shaking speed of 100 to 150 r / min. In this embodiment, the initial pH of the liquid culture medium was 7.5, the inoculation amount was 0.12%, the culture temperature was 37 °C, and the shaking speed was 120 r / min.

[0030] When the bacteria are activated to the logarithmic growth phase, OD 600 The value reaches 0.4~0.8, and the pure strain is placed under a transmission electron microscope to observe the morphology of the bacteria. Figure 2 , transmission electron microscopy images show that the bacterial cells are surrounded by a dense shell, and the internal contents are separated by an inner membrane. The cells are long rod-shaped, 3.07 mm long and 431 nm wide.

[0031] 3. Identification and naming of bacteria The phylogenetic position of this bacterium was determined by 16S rRNA gene sequence analysis. The DNA of this strain was isolated and the 16S rRNA gene was amplified by PCR using universal bacterial primers 27F and 1492R. The 16S rRNA sequence of this bacterium was compared with the GeneBank online database using the BLASTN program, and a phylogenetic tree was constructed using Mega 7.0 software. Figure 3 .

[0032] Rossellomorea marisflavi 16S rRNA sequence:

[0033] 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 .

[0034] 4. Determination of bacterial growth curve 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.

[0035] 5. Determination of bacterial endotoxicity 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.

[0036] 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.

[0037] 6. Determination of carotenoid production content of bacteria 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.

[0038] 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.

[0039] 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.

[0040] 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).

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A marine bacterium for producing carotenoids, characterized in that: The strain was named Rosella yellowensis NDS ( R. marisflavi NDS ), deposited in the China General Microbiological Culture Collection Center (CGMCC), its deposit number is CGMCCNO. 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.

3. A method for separating and cultivating marine bacteria according to claim 1, characterized in that: A1. Isolation of marine bacteria from seawater 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; 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. 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; A2. Cultivation of marine bacteria 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.

4. The method for separating and cultivating marine bacteria according to claim 3, characterized in that: The liquid culture medium is a liquid beef extract peptone culture medium, which contains the following components: 0.3% beef extract, 1% peptone, 2% sodium chloride, and the rest is water.

5. The method for separating and cultivating marine bacteria according to claim 3, characterized in that: The plate culture medium is a solid beef extract peptone culture medium, which contains the following components: 0.3% beef extract, 1% peptone, 2% sodium chloride, 2% agar, and the rest is water.

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