Anti-ros probiotics, bacterial agent screening and methods of use and applications for coral farming

Abstract

The present application belongs to the technical field of marine probiotics, and discloses an anti-ROS probiotic for coral culture, a screening method of an anti-ROS probiotic agent for coral culture, a use method of the anti-ROS probiotic agent for coral culture, and application of the anti-ROS probiotic agent for coral culture. Based on the great potential of DMSP active microflora in resisting ROS, the present application obtains an anti-ROS probiotic for coral culture, a screening and use method of the probiotic, and application. By using microecological preparations, the activity of probiotics with antioxidant capacity of marine organisms is improved, an antioxidant function system of coral symbiont is constructed and cultured, the peroxide level of coral water is reduced, the immunity and stress resistance potential of coral symbiont are improved, the quality of the environment of the marine aquarium is improved, the risk of coral whitening and disease is effectively reduced, and the treatment and prevention effects on various diseases caused by "oxidative stress" stress response are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of marine probiotics, in particular to an ROS-resistant probiotic for coral cultivation, and further relates to a screening method of an ROS-resistant probiotic for coral cultivation, a use method of an ROS-resistant probiotic for coral cultivation, and an application of an ROS-resistant probiotic for coral cultivation. BACKGROUND

[0002] Reactive oxygen species (ROS) is a kind of single electron reduction product of oxygen in vivo, which is generally referred to as a general term of oxygen-containing, active substances in the body or natural environment, mainly including one kind of excited state oxygen molecule (singlet oxygen molecule or single line oxygen molecule O2), three kinds of oxygen-containing free radicals (superoxide anion free radical, hydroxyl radical-OH and hydrogen peroxide radical HO2), two kinds of peroxides (hydrogen peroxide H2O2 and peroxide lipid ROOH), and one kind of nitrogen-containing oxide NO, etc.

[0003] The coral symbiotic body is composed of coral polyps and zooxanthellae. Once the zooxanthellae leave the coral, the coral polyps will become white and then slowly die, i.e. coral bleaching. The zooxanthellae that symbiotically coexist with the coral polyps use light for photosynthesis, which can provide up to 90% of the energy and oxygen required for the growth of the coral skeleton. The zooxanthellae not only stably grow in the coral polyps, but also obtain plant nutrients such as carbon dioxide, nitrogen and phosphorus required for photosynthesis from the coral polyps. This symbiotic relationship between the coral polyps and the zooxanthellae not only removes the metabolic waste of the coral polyps, creating a better living environment for the coral polyps, but also helps purify the seawater environment for the coral ecosystem. The frequent occurrence of coral bleaching is mainly due to the weak self-regulation ability of the micro seawater ecosystem, and the fact that the ornamental coral (especially the stony coral) is very sensitive to fluctuations in seawater environment, such as fluctuations in parameters such as temperature, water quality, light, pH, water flow, nutrition and ions in the tank, which may cause the coral to "bleach" overnight. From a biological point of view, the most direct biological mechanism of coral bleaching is "oxidative stress", i.e. when the temperature rises or the light radiation increases, the photosynthetic system of the symbiotic algae is damaged, resulting in excessive ROS free radicals, which are leaked into the host cells, causing ROS damage, leading to a decline in immune function and triggering bleaching and diseases. Most ornamental organisms grow by photosynthesis, and to some extent, the "oxidative stress" state in the aquarium system is inevitable. At present, there is no good method to effectively prevent coral bleaching. Research has found that it is a major technical bottleneck for marine microecological preparations to quickly and effectively reduce the ROS free radical level in the aquarium system and achieve stable state control of the ROS balance of marine ornamental organisms.

[0004] Coral symbionts have developed a unique set of mechanisms to resist environmental changes, bleaching, and pests and diseases through long-term evolution—DMSP (dimethylthiopropionic acid) metabolic active substances, including DMS (dimethyl malonate), AA (acrylic acid), DMSO (dimethyl sulfoxide), TDA (m-toluenediamine), and other compounds. These active substances play important roles in resisting bleaching, antibacterial activity, acquired immunity, and abiotic stress. However, there are currently no microecological preparations based on DMSP metabolic active substances in the marine aquarium market.

[0005] Because DMSP reacts rapidly with ROS, it is considered an effective cellular scavenger that destroys free radicals. Furthermore, the decomposition products of DMSP, DMS and acrylates, are even more reactive than DMSP under laboratory conditions, with an oxidation efficiency 20-60 times higher. These three compounds together constitute a highly effective antioxidant system. This invention addresses coral bleaching by leveraging the significant potential of DMSP-active microbial communities in combating ROS. It provides a method for screening and using ROS-resistant probiotics and agents in coral aquaculture, along with their application. By employing microecological preparations to enhance the activity of probiotic communities with antioxidant capabilities in marine organisms, an antioxidant functional system is constructed and cultivated within the coral symbiont, reducing peroxide levels in the coral water, improving the immunity and stress resistance of the coral symbiont, improving the quality of the marine aquarium environment, and reducing the risk of coral bleaching and disease. Summary of the Invention

[0006] The present invention aims to provide a method and application of anti-ROS probiotics, bacterial agents screening and use for coral farming, in order to solve the problems of coral bleaching and disease in existing marine aquariums.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] ROS-resistant probiotics for coral aquaculture, wherein the ROS-resistant probiotics are composed of *A. shortwave monocytogenes* strain D6 cells at a final concentration of not less than 1 × 10⁻⁶. 12 Zooxanthindella type D cells / mL, final concentration not less than 1×10⁻⁶ 8 The cell / mL composition includes DMSP / DMS / AA, vitamins, amino acids, trace elements, CaCl2, and artificial seawater. The shortwave monocellular strain D6 was isolated from the tissue of *Haloxylon ammodendron*, and deposited on March 18, 2021, at the China General Microbiological Culture Collection Center (CGMCC), No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The depositor is the Institute of Microbiology, Chinese Academy of Sciences, and the accession number is CGMCC NO.: 22031.

[0009] Furthermore, the culture temperature of the shortwave monocellular strain D6 is 25–28°C, and the salinity is 30–35‰.

[0010] Further, the culture pH value of the Brevundimonas strain D6 is 7.85-8.45.

[0011] The screening method of the ROS-resistant probiotic agent for coral culture uses DMSP active microorganisms to screen the ROS-resistant probiotic agent for coral culture, and comprises the following steps:

[0012] S1, isolation, purification and identification of DMSP metabolic active bacteria: through in-situ culture of endogenous bacteria in ornamental hard and soft coral tissues, skeletons and seawater, using MAMS and MASW basic culture media for improvement, using DMSP or DMS or AA as the sole carbon source, DMSP metabolic active bacteria are cultured, isolated and purified, and the 16S-rDNA fragments of the purified bacteria are subjected to PCR amplification, and the bacterial species are identified through sequence comparison analysis combined with morphological analysis;

[0013] S2, trait screening and function analysis of microorganisms: the DMSP degradation and nitrogen cycle function genes of the above-mentioned isolated strains are amplified by PCR, and endogenous probiotic bacteria with sulfur cycle and nitrogen cycle metabolic potential are screened out;

[0014] S3, ROS-resistant strain screening: the hydrogen peroxidase test is used to screen strains with ROS-resistant strain potential, and the Solaibo hydrogen peroxidase activity detection kit is used for detection to further screen and verify strains with high hydrogen peroxidase activity value, finally, the Brevundimonas strain D6 has the strongest hydrogen peroxidase activity, and the hydrogen peroxidase activity is as high as 0.3768u / 10 5 cells, and the strain D6 is used for the development of coral ROS-resistant agents.

[0015] Further, in S2, dmdA, dddD, dddL and dddP are obtained by DMSP degradation, and nifH and nirK are obtained by nitrogen cycle.

[0016] The use method of the ROS-resistant probiotic agent for coral culture comprises adding 10mL of bacterial liquid in each 100L of water body, so that the total number of active microbial flora is ≥5×10 10 / L; the agent is added once every 10-15 days to keep the number of microbial flora in the water body stable; the low-temperature storage is carried out at 4-8℃; the effective period is 6 months; before use, the bacterial liquid is shaken to make the microbial flora evenly distributed.

[0017] The ROS-resistant probiotic agent for coral culture is applied in the cultivation of seawater breeding corals, fish, shrimps and shellfish, and in the repair of coral reefs.

[0018] The beneficial effects of the technical solutions are:

[0019] The present application is based on the great potential of DMSP active microorganisms in resisting ROS, and a ROS-resistant probiotic for coral cultivation, a screening method and use method and application are prepared. By using microecological preparations to improve the activity of antioxidant probiotic groups of marine organisms, the antioxidant function system of coral symbiotic body is constructed and cultured, the peroxide level of coral water body is reduced, the immunity and stress resistance potential of coral symbiotic body are improved, the quality of marine aquarium water environment is improved, the risk of coral whitening and disease is effectively reduced, and the treatment and prevention effects on various diseases caused by "oxidative stress" stress response are achieved. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A pH sensitivity test curve of the brevibacterium strain D6 in embodiment 2 of the present application;

[0021] Figure 2 A test curve of the activity half-life of the brevibacterium strain D6 in embodiment 3 of the present application;

[0022] Figure 3 A packaging diagram of the ROS-resistant probiotic product for coral cultivation prepared in embodiment 4 of the present application. DETAILED DESCRIPTION

[0023] The present application will be further described in detail below in combination with the drawings and embodiments:

[0024] Embodiment 1

[0025] Catalase activity test and strain screening

[0026] In this experiment, the Solaibo catalase (CAT) activity detection kit was used for detection, and the detection method steps were as follows:

[0027] (1) Crude enzyme solution extraction

[0028] The third generation of bacteria was collected into a centrifuge tube, and the supernatant was discarded after centrifugation; 1 mL of extraction solution was added for every 50 million bacteria, and the bacteria were broken by ultrasonic wave (power 20%, ultrasonic 3s, interval 10s, repeated 30 times); centrifuged at 8000r / min, 4℃ for 10min, and the supernatant was taken and placed on ice for detection.

[0029] (2) Enzyme label preheating

[0030] 1. Adjust the wavelength to 240nm for more than 30min, and adjust the zero with distilled water;

[0031] 2. Preparation of CAT detection working solution: add 25mL of reagent one to reagent two, mix well, and use as working solution;

[0032] 3. CAT detection working solution was placed in water bath at 25℃ for more than 10 minutes before determination;

[0033] 4. 10 μL sample and 190 uL working solution were added into micro quartz cuvette, mixed immediately and timed, and the initial absorbance value A1 at 240 nm and the absorbance value A2 after 1 minute were recorded.

[0034] (3) CAT activity calculation

[0035] Definition of enzyme activity unit (u): 1 nmol H2O2 degraded per minute in reaction system per 10,000 bacteria or cells is defined as one enzyme activity unit (u).

[0036] CAT (u / 104 cell) = [ΔA x V 反总 ÷ (ε x d) x 109] ÷ (V 样 ÷ V 样总 x 500 ÷ T) = 1.529 x ΔA

[0037] ΔA = A1 - A2

[0038] V 反总 : total volume of reaction system, 2 x 10 -4 L;

[0039] ε: molar extinction coefficient of H2O2, 4.36 x 10 4 L / mol / cm;

[0040] d: 96-well plate, optical path: 0.6 cm;

[0041] V 样 : sample volume, 0.01 mL;

[0042] V 样总 : extraction liquid volume, 1 mL;

[0043] T: reaction time, 1 min;

[0044] 500: total number of bacteria, 5 million;

[0045] 10 9 : unit conversion factor, 1 mol = 10 9 nmol

[0046] As shown in Table 1, among the 75 strains screened, the CAT activity values of most strains were basically between 0.01 and 0.03, for example, the CAT value of Alteromonas sp. ZD22-1 (GDMCC 61647) was 0.029 ± 0.0064, and the CAT activity of some strains was even lower than 0.001 u / 10 5. The Brevundimonas sp strain D6 showed the strongest antioxidant capacity, with CAT activity reaching 0.3768 u / 10 5 Therefore, the Brevundimonas sp strain D6 was selected for the development of a coral ROS-resistant agent.

[0047] Table 1 Hydrogen peroxidase activity of different strains

[0048]

[0049]

[0050]

[0051] Note: ND indicates below the detection limit

[0052] Example 2

[0053] As shown in Figure 1 , the pH sensitivity test of Brevundimonas sp D6 strain

[0054] Sterile 50 mL conical flasks were prepared, and 25 mL of previously sterilized and adjusted Zobell 2216E medium with pH 7.85, pH 8.15, and pH 8.45 was poured into each conical flask, respectively. Then 20 uL of Brevundimonas sp D6 strain solution was added and mixed thoroughly. After inoculation, the conical flasks were placed in a 28°C shaker and the absorbance at 600 nm was measured at different times, and the data was recorded. The results showed that the growth of Brevundimonas sp strain D6 at pH 7.85 was better than that at pH 8.15 and pH 8.45. Therefore, strain ZD71 is more suitable for growing in acidic seawater conditions.

[0055] Example 3

[0056] As shown in Figure 2 , the viability cycle determination of Brevundimonas sp D6 strain

[0057] Brevundimonas sp strain D6 was cultured to the stationary phase, and the bacterial cells were collected by centrifugation at 6000 r / min. The bacterial cells were diluted with artificial seawater (35‰) to a target concentration of OD600 = 1.0, with a concentration of about 1 × 10 9The target concentration of the bacteria is put into a coral tank containing sterilized seawater. The residual concentration of the microorganisms put into the coral tank is detected by an ATP fluorescence detector, and the detection frequency is 2-3 times per day. The ATP fluorescence detector is based on the firefly light-emitting principle and uses the "luciferase-luciferin system" to quickly detect adenosine triphosphate (ATP). Since all living cells contain a constant amount of ATP, the ATP content can clearly indicate the concentration or biomass of microorganisms in the sample. The specific operation method is described in the instrument manual. An equal amount of sterile water is set as a blank control.

[0058] The experimental results show that the average ATP fluorescence intensity (RLU) of Brevundimonas.sp strain D6 after inoculation is 170, and the average RLU on the 22nd day is 90, indicating that the half-life of Brevundimonas.sp strain D6 is 17 days, and the average RLU on the 25th day is close to the blank control. To ensure that Brevundimonas.sp strain D6 has strong biological activity in the developed seawater, the inoculation frequency is set to 15-20 days per cycle based on the experimental results. The viability cycle determination experiment of Brevundimonas.sp strain D6 will provide data support for future development of coral ROS-resistant bacterial agents.

[0059] Example 4

[0060] Formulation and use of ROS-resistant probiotics for coral farming

[0061] The main components of the ROS-resistant probiotics for coral farming are ROS-resistant active probiotics (Brevundimonas.sp strain D6), D-type zooxanthellae, DMSP / DMS / AA, vitamins, amino acids, trace elements, CaCl2, artificial seawater, etc. As shown in Figure 3 , the design specifications are 1 liter / bottle and 500 milliliters / bottle.

[0062] Specific preparation steps:

[0063] 1) Collect Brevundimonas.sp strain D6 bacteria by centrifugation at 5000-7000 rpm.

[0064] 2) Sterilize seawater with a salinity of 30-35 under high temperature and pressure conditions;

[0065] 3) Add Brevundimonas.sp strain D6 bacteria to the sterilized seawater, and the final concentration should not be less than 1x10 12Cells / mL, D-type of green algae, final concentration not less than 1 x 10 8 Cells / mL, DMSP / DMS0 / AA, final concentration of 100-1000 μm, appropriate amount of multi-dimensional amino acids, appropriate amount of vitamins, and appropriate amount of trace elements.

[0066] Method of use:

[0067] Notes for the use of marine micro-ecological preparation-anti-vibrio pathogenic probiotic agent:

[0068] 1) Method of use: add 10 mL of bacterial solution to every 100 L of water body, so that the total number of active microbial flora is ≥5 x 10 10 / L;

[0069] 2) Period of use: add the bacterial agent once every 10-15 days to keep the number of flora stable in the water body;

[0070] 3) Storage conditions: should be stored at 4-8°C for low-temperature preservation;

[0071] 4) Validity period: 6 months;

[0072] 5) Note that it needs to be shaken well before use to ensure uniform distribution of the flora.

[0073] Example 5

[0074] Effect evaluation of anti-ROS probiotic bacterial agent for coral cultivation

[0075] a. Active bacteria introduction test and coral temperature stress experiment

[0076] Set up two temperature (25-26°C and 29-30°C) Brevundimonas.sp strain D6 introduction treatment experimental groups of Pocillopora damicornis for comparison:

[0077] (1) Non-inoculated probiotic control group;

[0078] (2) Inoculated probiotic experimental group. Specific experimental steps: first, acclimate the corals in the above 1-3 treatment experimental groups at normal temperature 25°C for 10 days, and take samples for detection analysis. Then, increase the temperature to 29°C for feeding, and take samples for detection analysis on the 15th day and the 20th day. The experimental results show that the white coral rate of the non-inoculated probiotic control group is significantly higher than that of the inoculated probiotic experimental group after 20 days of feeding at 29°C. This shows that Brevundimonas.sp strain D6 has a therapeutic and preventive effect on high temperature stress.

[0079] b. Detection of physiological and health indicators of experimental corals

[0080] The samples in the above experimental stage are detected, the DNA of the coral tissue and the marine sample is extracted, the microbial community structure changes of the coral tissue and the seawater in each experimental treatment are analyzed by 16S tag sequencing analysis; the coral tissue is taken and the ROS level of each treated coral is detected by DCFH-DA active oxygen ROS fluorescent probe under a laser confocal microscope. The experimental results show that the relative abundance of Brevundimonas.sp in the inoculation of probiotics experimental group is obviously higher than that in the non-inoculation of probiotics control group. The active oxygen ROS level of the non-inoculation of probiotics control group is obviously higher than that of the inoculation of probiotics experimental group, which indicates that the Brevundimonas.sp strain D6 has an anti-ROS effect.

[0081] The above is only an embodiment of the present application, and the specific technical solutions or common knowledge of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the technical solutions of the present application, some modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect and practicality of the present application. The protection scope of the present application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. ROS-resistant probiotics for coral aquaculture, characterized by: The anti-ROS probiotics are derived from strain D6 of the genus *Shortwave Monota* (… Brevundimonas sp Composed of bacterial cells, Zooxanthus type D, DMSP / DMS / AA, vitamins, amino acids, trace elements, CaCl2, and artificial seawater; among which, the strain D6 of the genus *Shortwave Monoclinicia* (Syntrophus spp.) Brevundimonas sp The final concentration of the bacteria should not be less than 1×10⁻⁶. 12 Cells / mL, final concentration of Zooxanthus type D not less than 1×10⁻⁶ cells / mL 8 Cells / mL, final concentration of DMSP / DMS / AA was 100~1000 μmol; the shortwave monocellular strain D6 was isolated from the tissue of *Archiveia cumingii*, and deposited on March 18, 2021 at the China General Microbiological Culture Collection Center (CGMCC), No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.: 22031.

2. The method of using the anti-ROS probiotics according to claim 1, characterized in that: This includes adding 10 mL of bacterial solution per 100 L of water to ensure a total number of active microorganisms ≥ 5 × 10⁻⁶. 10 / L; add the bacterial agent every 10 to 15 days to keep the number of bacteria in the water stable; store at low temperature of 4 to 8℃; shelf life of 6 months; shake well before use to ensure even distribution of bacteria.

3. The application of the anti-ROS probiotics as described in claim 1 in coral reef restoration.

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