Bergeri bivaleriei CB24031-XJ12 and application thereof
By co-culturing the bivalve Berrugella CB24031-XJ12 with sea grapes and utilizing its IAA production ability, the problem of unstable development of upright branches of sea grapes was solved, the growth of sea grapes was promoted and the antioxidant properties were improved, thereby increasing the yield and quality of sea grapes.
Patent Information
- Application Number
- CN202511096955.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-06
- Publication Date
- 2025-09-26
AI Technical Summary
In the existing sea grape cultivation model, the number and length of upright branches are greatly affected by environmental factors, resulting in a decrease in the stability of raw material supply, weakening the economic benefits of the product, and a lack of screening of functional strains that promote the development of upright branches and development of related preparations.
The bivalve strain Berrujeira bivalves CB24031-XJ12 was co-cultured with sea grapes to utilize its ability to produce 3-indoleacetic acid (IAA) to promote the growth and upright branch germination of sea grapes and improve their antioxidant properties.
Significantly promote the growth of sea grapes and the germination of upright branches, increase the yield and quality of sea grapes, improve antioxidant capacity, and enhance the stability of cultivation technology.
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Figure CN120699848A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microorganisms, in particular to a strain of B. bivalvata CB24031-XJ12 and an application thereof. Background Art
[0002] Sea grapes, scientifically known as Caulerpa lentillifera, are a large green algae with important economic and ecological value. Rich in polysaccharides, minerals and antioxidants, sea grapes are widely used in the fields of food, medicine and cosmetics. Sea grapes are named for the emerald green color of the algae and the crystal clear spherical branches on the upright branches, which resemble bunches of grapes. Although the entire algae of sea grapes has edible value, the upright branches have become the core commodity part in commercial development because of their unique crisp and elastic texture and ornamental morphological characteristics. However, in the existing sea grape cultivation model, the number and length of upright branches are greatly affected by environmental factors (such as light, temperature, salinity, and nutrient supply), resulting in a decrease in the stability of raw material supply, thereby weakening the economic benefits of the product.
[0003] Studies have shown that symbiotic bacterial communities can regulate the morphological development of large seaweeds through the exchange of metabolites or the secretion of essential growth factors and exopolysaccharides. However, existing research has largely focused on the fundamental theories of seaweed-microbe interactions, with limited reports on the screening of functional strains specifically regulating the development of upright shoots in sea grapes and the development and application of related agents. Therefore, the development of microbial agents that promote upright shoot development and increase the yield and quality of commercial parts is crucial for improving the stability of cultivation processes and establishing a standardized cultivation technology system. Summary of the Invention
[0004] In view of this, the present invention provides a strain of B. bivalvae CB24031-XJ12 and its application to solve the above problems.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides a strain of B. bivalvae CB24031-XJ12, which is deposited in Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No: 66493, a deposit date of June 10, 2025, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0007] The present invention provides a microbial agent, which contains Berugelia bivalvae CB24031-XJ12.
[0008] The present invention also provides a method for preparing the microbial agent, which comprises inoculating the B. bivalvae CB24031-XJ12 into a 2216E culture medium for fermentation to obtain the microbial agent.
[0009] Preferably, the fermentation temperature is 25-30° C., the rotation speed is 130-200 rpm, and the fermentation time is 16-48 h.
[0010] Preferably, the 2216E liquid culture medium comprises components with the following concentrations: peptone 5.0 g / L, yeast extract powder 1.0 g / L, ferric citrate 0.1 g / L, NaCl 19.45 g / L, MgCl2 5.98 g / L, Na2SO4 3.24 g / L, CaCl2 1.8 g / L, KCl 0.55 g / L, Na2CO3 0.16 g / L, KBr 0.08 g / L, SrCl2 0.034 g / L, H3BO3 0.022 g / L, Na2SiO3 0.004 g / L, NaF 0.0024 g / L, NaNO3 0.0016 g / L, and NaH2PO4 0.008 g / L.
[0011] The present invention also provides the use of the bivalve B. bivalviae CB24031-XJ12 and / or the microbial agent in at least one of the following:
[0012] (1) Application in promoting the growth of sea grapes and the germination of upright branches;
[0013] (2) Application in improving the antioxidant properties of sea grapes;
[0014] (3) Application in the preparation of bacterial agents for promoting the growth of sea grapes;
[0015] (4) Application in the preparation of bacterial agents for improving the antioxidant properties of sea grapes.
[0016] The present invention also provides a method for promoting the growth of sea grapes, comprising co-culturing the sea grapes with the bivalve Berrujea sp. CB24031-XJ12.
[0017] Preferably, in the culture system of sea grapes, the concentration of B. bivalvae CB24031-XJ12 is 5×10 4 ~5×10 6 cfu / mL.
[0018] Preferably, the culture conditions of the co-cultivation are as follows:
[0019] Temperature 26-28℃, salinity 28-33‰, photon flux density 30-60μmol / (m 2s), with a photoperiod of 12L:12D; the culture medium was replaced every 64–72 h, and CB24031-XJ12 was added until the effective viable count reached 5 × 10 4 ~5×10 6 cfu / mL.
[0020] By adopting the above technical solution, the present invention has the following beneficial effects: the bivalve Berruje's bacterium CB24031-XJ12 of the present invention has the ability to produce 3-indoleacetic acid (IAA). Co-cultivation of the bivalve Berruje's bacterium CB24031-XJ12 with sea grapes can significantly promote the growth and upright branch germination of sea grapes, and improve their antioxidant capacity, which is of great significance in improving the yield and quality of sea grapes.
[0021] Biological Deposit Description
[0022] The bivalve Ruegeria conchae CB24031-XJ12 of the present invention is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 66493, a deposit date of June 10, 2025, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is the 16S rRNA phylogenetic tree of B. bivalviae CB24031-XJ12.
[0024] Figure 2 This is the growth-promoting effect of B. bivalviae CB24031-XJ12 on the growth and development of sea grapes after 7 days of co-culture.
[0025] Figure 3 The promoting effect of B. bivalvae CB24031-XJ12 on the growth and stress resistance of sea grapes after 7 days of co-culture (CK was without bacteria, L1 to L3 were added with 5×10 2 , 5×10 4 and 5×10 6 cfu / mL of bacterial solution and co-cultured with sea grapes). DETAILED DESCRIPTION
[0026] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0027] Example 1. Isolation and screening of sea grape symbionts
[0028] Sea grape samples were collected from different cultivation environments in Changpo Town, Qionghai City, Hainan Province, placed in sterile sampling bags, and brought back to the laboratory within 2 hours. The samples were repeatedly rinsed with sterile seawater to remove algae, small animals, and impurities on the surface of the algae.
[0029] Weigh an appropriate amount of algal sample, add 2 mL of sterile seawater and grind in a sterile mortar, take 1 mL of the uniform grinding solution and perform gradient dilution to obtain 10 -1 ~10 -5 Prepare a bacterial suspension with a concentration of cfu / mL. Evenly spread an appropriate amount of the suspension onto a 2216E agar plate and incubate in a 28°C incubator for 48 hours to obtain single colonies. Use an inoculating needle to pick individual colonies of varying morphology and streak them onto the surface of the 2216E agar plate. Repeat the purification process 3-5 times to obtain a purified strain containing a single colony.
[0030] Single colonies with different morphologies were randomly picked and the IAA production ability of the strains was preliminarily screened using the Salkowski colorimetric method. One strain with IAA production ability was screened out: CB24031-XJ12.
[0031] The purified strain CB24031-XJ12 was frozen in glycerol tubes (-80°C).
[0032] Example 2. Identification of CB24031-XJ12
[0033] (1) Morphological characteristics: On 2216E agar medium, the colonies of CB24031-XJ12 were small round particles, white, translucent, and had a smooth and moist surface.
[0034] (2) 16S rRNA sequence analysis: DNA of the strain was extracted and the sequence was analyzed using universal bacterial primers.
[0035] 27F(5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO:1);
[0036] 1492R (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2) was amplified by PCR, and the sequencing results were compared with strains with known sequences in the EzBioCloud database (https: / / www.ezbiocloud.net / identify). The identification results showed that the bacterium was Ruegeria conchae.
[0037] The 16S rRNA sequence of B. bivalviae CB24031-XJ12 is shown in SEQ ID NO: 3.
[0038] SEQ ID NO:3:
[0039]
[0040] (3) Phylogenetic tree was constructed based on the 16S rRNA sequences of B. bivalviae CB24031-XJ12 and related bacteria. Figure 1 shown.
[0041] Example 3. Determination of the ability of B. bivalvae to secrete IAA
[0042] The IAA production capacity of the strain was determined using the Salkowski colorimetric method. The specific steps are as follows:
[0043] (1) B. bivalvata CB24031-XJ12 was activated on 2216E solid medium. A single colony was picked and inoculated into 2216E liquid medium containing 0.1 g / L L-tryptophan. The culture was shaken at 28°C and 130-200 rpm for 1-2 days. After the culture was completed, the supernatant was collected by centrifugation at 8000 rpm for 10 minutes.
[0044] (2) Aspirate an appropriate amount of the supernatant, add an equal volume of Salkowski colorimetric solution, and mix thoroughly. Incubate the reaction tube at room temperature in the dark for 30 minutes. Simultaneously, set up an equal volume of uninoculated L-tryptophan-containing culture medium as a blank control. Three biological replicates were performed for all treatments.
[0045] (3) The absorbance of each reaction mixture was measured at a wavelength of 530 nm. Based on the measured values and the standard curve drawn using the IAA standard, the IAA content per unit volume of the fermentation broth of CB24031-XJ12 was calculated.
[0046] The results showed that the IAA produced by B. bivalvae CB24031-XJ12 was 309.13±6.75 mg / L, which had growth-promoting potential.
[0047] Example 4. Verification of the growth-promoting function of B. bivalviae CB24031-XJ12
[0048] Preparation of 2216E liquid culture medium: distilled water 1000 mL, peptone 5.0 g, yeast extract powder 1.0 g, ferric citrate 0.1 g, NaCl 19.45 g, MgCl2 5.98 g, Na2SO4 3.24 g, CaCl2 1.8 g, KCl 0.55 g, Na2CO3 0.16 g, KBr 0.08 g, SrCl2 0.034 g, H3BO3 0.022 g, Na2SiO3 0.004 g, NaF 0.0024 g, NaNO3 0.0016 g, NaH2PO4 0.008 g, pH = 7.6 ± 0.2, autoclave at 121°C for 15 min.
[0049] A single colony of B. bivalvata CB24031-XJ12 was selected and inoculated into 2216E liquid culture medium. Culture was performed at 28°C and 180 rpm for 24–36 hours to obtain a seed solution. The seed solution was then inoculated into fresh 2216E liquid culture medium at a 1% inoculum rate and cultured for approximately 24 hours to obtain a fermentation broth. The number of viable cells in the fermentation broth was calculated using the plate count method.
[0050] The cells were collected by centrifugation and resuspended in an appropriate amount of sterile seawater to prepare a total concentration of 10 viable bacteria of B. bivalviae CB24031-XJ12. 8 cfu / mL of bacterial agent.
[0051] Sea grapes were placed in 5×10 2 (L1), 5×10 4 (L2) and 5×10 6 cfu / mL(L3) of B. bivalvei CB24031-XJ12 was sterilized in a natural seawater culture system with effective live bacteria at 27±1℃, salinity 32‰, and light quantum flux density 40μmol / (m 2 s) and cultured under a photoperiod of 12L:12D; the culture medium was replaced every 72 h, and bacterial agents were added until the effective viable count was the same as the initial concentration. The group without the addition of B. bivalviae was used as a control.
[0052] The growth of sea grapes was observed regularly, including indicators such as runner elongation, new upright branch sprouting, and growth rate. After 7 days of cultivation, the photosynthetic capacity and physiological indicators of sea grapes were measured, such as specific growth rate, maximum light quantum yield (Fv / Fm), runner length, number of new upright branches sprouting, total antioxidant capacity, and superoxide dismutase (SOD) activity. The results are as follows: Figure 2 and Figure 3 shown.
[0053] The results showed that adding an appropriate amount (final concentration of about 5×10 4 ~5×10 6 cfu / mL) of the bivalve Berrujea CB24031-XJ12 bacterial solution can significantly promote the growth and development of sea grapes, promote the germination of upright branches, and improve its photosynthesis and antioxidant capacity.
[0054] As can be seen from the above examples, the present invention provides a strain of B. bivalvae CB24031-XJ12 and its application. The B. bivalvae CB24031-XJ12 of the present invention can promote the growth of sea grapes and the germination of upright branches, and improve their photosynthesis and antioxidant capacity.
[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A strain of B. bivalvae CB24031-XJ12, characterized in that: It is deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCC No: 66493, the deposit date is June 10, 2025, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. A microbial agent, characterized in that: The microbial preparation comprises Berrugella bivalvata CB24031-XJ12.
3. The method for preparing the microbial agent according to claim 2, characterized in that: The bivalve Berrugella sp. CB24031-XJ12 according to claim 1 is inoculated into 2216E culture medium for fermentation to obtain the microbial agent.
4. The preparation method according to claim 3, characterized in that The fermentation culture temperature is 25-30° C.; the rotation speed is 130-200 rpm; and the fermentation culture time is 16-48 hours.
5. The method according to claim 3, characterized in that The 2216E liquid culture medium includes components with the following concentrations: peptone 5.0 g / L, yeast extract powder 1.0 g / L, ferric citrate 0.1 g / L, NaCl 19.45 g / L, MgCl2 5.98 g / L, Na2SO4 3.24 g / L, CaCl2 1.8 g / L, KCl 0.55 g / L, Na2CO3 0.16 g / L, KBr 0.08 g / L, SrCl2 0.034 g / L, H3BO3 0.022 g / L, Na2SiO3 0.004 g / L, NaF 0.0024 g / L, NaNO3 0.0016 g / L, and NaH2PO4 0.008 g / L.
6. Use of the bivalve B. CB24031-XJ12 according to claim 1 and / or the microbial agent according to claim 2 in at least one of the following: (1) Application in promoting the growth of sea grapes and the germination of upright branches; (2) Application in improving the antioxidant properties of sea grapes; (3) Application in the preparation of a fungal agent for promoting the growth of sea grapes and the germination of upright branches; (4) Application in the preparation of bacterial agents for improving the antioxidant properties of sea grapes.
7. A method for promoting the growth of sea grapes, characterized in that: The sea grapes are co-cultured with the bivalve Berrujea sp. CB24031-XJ12 according to claim 1.
8. The method according to claim 7, characterized in that In the culture system of sea grapes, the concentration of B. bivalviae CB24031-XJ12 was 5×10 4 ~5×10 6 cfu / mL.
9. The method according to claim 7, characterized in that The co-culture conditions are as follows: co-culture temperature 26-28°C, salinity 28-33‰, light quantum flux density 30-60 μmol / (m 2 s), with a photoperiod of 12L:12D; the culture medium was replaced every 64–72 h, and CB24031-XJ12 bacteria were added until the effective viable count reached 5 × 10 4 ~5×10 6 cfu / mL.