Bacillus desiccabilis strain xt23 against vibrio parahaemolyticus, culture method and application thereof

Abstract

This invention provides a strain of *Bacillus desertica* XT23 resistant to *Vibrio parahaemolyticus*, its culture method, and its applications, belonging to the field of functional microbial technology. A strain of *Bacillus desertica* (… Bacillus inaquae Bacillus desertis strain XT23, with accession number GDMCC No: 67779, exhibits significant inhibitory effects against Vibrio parahaemolyticus, achieving an inhibition rate of 66.67%, demonstrating good potential for biocontrol. Optimization of the culture medium composition and fermentation process significantly increased bacterial biomass and the yield of antimicrobial active substances. The fermentation cycle was stable and reproducible, which is beneficial for further enhancing its activity against Vibrio parahaemolyticus.

Description

Technical Field

[0001] This application belongs to the field of functional microbial technology, specifically relating to a strain of *Bacillus desertica* resistant to *Vibrio parahaemolyticus*. Bacillus inaquosorum ) strain XT23 and its culture methods and applications. Background Technology

[0002] Vibrio parahaemolyticus is a common aquatic pathogen that easily causes diseases in aquatic animals and may endanger human health through the food chain. Current methods for controlling this bacterium mainly include chemical disinfectants and antibiotics, but these can easily lead to residues, drug resistance, and environmental pollution.

[0003] Currently, existing technology discloses a *Pediococcus pentosaceus* strain with high aggregation activity, which can be used for the reduction and purification of *Vibrio parahaemolyticus* in water bodies, avoiding the use of chemical bacteriostatic agents and antibiotics for water reduction and purification, providing a new means for the biological control of *Vibrio parahaemolyticus* in aquaculture and temporary holding water bodies (CN 104611255 A). However, there are still reports on the use of more types of functional microorganisms for the inhibition of *Vibrio parahaemolyticus* and the prevention and control of its diseases. Summary of the Invention

[0004] The purpose of this application is to provide a strain of Bacillus desertica XT23 that has good antibacterial activity against Vibrio parahaemolyticus.

[0005] This invention provides a strain of Bacillus desertis ( Bacillus inaquosorum The strain XT23, with accession number GDMCC No: 67779.

[0006] This invention provides a fermentation method for the aforementioned Bacillus desertis strain XT23, comprising the following steps: The seed culture of Bacillus desertis strain XT23 was inoculated into a fermentation medium for fermentation culture to obtain the fermentation broth; The fermentation medium comprises the following components in the indicated mass concentrations: 6%~12% carbon source, 2%~5.5% nitrogen source, 0.1%~0.5% light calcium carbonate, 0.2%~0.8% sodium chloride, 0.2%~0.5% antifoaming agent, and the remainder being water; During fermentation, the pH value was 6.3-6.8, the rotation speed was 180-250 rpm, the temperature was 27-33℃, and the fermentation time was 24-48h.

[0007] Preferably, the carbon source includes a single carbon source and a mixed carbon source with a mass ratio of 1.8~2.2:0.8~1.2; The single carbon source includes at least one of the following: glucose, sucrose, and maltose; The mixed carbon source includes molasses.

[0008] Preferably, the nitrogen source includes at least one of the following: peptone, yeast powder, fish meal, soybean meal hydrolysate, and corn steep liquor.

[0009] Preferably, the pH value is 6.5~6.6, the rotation speed is 200~250 rpm, the temperature is 29~31℃, and the fermentation time is 28~42h.

[0010] The present invention provides a biological agent comprising at least one of the following: the Bacillus desertica strain XT23, metabolites of the Bacillus desertica strain XT23, and fermentation broth obtained by the fermentation method.

[0011] Preferably, the desert Bacillus strain XT23 is in the form of cells and / or spores.

[0012] Preferably, the viable bacterial concentration of the *Bacillus desertica* strain XT23 is 5 × 10⁻⁶. 8 CFU / mL.

[0013] This invention provides the application of the aforementioned Bacillus desertis strain XT23, the fermentation broth obtained by the fermentation method, or the biological agent in the in vitro inhibition of Vibrio parahaemolyticus.

[0014] This invention provides the use of the aforementioned Bacillus desertis strain XT23, the fermentation broth obtained by the fermentation method, or the biological agent in the preparation of drugs for the prevention and / or treatment of diseases caused by Vibrio parahaemolyticus.

[0015] This invention provides a strain of Bacillus desertis, XT23, with accession number GDMCC No: 67779. Strain XT23 was screened from a marine aquaculture environment and fermented using optimized culture medium and processes, significantly improving the fermentation efficiency of the fermentation broth. Simultaneously, the fermentation broth exhibits inhibitory effects against Vibrio parahaemolyticus and demonstrates significant advantages compared to other conventional Bacillus strains, providing a new approach for the green control of Vibrio diseases in aquaculture. Attached Figure Description

[0016] Figure 1 The antibacterial results of Bacillus desertis strain XT23; Figure 2 The result indicates the antibacterial activity of Lactococcus lactis; Figure 3 The results show the antibacterial effect of Bacteroides; Figure 4 The results show the antibacterial effect of Bacillus subtilis.

[0017] Information on the preservation of biological materials Desert Bacillus ( Bacillus inaquosorumStrain XT23 was deposited on February 2, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with accession number GDMCC No: 67779. Detailed Implementation

[0018] This invention provides a strain of Bacillus desertis XT23, with accession number GDMCC No: 67779.

[0019] In this invention, the *Bacillus desertica* strain XT23 was screened from a marine aquaculture environment. The morphological characteristics of *Bacillus desertica* strain XT23 are elongated rod-shaped Gram-positive bacteria; the colonies are round, white, and have a wrinkled surface. *Bacillus desertica* strain XT23 was identified as *Bacillus desertica* by 16S rRNA molecular identification. Bacillus inaquosorum ).

[0020] This invention provides a fermentation method for the aforementioned Bacillus desertis strain XT23, comprising the following steps: The seed culture of Bacillus desertis strain XT23 was inoculated into a fermentation medium for fermentation culture to obtain the fermentation broth; The fermentation medium comprises the following components in the indicated mass concentrations: 6%~12% carbon source, 2%~5.5% nitrogen source, 0.1%~0.5% light calcium carbonate, 0.2%~0.8% sodium chloride, 0.2%~0.5% antifoaming agent, and the remainder being water; During fermentation, the pH value was 6.3-6.8, the rotation speed was 180-250 rpm, the temperature was 27-33℃, and the fermentation time was 24-48h.

[0021] In this invention, the fermentation medium preferably comprises the following components by mass concentration: 7.5%~10.5% carbon source, 2.5%~3.5% sweetener, 2.5%~5.0% nitrogen source, 0.2%~0.4% light calcium carbonate, 0.3%~0.7% sodium chloride, 0.3%~0.4% antifoaming agent, and the balance being water; or it can be 8.7% carbon source, 2.7% sweetener, 3.5% nitrogen source, 0.2% light calcium carbonate, 0.6% sodium chloride, 0.3% antifoaming agent, and the balance being water. The carbon source includes a single carbon source and a mixed carbon source in a mass ratio of 1.8~2.2:0.8~1.2, or a single carbon source and a mixed carbon source in a mass ratio of 2:1. The single carbon source preferably includes at least one of the following: glucose, sucrose, and maltose. The mixed carbon source preferably includes molasses. The glucose is preferably glucose monohydrate. The nitrogen source preferably includes at least one of the following: peptone, yeast extract, fish meal, soybean meal hydrolysate, and corn steep liquor. In one embodiment of the invention, the peptone concentration in the fermentation medium is preferably 0.5%~2% (1.0%, 1.2%, 1.5%) and the yeast extract concentration is preferably 1.5%~3.5% (2.0%, 2.5%, and 3.0%). In another embodiment of the invention, the fermentation medium contains 1% fish meal and 2.5% yeast extract. In yet another embodiment of the invention, the fermentation medium contains 1% soybean meal hydrolysate and 2.5% corn steep liquor.

[0022] In this invention, the pH value is preferably 6.5-6.6. The rotation speed is preferably 200-250 rpm, and can be 210-230 rpm. The temperature is preferably 29-31℃, and can be 30℃. The fermentation time is preferably 28-42 hours, and can be 30-36 hours.

[0023] In this invention, by optimizing the composition of the fermentation medium and the fermentation parameters, the biomass of the cells and the yield of antibacterial active substances are significantly increased, the fermentation cycle is stable, and the repeatability is good.

[0024] The present invention provides a biological agent comprising at least one of the following: the Bacillus desertica strain XT23, metabolites of the Bacillus desertica strain XT23, and fermentation broth obtained by the fermentation method.

[0025] In this invention, the form of the *Bacillus desertica* strain XT23 preferably includes bacterial cells and / or spores. The bacterial cells are vegetative XT23 cells, representing the cellular form of the XT23 strain in a state of active growth, reproduction, and metabolism; they are characterized by intact structure, vigorous metabolism, and sensitivity to environmental conditions, and are the main form in which the bacteria exert their physiological and pathogenic effects. The spores are dormant structures formed inside the bacterial cells under stress conditions such as nutrient deficiency and harsh environments. They are round or oval, thick-walled, low in water content, highly heat- and dryness-resistant, and extremely resilient; they lack reproductive capacity, their metabolism is essentially quiescent, and their resistance to adverse external environments (high temperature, radiation, chemical disinfectants, etc.) is far stronger than that of vegetative bacterial cells.

[0026] In this invention, the biological agent comprises at least one of the following: lyophilized powder, aqueous solution or suspension, wettable powder, granules, and microcapsules. This invention does not impose any particular limitation on the preparation method of the biological agent; any preparation method of biological agents well known in the art can be used. The preferred viable cell concentration of the *Bacillus desertica* strain XT23 is 3 × 10⁻⁶. 8 CFU / mL ~8×10 8 CFU / mL, which can be 5 × 10 8 CFU / mL.

[0027] This invention provides the application of the aforementioned Bacillus desertis strain XT23, the fermentation broth obtained by the fermentation method, or the biological agent in the in vitro inhibition of Vibrio parahaemolyticus.

[0028] In this invention, in vitro inhibition experiments on Vibrio parahaemolyticus showed that the Bacillus desertica strain XT23 or its metabolites had a significant antibacterial effect on Vibrio parahaemolyticus, with an inhibition zone diameter ≥15 mm, demonstrating good potential for biocontrol.

[0029] Given that Vibrio parahaemolyticus is a common pathogenic bacterium in aquatic animals, causing diseases in aquatic animals or infecting humans, the present invention provides the use of the aforementioned Bacillus desertica strain XT23, the fermentation broth obtained by the fermentation method, or the aforementioned biological agent in the preparation of drugs for the prevention and / or treatment of diseases caused by Vibrio parahaemolyticus.

[0030] The following detailed description, in conjunction with embodiments, illustrates a Desert Bacillus strain XT23 resistant to Vibrio parahaemolyticus provided by the present invention, its culture method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 Methods for the isolation and identification of a strain of Bacillus desertis XT23 Samples were collected from a marine aquaculture farm in Tangshan in December 2023. The samples were inoculated into LB medium for screening and culture. The resulting single colonies were then inoculated into liquid medium for screening and cultured to obtain a strain XT23.

[0032] Morphological identification of strain XT23: long rod-shaped Gram-positive bacteria, with round, white colonies and wrinkled surfaces.

[0033] Molecular identification of strain XT23: Molecular identification was performed based on 16S rRNA. Genomic DNA was extracted from the strain, and PCR amplification was performed using universal primers for bacterial 16S rRNA with the genomic DNA as a template. The PCR products were then recovered and purified using a gel extraction kit, followed by cloning, transformation, and screening for positive clones. After amplification culture, the colonies were sequenced by Beijing Ruiboxingke Biotechnology Co., Ltd. The 16S rRNA sequence of strain XT23 is shown in SEQ ID NO:1.

[0034] The sequencing results were compared with the 16S rRNA sequences in GenBank to determine the species relationship of the strains. The comparison result of strain XT23 was closest to *Bacillus desertica*. The identification result of strain XT23 was *Bacillus desertica* (…). Bacillus inaquosorum The strain XT23 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 2, 2026, with accession number GDMCC No: 67779.

[0035] Example 2 A method for preparing fermentation broth of Bacillus desertis strain XT23 Bacillus desertica strain XT23 was inoculated at a 10% inoculum in seed culture medium (10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride) and cultured at 27°C for 12 h to obtain seed liquid. The seed liquid was then inoculated at a 5% inoculum in sterile fermentation medium (4% glucose monohydrate, 4% molasses, 0.5% peptone, 3.5% yeast extract, 0.1% light calcium carbonate, 0.8% sodium chloride, 0.2% antifoaming agent, and the remainder being water). Fermentation was carried out at 27°C and 180 rpm for 24 h, with the pH of the fermentation system controlled at 6.5–6.6. After fermentation, the fermentation broth was obtained.

[0036] Take a portion of the fermentation broth and transfer it to a centrifuge tube. Centrifuge at 5000 rpm for 10 minutes, collect the solid phase, dry the solid phase cells, weigh them, and calculate the cell concentration.

[0037] The concentration of Bacillus desertis strain XT23 in the fermentation broth was calculated to be 12.5 g / L.

[0038] Example 3 A method for preparing fermentation broth of Bacillus desertis strain XT23 Bacillus desertica strain XT23 was inoculated at 8% in seed culture medium (10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride) and cultured at 27°C for 12 h to obtain seed liquid. The seed liquid was then inoculated at 6% in sterile fermentation medium (8% glucose monohydrate, 2% molasses, 2% peptone, 1.5% yeast extract, 0.5% light calcium carbonate, 0.2% sodium chloride, 0.5% antifoaming agent, and the remainder being water) and fermented at 33°C and 250 rpm for 48 h. The pH of the fermentation system was controlled at 6.5-6.6. After fermentation, the fermentation broth was obtained.

[0039] The bacterial cells in the fermentation broth were isolated according to the method in Example 2. The concentration of *Bacillus desertica* strain XT23 in the fermentation broth was calculated to be 10.5 g / L.

[0040] Example 4 A method for preparing fermentation broth of Bacillus desertis strain XT23 Bacillus desertica strain XT23 was inoculated at 8% in seed culture medium (10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride) and cultured at 27°C for 12 h to obtain seed liquid. The seed liquid was then inoculated at 6% in sterile fermentation medium (5.7% glucose monohydrate, 2.7% molasses, 1% peptone, 2.5% yeast extract, 0.2% light calcium carbonate, 0.6% sodium chloride, 0.3% antifoaming agent, and the remainder being water) and fermented at 30°C and 250 rpm for 24 h. The pH of the fermentation system was controlled at 6.5-6.6. After fermentation, the fermentation broth was obtained.

[0041] The bacterial cells in the fermentation broth were isolated according to the method in Example 2. The concentration of *Bacillus desertica* strain XT23 in the fermentation broth was calculated to be 11.2 g / L.

[0042] Example 5 A method for preparing fermentation broth of Bacillus desertis strain XT23 Bacillus desertis strain XT23 was inoculated at 8% in seed culture medium (10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride) and cultured at 27°C for 12 h to obtain seed liquid. The seed liquid was then inoculated at 6% in sterile fermentation medium (5.7% sucrose, 2.0% molasses, 1% fish meal, 2.5% yeast extract, 0.2% light calcium carbonate, 0.6% sodium chloride, 0.3% antifoaming agent, and the remainder being water) and fermented at 30°C and 250 rpm for 24 h. The pH of the fermentation system was controlled at 6.5–6.6. After fermentation, the fermentation broth was obtained.

[0043] The bacterial cells in the fermentation broth were isolated according to the method in Example 2. The concentration of *Bacillus desertica* strain XT23 in the fermentation broth was calculated to be 10.2 g / L.

[0044] Example 6 A method for preparing fermentation broth of Bacillus desertis strain XT23 Bacillus desertica strain XT23 was inoculated at 8% in seed culture medium (10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride) and cultured at 27°C for 12 h to obtain seed liquid. The seed liquid was then inoculated at 6% in sterile fermentation medium (5.7% maltose, 2.7% molasses, 1% soybean meal hydrolysate, 2.5% corn steep liquor, 0.2% light calcium carbonate, 0.6% sodium chloride, 0.3% antifoaming agent, and the remainder being water) and fermented at 28°C and 250 rpm for 24 h. The pH of the fermentation system was controlled at 6.5-6.6. After fermentation, the fermentation broth was obtained.

[0045] The bacterial cells in the fermentation broth were separated according to the method in Example 2. The concentration of *Bacillus desertica* strain XT23 in the fermentation broth was calculated to be 11 g / L.

[0046] Comparative Example 1 Preparation of fermentation broth for Bacillus desertica strain XT23 based on commonly used Bacillus subtilis fermentation media The XT23 strain was fermented using LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride) under the same fermentation conditions as in Example 4, and the cells were isolated from the fermentation broth according to the method in Example 2.

[0047] The concentration of Bacillus desertis strain XT23 in the fermentation broth was calculated to be 8.3 g / L.

[0048] Comparative Example 2 A method for preparing fermentation broth of Bacillus desertica strain XT23 was developed by adjusting the fermentation pH to 7.2. The experiment was conducted according to the method of Example 4, except that the pH value was controlled at 7.2 during the fermentation process.

[0049] Comparative Example 3 Bacillus subtilis ( Bacillus subtilis Replace strain XT23 in Example 4 as the inoculum, and keep other parameters the same as in Example 4.

[0050] Comparative Example 4 Bacillus berberis ( Bacillus velezensis Replace strain XT23 in Example 4 as the inoculum, and keep other parameters the same as in Example 4.

[0051] Comparative Example 5 Lactococcus lactis ( Lactococcus lactis Replace strain XT23 in Example 4 as the inoculum, and keep other parameters the same as in Example 4.

[0052] Comparative Example 6 Bacteroides ( Bacillus stercoris Replace strain XT23 in Example 4 as the inoculum, and keep other parameters the same as in Example 4.

[0053] Example 7 In vitro antibacterial test 1. Preparation of bacterial plates: Vibrio parahaemolyticus activated 2-3 times was inoculated onto LB agar and incubated at 37°C for 1-2 days. Using a hemocytometer, 5 mL of physiological saline was added to a slant agar plate containing Vibrio parahaemolyticus, and the plate was thoroughly shaken. A single drop was placed in the counting chamber between the slide and coverslip of the hemocytometer and counted under a microscope. The plate was then diluted to a bacterial concentration of 3 × 10⁻⁶. 7 CFU / mL was used to obtain a bacterial suspension of Vibrio parahaemolyticus; the bacterial suspension was added to LB medium at a ratio of 1 mL of bacterial suspension per 100 mL of medium, mixed well, and then quickly dispensed into sterilized empty plates and cooled to prepare plates containing Vibrio parahaemolyticus.

[0054] 2. Antibacterial experiments were conducted using the inhibition zone method. Six wells were punched in the center of each Vibrio parahaemolyticus plate using a sterile punch. 200 μl of the fermentation broth prepared in Examples 2-6 and Comparative Examples 1-6 was added to each well. Three replicates were set up for each experimental group. Uninoculated fermentation media from the corresponding examples and comparative examples served as negative controls. The plates were then incubated at 30°C for 24 hours. The diameter of the inhibition zone was measured using calipers to record the antibacterial effect. The results are shown in Table 1 and [Table data would be inserted here]. Figures 1-4 As shown.

[0055] Table 1. In vitro antibacterial results of different fermentation broths

[0056] Note: The formula for calculating the inhibition rate is: Inhibition rate (%) = (Average inhibition zone diameter - Initial inhibition zone diameter) / Average inhibition zone diameter) × 100%.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of desert Bacillus ( Bacillus inaquosorum strain XT23, characterized in that, It is deposited at the Guangdong Institute of Microbiology, with accession number GDMCC No: 67779.

2. A fermentation method for the Bacillus desertis strain XT23 according to claim 1, characterized in that, Includes the following steps: The seed culture of Bacillus desertis strain XT23 was inoculated into a fermentation medium for fermentation culture to obtain the fermentation broth; The fermentation medium comprises the following components in the indicated mass concentrations: 6%~12% carbon source, 2%~5.5% nitrogen source, 0.1%~0.5% light calcium carbonate, 0.2%~0.8% sodium chloride, 0.2%~0.5% antifoaming agent, and the remainder being water; During fermentation, the pH value was 6.3-6.8, the rotation speed was 180-250 rpm, the temperature was 27-33℃, and the fermentation time was 24-48h.

3. The fermentation method according to claim 2, characterized in that, The carbon source includes a single carbon source and a mixed carbon source with a mass ratio of 1.8~2.2:0.8~1.2; The single carbon source includes at least one of the following: glucose, sucrose, and maltose; The mixed carbon source includes molasses.

4. The fermentation method according to claim 2, characterized in that, The nitrogen source includes at least one of the following: peptone, yeast powder, fish meal, soybean meal hydrolysate, and corn steep liquor.

5. The fermentation method according to any one of claims 2 to 4, characterized in that, The pH value is 6.5~6.6, the rotation speed is 200~250 rpm, the temperature is 29~31℃, and the fermentation time is 28~42h.

6. A biological agent, characterized in that, It includes at least one of the following: the desert Bacillus strain XT23 of claim 1, the metabolites of the desert Bacillus strain XT23, and the fermentation broth obtained by the fermentation method of any one of claims 2 to 5.

7. The biological agent according to claim 6, characterized in that, The desert Bacillus strain XT23 exists in the form of cells and / or spores.

8. The biological agent according to claim 6, characterized in that, The viable cell concentration of the *Bacillus desertica* strain XT23 was 5 × 10⁻⁶. 8 CFU / mL.

9. The use of the Desert Bacillus strain XT23 of claim 1, the fermentation broth obtained by the fermentation method of any one of claims 2 to 5, or the biological agent of any one of claims 6 to 8 in the in vitro inhibition of Vibrio parahaemolyticus.

10. The use of the Bacillus desertis strain XT23 of claim 1, the fermentation broth obtained by the fermentation method of any one of claims 2 to 5, or the biological agent of any one of claims 6 to 8 in the preparation of a medicament for the prevention and / or treatment of diseases caused by Vibrio parahaemolyticus.

Images