Geobacillus saccharophilus JX-8 and application thereof

Abstract

This application belongs to the field of microbial technology, specifically relating to a strain of *Bacillus saccharophilus* JX-8 and its applications. This *Bacillus saccharophilus* (… Terribacillus saccharophilus JX-8 was deposited on May 8, 2025, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2025996. The *Bacillus saccharophilus* JX-8 provided by this invention has simple culture conditions, rapid growth, and is easy to prepare on a large scale. Inoculants based on this JX-8 can be used for biological detoxification of feed, food ingredients, etc.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, specifically relating to a strain of Bacillus saccharophilus JX-8 and its applications. Background Technology

[0002] Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus species (…). Aspergillus ) and Penicillium ( Penicillium The toxic secondary metabolites produced by fungi widely contaminate various agricultural products and foods, including grains, beans, coffee, and wine. OTA (Anti-Oxygen sulfide) exhibits strong nephrotoxicity, hepatotoxicity, teratogenicity, and immunotoxicity in humans and animals, and is classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC), posing a serious threat to human health.

[0003] Currently, detoxification methods for OTA (Ointment toxins) mainly include physical, chemical, and biological methods. Physical and chemical methods often suffer from problems such as unstable detoxification efficiency, incomplete detoxification, loss of nutrients, chemical reagent residues, and high costs. Biodegradation methods utilize microorganisms or their enzyme systems to degrade toxins. They have advantages such as mild operating conditions, high specificity, high safety, and no impact on product quality, and are considered the most promising detoxification method.

[0004] Currently reported OTA-degrading microorganisms include several genera such as lactic acid bacteria, Bacillus, and Acinetobacter. However, these strains vary in degradation efficiency, stability, and applicability, and there are differences in the genus *Agrobacterium* (Saccharophilus). Terribacillus The ability of strains to degrade OTA has not been reported. Therefore, discovering strains with efficient and stable OTA degradation capabilities is of great significance for developing novel biological detoxification products and ensuring food and feed safety. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a strain of Bacillus saccharophilus JX-8 and its applications, specifically adopting the following technical solution:

[0006] In a first aspect, the present invention provides a strain of *Bacillus saccharophilus* JX-8, wherein *Bacillus saccharophilus* ( Terribacillus saccharophilus JX-8 was deposited on May 8, 2025 at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2025996.

[0007] The *Bacillus saccharophilus* JX-8 provided by this invention was isolated and screened from Chinese pickled vegetable juice. The 16S rRNA sequence of *Bacillus saccharophilus* JX-8 is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009]

[0010] Secondly, the present invention provides a microbial inoculant, characterized in that it comprises the aforementioned Bacillus saccharophilus JX-8 and / or its fermentation products. Preferably, the fermentation products include its whole-cell fermentation broth, bacterial cells, or cell lysates.

[0011] Thirdly, the present invention provides the application of the above-mentioned Bacillus saccharophilus JX-8 or the above-mentioned microbial agent in the degradation of ochratoxin A or in the preparation of ochratoxin A degrading agent.

[0012] As a further preferred embodiment, the degrading agent includes feed additives, food additives, or biological detoxifiers.

[0013] Fourthly, the present invention provides an ochratoxin A degrading agent, comprising the above-mentioned Bacillus saccharophilus JX-8 or its fermentation product.

[0014] As a further preferred embodiment, the concentration of live Bacillus saccharophilus in the degrading agent is 1×10⁻⁶. 8 CFU / mL - 1×10 11 CFU / mL.

[0015] As a further preferred embodiment, the ochratoxin A degrading agent is in the form of a liquid or a solid.

[0016] As a further preferred embodiment, the solid dosage form is prepared by collecting the bacterial cells after centrifuging the fermentation broth, and then freeze-drying or adding a carrier for adsorption.

[0017] Fifthly, the present invention provides a method for degrading ochratoxin A, wherein the above-mentioned Bacillus saccharophilus JX-8 or the above-mentioned microbial agent or the above-mentioned ochratoxin A degrading agent is placed in the material contaminated with ochratoxin A, and the temperature is controlled at 10℃-50℃ and the pH is 6.0-9.0.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) The present invention provides a strain of Bacillus saccharophilus JX-8, which has the ability to degrade OTA and has a high OTA degradation efficiency. Its bacterial cells or cell lysates can completely degrade 12.4 μM OTA within 12 hours, which has practical application value.

[0020] (2) The saccharophilic land bacillus JX-8 provided by the present invention degrades OTA into non-toxic ochratoxin α (OTα) and L-β-phenylalanine (L-β-Phe) by hydrolyzing the amide bond of OTA, which is an ideal way to achieve true detoxification.

[0021] (3) The Bacillus saccharophilus JX-8 provided by the present invention has simple culture conditions, fast growth and is easy to prepare on a large scale. The bacterial agent with it as the core can be used for biological detoxification of feed, food raw materials and so on. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The images show the colony morphology (A), Gram staining micrograph (B), transmission electron micrograph (C), and phylogenetic tree (D) of Bacillus saccharophilus JX-8 based on the 16S rRNA gene sequence.

[0024] Figure 2 The following are the time-dynamic curves of OTA degradation by strain JX-8 (A), the comparison of degradation capacity of different cell components (B), and the effects of temperature (C) and pH (D) on the OTA degradation activity of cell lysate.

[0025] Figure 3 The figures show the metabolites of OTA degradation by strain JX-8 as analyzed by HPLC; (A) are OTA and OTα standards; (B) is the OTA degradation result at 0 hours; (C) is the OTA degradation result at 6 hours; and (D) is the OTA degradation result at 12 hours.

[0026] Figure 4 The image shows the chromatograms of OTA degradation products analyzed by UPLC-QTOF-MS / MS; where (A) is OTA; (B) is product A, OTα; (C) is product B, L-β-phenylalanine; and (D) is product C, phenylpyruvic acid.

[0027] Figure 5 The figure shown is a graph illustrating the performance of Bacillus saccharophilus JX-8 in detoxifying OTA-contaminated feed. Detailed Implementation

[0028] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0029] Example 1

[0030] Bacillus saccharophilus ( Terribacillus saccharophilus Isolation, screening and identification of JX-8

[0031] (1) Screening and purification of Bacillus saccharophilus JX-8

[0032] Five mL of sauerkraut juice sample was inoculated into 50 mL of MSM medium containing 12.4 μM OTA and cultured at 30 °C with shaking at 180 rpm for 3 days. Five mL of the culture was then transferred to fresh MSM medium, and the enrichment was repeated five times. The final enriched solution was subjected to 10... -1 ~10 -6 Gradient dilution, take 10 -4 ~10 -6 0.1 mL of each dilution was spread onto LB agar plates containing 12.4 μM OTA and incubated at 30°C for 5 days. Single colonies with different morphologies were picked and inoculated into LB liquid medium and incubated at 37°C and 180 rpm for 24 h. The strain was purified three times using the streak plate method to obtain pure strains. The pure strains were inoculated into LB liquid medium containing 12.4 μM OTA and incubated at 37°C and 180 rpm for 24 h. The OTA residue was detected by HPLC, and strain JX-8 with the highest degradation rate was screened.

[0033] (2) Identification of Bacillus saccharophilus JX-8

[0034] After culturing the above-screened strain JX-8 on LB agar for 2 days, the colonies were round, raised, and pale yellow. Figure 1 A). The cells of strain JX-8 are Gram-positive, sporulating, and rod-shaped (0.6-1.0 × 2.0-3.8 µm). Figure 1 B and Figure 1 C). Strains JX-8 are catalase-positive but urease- and oxidase-negative. They can utilize glucose, galactose, raffinose, trehalose, mannose, cellobiose, salicin, and N-acetylglucosamine, but not L-arabinose, ribose, sorbitol, glycogen, and D-fructose. PCR amplification was performed using universal primers 27F (5´-GAGTTTGATCCTGGCTCAG-3´) and 1492R (5´-ACG GCTACCTTGTTACGACTT-3´) to obtain the 16S rRNA gene fragment of strain JX-8. Its sequence is shown in SEQ ID NO.1, approximately 1500 bp in length. Homology alignment of the 16S rRNA gene sequence yielded a phylogenetic tree of the strain, as shown below. Figure 1 As shown in D. Based on the morphological, physiological and biochemical characteristics and molecular biological identification results of the strain, it was determined that this strain belongs to *Bacillus saccharophilus* (…). Terribacillus saccharophilus), named Terribacillus saccharophilus JX-8 。

[0035] Example 2

[0036] Bacillus saccharophilus ( Terribacillus saccharophilus Study on the degradation characteristics of OTA by JX-8

[0037] Study on the OTA degradation ability and degradation active components of strain JX-8

[0038] (1) Strain JX-8 was cultured in LB medium to the logarithmic growth phase; subsequently, cells were collected by centrifugation, washed, and resuspended in 20 mL of MSM to allow OD to reach the target growth phase. 600 Once the concentration reached 1.0, 12.4 µM of OTA was added, and the mixture was incubated at 37 °C and 180 rpm. MSM containing the same concentration of OTA but without inoculation of strain JX-8 was used as a control (OTA control). Samples were taken every 3 hours.

[0039] The result is as follows Figure 2 As shown, HPLC analysis results indicate that strain JX-8 completely degraded 12.4 µM OTA within 12 hours. Figure 2 A). The fermentation broth of strain JX-8 was centrifuged at 8,000 × g for 10 min at 4 °C to separate the culture supernatant and cells. The culture supernatant was then filtered through a 0.22 µm sterile filter to prepare a cell-free supernatant. The cell pellet was resuspended in PBS (50 mM, pH 7.4) to prepare a cell suspension. After washing three times with PBS, the cell suspension was disrupted by sonication and then centrifuged at 12,000 × g for 10 min. The supernatant was filtered through a 0.22 µm sterile filter to prepare a cell lysate. The heat-inactivated cell lysate was prepared by boiling at 100 °C for 10 min. Subsequently, OTA was added to each prepared component to achieve a final concentration of 12.4 µM, and incubated at 37 °C for 12 h. The concentration of residual OTA was analyzed by HPLC. The results are as follows. Figure 2 As shown in Figure B, cell suspension and cell lysate were able to completely degrade OTA within 12 hours, but cell-free supernatant showed no degradation ability. After heat inactivation, the cell lysate significantly lost its OTA-degrading activity. These results indicate that the degradation components of strain JX-8 are intracellular.

[0040] (2) Investigate the effects of environmental factors (temperature, pH, metal ions, chemical reagents and organic solvents) on the degradation of OTA by cell lysate.

[0041] 12.4 μM OTA and 100 µL cell lysis buffer were added to 50 mM PBS buffer (pH 7.4), for a total volume of 1 mL. The enzyme reaction samples were incubated at temperatures ranging from 10℃ to 80℃ to determine enzyme activity. Relative enzyme activity was calculated with the enzyme activity at the optimal reaction temperature defined as 100%. The results show that the cell lysis buffer exhibited high OTA degradation activity within the 10-50℃ range, with the highest activity observed at 50℃. Figure 2 C).

[0042] Enzyme activity was measured at 50°C after adding 12.4 μM OTA and 100 µL of cell lysis buffer to different pH buffer systems (pH 4.0–pH 10.0). The enzyme activity at the optimal pH was taken as 100%, and relative enzyme activity was calculated. The activity of the cell lysis buffer was detectable between pH 6.0 and 9.0, but completely lost at pH < 5.0 or pH 10.0. The optimal pH was 8.0. Figure 2 D).

[0043] Example 3

[0044] For Bacillus saccharophilus ( Terribacillus saccharophilus Identification of OTA degradation products by JX-8

[0045] The OTA-degrading sample from strain JX-8 was thoroughly mixed with an equal volume of acetonitrile, centrifuged at 12,000 × g for 10 min, and filtered through a 0.22 µm Millipore membrane. OTA and its degradation products were analyzed by HPLC (Waters Alliancee 2695) using a ZORBAX Eclipse Plus C18 column (5 µm, 150 mm × 4.6 mm). The mobile phase was acetonitrile, water, and acetic acid (45:54:1, v / v), with an injection volume of 10 µL and a flow rate of 1.0 mL / min. Detection was performed using a UV detector at 330 nm.

[0046] HPLC analysis results of OTA degradation by strain JX-8 are as follows: Figure 3 As shown, the retention times for OTA and OTα are 10.1 and 2.5 minutes, respectively. Figure 3 A). Strain JX-8 degrades OTA to generate a new product with a retention time consistent with the OTα standard. Figure 3 (BD). As degradation time increased, the chromatographic peak of OTA gradually decreased, while the product peak continued to increase. After 12 hours of reaction, OTA was completely converted into the product, but the product was not further degraded (BD). Figure 3 D).

[0047] The degradation products were identified and analyzed using liquid chromatography-mass spectrometry (LC-MS / MS). The mass spectrometry used was an AB Sciex high-resolution tandem mass spectrometer with a Triple TOF 5600 + LC / MS system, an ESI ion source, positive ion detection mode, and a mass scan range (m / z) of 100–550. Figure 4 As shown in Figure A, OTA produces a significant deprotonated molecular ion [MH] at m / z 402.0773. - Its key fragment ion appears at m / z 358.1119 [M-CO2-H]. - 211.0323 [M-C9H9NO2-CO-H] - And 167.0026 [M-C9H9NO2-CO-HCl-H2O-H] - This is consistent with its structural fragmentation mode. For product A (… Figure 4 B), whose main deprotonated molecular ion appears at m / z 255.0076 [MH]. - It is accompanied by characteristic fragment ions at m / z 211.0323 [M-CO2-H]. - And 167.0026 [M-CO2-HCl-H2O-H] - Thus, it was identified as OTα. Product B exhibits a unique deprotonated molecular ion at m / z 164.0723 [MH]. - ( Figure 4 C). Its characteristic fragment ion is [M-CO2-H] at m / z 120.0617. - The presence of [a specific substance] strongly supports its identification as L-β-Phe. Similarly, product C is characterized by its significantly deprotonated molecular ion at m / z 163.0407 [MH]. - Its diagnostic fragment ion is [M-CO2-H] at m / z 119.0603. - It was definitively identified as phenylpyruvic acid (PPA) Figure 4 D). The above analysis indicates that strain JX-8 degrades OTA into OTα and L-β-Phe by hydrolyzing amide bonds. L-β-Phe is further metabolized into PPA or participates in protein synthesis.

[0048] The metabolic pathway by which the above-mentioned strain JX-8 degrades OTA is speculated to be as follows:

[0049] .

[0050] Example 4

[0051] Application of Bacillus saccharophilus JX-8 in detoxification of contaminated feed

[0052] Bacillus saccharophilus JX-8 was inoculated into LB liquid medium and cultured at 37°C with shaking until the early stationary phase. The bacterial cells were collected by centrifugation and resuspended in sterile physiological saline to obtain a viable count of approximately 1 × 10⁻⁶. 10 A liquid microbial agent at CFU / mL was prepared. Under laboratory conditions, OTA standard was added to crushed corn feed to achieve an initial concentration of approximately 100 μg / kg. The prepared liquid microbial agent (with a mass ratio of microbial agent to feed of 1:10) was added to the contaminated feed, mixed thoroughly, and incubated at 37 °C for 24 hours. An identical system without inoculation was used as a control.

[0053] HPLC analysis showed that the degradation rate of OTA in the treated group exceeded 99%. Figure 5 This indicates that the microbial agent can effectively remove OTA contamination from feed.

[0054] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A strain of Bacillus saccharophilus ( Terribacillus saccharophilus JX-8, characterized in that, The saccharophilic Bacillus agronomycetes JX-8 was deposited on May 8, 2025 at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2025996.

2. The *Bacillus saccharophilus* JX-8 according to claim 1, characterized in that, The 16S rRNA sequence of the saccharophilic Bacillus landsporum JX-8 is shown in SEQ ID NO.

1.

3. A microbial inoculant, characterized in that, Contains Bacillus saccharophilus JX-8 as described in any one of claims 1-2 and / or its fermentation products; The fermentation product is a cell suspension of Bacillus saccharophilus JX-8 or a cell lysate of Bacillus saccharophilus JX-8.

4. The use of Bacillus saccharophilus JX-8 as described in any one of claims 1-2 or the microbial agent as described in claim 3 in the preparation of ochratoxin A degrading agent.

5. The application according to claim 4, characterized in that, The degradation agent includes feed additives, food additives, or biological detoxifiers.

6. An ochratoxin A degrading agent, characterized in that, Includes Bacillus saccharophilus JX-8 or its fermentation product as described in any one of claims 1-2; The fermentation product is a cell suspension of Bacillus saccharophilus JX-8 or a cell lysate of Bacillus saccharophilus JX-8.

7. The ochratoxin A degrading agent according to claim 6, characterized in that, When the degrading agent is *Bacillus saccharophilus* JX-8, the concentration of live *Bacillus saccharophilus* is 1×10⁻⁶. 8 CFU / mL - 1×10 11 CFU / mL.

8. The ochratoxin A degrading agent according to claim 7, characterized in that, The ochratoxin A degrading agent is available in liquid or solid form.

9. The ochratoxin A degrading agent according to claim 8, characterized in that, The solid dosage form is prepared by collecting the bacterial cells after centrifuging the fermentation broth, and then freeze-drying or adding a carrier for adsorption.

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