Application of lactococcus gasseri ZB15 in degradation of kitasamycin

Abstract

The invention discloses application of lactococcus gasseri ZB15 in degradation of kitasamycin, and belongs to the technical field of microorganism application. The invention discovers and verifies that the lactococcus gasseri ZB15 strain has the capability of efficiently degrading kitasamycin for the first time. Specific experimental data show that the strain can maintain growth in a culture medium containing kitasamycin with a gradient concentration of 0-6 [mu] g / mL, and shows good tolerance. High performance liquid chromatography analysis proves that in a set culture period, the removal effect of the strain on kitasamycin mainly depends on a biodegradation effect instead of a pure adsorption effect. Furthermore, the influence of key parameters such as inoculum size, initial concentration, temperature and pH on the degradation efficiency is systematically researched, and the degradation characteristic and the optimal working window of the kitasamycin degrading bacteria are completely analyzed, so that a microbial resource with definite performance is obtained, and the blank of lacking of safe and efficient kitasamycin degrading bacteria in the prior art is filled.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to the application of Lactococcus gasseri ZB15 in the degradation of tylosin. Background Technology

[0002] Tadalafil, a macrolide antibiotic, exhibits excellent inhibitory effects against a variety of Gram-positive bacteria, and is therefore widely used in human medicine, veterinary medicine, and animal husbandry, particularly as a feed additive to promote animal growth. However, this antibiotic is highly stable in the natural environment and is difficult to rapidly degrade spontaneously, easily leaving residues in animal-derived foods, water bodies, soil, and other environmental media. These residues not only directly pose food safety risks but also exacerbate the spread and accumulation of antibiotic resistance genes in the environment, creating potential risks to public health.

[0003] Currently, conventional methods for treating tylosin residues still have significant shortcomings. Physical adsorption can only transfer the pollutant, not completely remove it, and may cause secondary pollution. While chemical oxidation has degradation capabilities, it is usually costly, complex, and may produce byproducts with unknown toxicity during the reaction. In contrast, biodegradation is considered a more promising green solution due to its environmental friendliness, strong substrate specificity, and potential application safety. However, existing technologies have very limited resources of microorganisms that efficiently and specifically degrade tylosin. Some reported degrading strains have shown low efficiency and weak adaptability in practical applications, especially in complex environments such as aquaculture wastewater and animal intestines, where their metabolic activity is easily inhibited and the degradation effect is unstable.

[0004] Furthermore, existing research largely focuses on screening for common degrading bacteria, often neglecting the safety of the strains themselves and their ecological compatibility with the host and environment. Currently, strains possessing both highly efficient degradation characteristics and beneficial microbial properties are particularly scarce. *Lactococcus gasseri*, as a recognized safe probiotic, is widely found in fermented foods and animal probiotic preparations, exhibiting good environmental adaptability and host affinity. However, to date, there are no reports of *Lactococcus gasseri* strains capable of specifically degrading tyrosine. Therefore, developing a new *Lactococcus gasseri* strain that retains beneficial bacterial characteristics while efficiently and specifically degrading tyrosine would not only help address antibiotic residue issues but also provide a safer and more compatible microbial tool for the farming environment and animal health. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Lactococcus gasseri ZB15 in the degradation of tylosin, so as to solve the problems existing in the prior art. This invention is the first to discover and confirm that Lactococcus gasseri ZB15 strain has the ability to efficiently degrade tylosin, filling the gap in the prior art of lacking safe and efficient tylosin-degrading bacteria.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides Lactococcus gasseri ( Lactococcus garvieae The application of ZB15 or its bacterial suspension in the degradation of tylosin, wherein the preservation number of Lactococcus gasseri ZB15 is GDMCC No: 65947.

[0007] The present invention also provides the application of Lactococcus gasseri ZB15 or its bacterial suspension in the preparation of a tylosin-degrading bacterial agent, wherein the preservation number of Lactococcus gasseri ZB15 is GDMCC No: 65947.

[0008] Optionally, the OD of the bacterial suspension 600 =1.0.

[0009] The present invention also provides a method for degrading tylosin, comprising the step of inoculating Lactococcus gasseri ZB15 or a suspension thereof in an environment containing tylosin; The preservation number of the Lactococcus gasseri ZB15 is GDMCC No: 65947.

[0010] Optionally, the following steps are included: Inoculate Lactococcus gasseri ZB15 suspension at an inoculum concentration of 3%-8% in an environment containing tylosin, and incubate at a temperature of 22℃-37℃ and a pH of 6.0-8.0.

[0011] Preferably, the inoculation amount is 5%.

[0012] Preferably, the OD of the bacterial suspension is... 600 =1.0.

[0013] Preferably, the temperature is 37°C.

[0014] Preferably, the pH is 8.0.

[0015] Optionally, the culture time is 24 h-48 h.

[0016] The present invention discloses the following technical effects: This invention constructs an HPLC detection system to systematically evaluate the tylosin degradation ability of Lactococcus gasseri ZB15 and three other lactic acid strains ZB1, ZB31 and ZB41, and successfully screens out one Lactococcus gasseri strain ZB15 with excellent tylosin degradation ability.

[0017] This invention is the first to discover and confirm the highly efficient ability of *Lactococcus gasseri* strain ZB15 to degrade tylosin. Specific experimental data show that this strain can maintain growth in culture media containing tylosin at gradient concentrations of 0-6 μg / mL, demonstrating good tolerance. High-performance liquid chromatography (HPLC) analysis confirmed that within the set culture period, the removal of tylosin by this strain mainly relies on biodegradation rather than simple adsorption. Furthermore, by systematically studying the effects of key parameters such as inoculum size, initial concentration, temperature, and pH on degradation efficiency, its degradation characteristics and optimal working window were fully elucidated, thus obtaining a microbial resource with well-defined performance, filling the gap in existing technologies for the lack of safe and highly efficient tylosin-degrading bacteria.

[0018] The technological achievements of this invention have significant application potential and social value. Since *Lactococcus gasseri* ZB15 originates from traditional fermented foods and is a recognized safe probiotic, it possesses unparalleled ecological safety and regulatory acceptance when directly applied to regulate the animal intestinal microenvironment or develop environmental remediation formulations. Its well-defined degradation performance and optimized process parameters provide solid core strains and key data support for the development of microbial products for livestock wastewater treatment, soil remediation, or green feed additives. Therefore, this invention not only provides an innovative technical method but also offers a safe, economical, and environmentally friendly practical solution for fundamentally reducing tylosin residues and controlling the spread of antibiotic resistance genes. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the 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.

[0020] Figure 1 To initially screen the tolerance of four lactic acid bacteria to tylosin using the plate method; Figure 2 To determine the tolerance range of tylosin to the primary screening strains using growth curve analysis; A: tylosin concentration of 0, B: tylosin concentration of 2 μg / mL, C: tylosin concentration of 3 μg / mL, D: tylosin concentration of 4 μg / mL, E: tylosin concentration of 5 μg / mL, F: tylosin concentration of 6 μg / mL; Figure 3 This is the standard curve for tylosin; Figure 4The results show the detection of the ability of strains ZB15 and ZB1 to remove tylosin at different inoculum amounts at 24 h and 48 h. Figure 5 To verify the ability of strains ZB15 and ZB1 to degrade tylosin; Figure 6 The effect of different inoculum amounts on the degradation of tylosin by strain ZB15 at 24 h and 48 h was investigated. Figure 7 The effect of different initial concentrations of tylosin on the degradation of tylosin by strain ZB15 at 24 h (A) and 48 h (B); Figure 8 The effect of different culture temperatures on the degradation of tylosin by strain ZB15 at 24 h (A) and 48 h (B); Figure 9 The effect of different initial pH values ​​of culture media on the degradation of tylosin by strain ZB15 at 24 h (A) and 48 h (B). Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] This invention aims to address the problem of scarce and inaccurate screening methods for efficient and safe microbial resources that degrade tylosin in existing technologies, including Lactococcus gasseri ZB15 (… Lactococcus garvieae Targeted screening and functional verification of various lactic acid bacteria, including *Lactococcus gasseri* ZB15, derived from cured meat, were conducted. The experiment was the first to discover and confirm that this strain has a significant degradation ability for tylosin. Plate and liquid culture verification showed that strain ZB15 can grow normally under tylosin stress and utilize it, while also exhibiting the safety and environmental adaptability of probiotics. Finally, its degradation efficiency was quantitatively determined by high-performance liquid chromatography, providing a novel microbial solution for the safe and efficient elimination of tylosin residues.

[0027] Example 1: Verification and Characterization of Lactococcus gasseri ZB15's Function in Degrading Tadalafil The Lactococcus gasseri ZB15 used in this embodiment has been disclosed in patent CN120118796A, "A strain of Lactococcus gasseri ZB15 and a cell-free fermented freeze-dried product with anti-inflammatory activity and its preparation method", with accession number GDMCC 65947.

[0028] The *Lactococcus lactis* ZB1, *Enterococcus faecalis* ZB31, and *Weissella viridescens* ZB41 used in this embodiment were provided by the Animal Reproduction and Development and Biotechnology Laboratory of Shaanxi University of Technology. *Lactococcus lactis* ZB1 is published in the literature “Jian Yuwen. Probiotic Characteristics and Whole Genome Analysis of *Lactococcus lactis* from Zhenba Cured Meat Source [D]. Shaanxi University of Technology, 2024.”, while *Enterococcus faecalis* ZB31 and *Weissella viridescens* ZB41 are published in the literature “Han Shuai. Study on Quality Formation and Cortisone Reduction Effect of Zhenba Cured Meat Based on Metabolomics [D]. Shaanxi University of Technology, 2025.”.

[0029] In this embodiment, the above four lactic acid bacteria strains were selected for functional verification, with tylosin as the target degradation product.

[0030] (1) All glycerol-preserved bacterial strains were inoculated onto MRS agar medium using the streak plate method and anaerobically cultured at 37℃ for 24 h for resuscitation. Single colonies were picked and inoculated onto MRS liquid medium and incubated statically at 37℃. The absorbance (OD) of the bacterial suspension at 600 nm was measured.600 When the bacterial cell density reaches approximately 0.6, the bacteria enter the logarithmic growth phase, yielding the first-generation activated bacterial suspension. This suspension is then transferred and cultured once using the same method to obtain a second-generation activated bacterial suspension with uniform growth, which is used for all subsequent experiments.

[0031] (2) Initial screening and assessment of strain tolerance to tylosin First, a preliminary screening using solid plates is performed: the OD of the four activated lactic acid bacteria suspensions is... 600 The value was adjusted to 1.0, and a series of dilutions were performed to obtain 10. 0 10 -1 10 - ²、10 -3 10 -4 10 -5 Six concentration gradients were used. 2 μL of each gradient was vertically inoculated onto MRS agar plates containing 0, 1, 2.5, 5.0, and 10.0 μg / mL of tylosin, and incubated at 37℃ for 24 h. 5 μL of methanol was used as a control. After incubation, *Lactococcus gasseri* ZB15 and *Lactococcus lactis* ZB1 were observed to form colonies normally on all plates with tylosin concentrations from 0 to 5.0 μg / mL, demonstrating good basic tolerance. Figure 1 ).

[0032] Furthermore, liquid culture growth curves were measured for quantitative evaluation: the four lactic acid bacteria strains were inoculated into MRS liquid medium containing a series of concentrations (0, 2, 3, 4, 5, 6 μg / mL) of tylosin and incubated statically at 37°C. The optical density (OD) of the culture medium at a wavelength of 600 nm was dynamically monitored using a microplate reader. 600 The specific monitoring time points were 0, 2, 4, 6, 8, 10, 12, 18, 24, 36, and 48 hours after the start of cultivation. Growth curves for each strain at different drug concentrations were plotted based on the measured data. By systematically comparing the growth kinetic parameters at each concentration, the tolerance range of each strain to tylosin could be quantitatively assessed and determined, providing crucial information for selecting the optimal initial drug concentration in subsequent degradation experiments targeting specific strains.

[0033] The results are as follows Figure 2 As shown, strain ZB15 was able to maintain growth in media containing 0-6 μg / mL gradient concentrations of tylosin, demonstrating good tolerance.

[0034] (3) Establishment of high performance liquid chromatography (HPLC) detection method The tylosin standard was thoroughly dissolved in methanol to obtain a 25 mg / mL tylosin standard stock solution. The stock solution was serially diluted with 0.1 mol / L ammonium acetate solution to obtain tylosin standard working solutions with concentration gradients of 2.0 μg / mL, 2.5 μg / mL, 3.0 μg / mL, 4.0 μg / mL, 5.0 μg / mL, and 6.0 μg / mL. High-performance liquid chromatography (HPLC) analysis was performed (detection conditions are shown in Table 1). The peak area of ​​tylosin in the chromatograms of each concentration standard solution was recorded at a detection wavelength of 238 nm. A linear regression was performed between the peak area (Y) and the standard solution concentration (X, μg / mL) to obtain the standard curve regression equation (results are shown in Table 1). Figure 3 The correlation coefficient (R² = 0.993) of Y = 0.106X + 0.0852 satisfies the quantitative analysis requirement of ≥0.99. The HPLC detection conditions are shown in Table 1.

[0035] Table 1 HPLC detection conditions (4) Detection of tylosin content in bacterial cell-free supernatant In a clean bench, activated bacterial suspensions (OD) of Lactococcus gasseri ZB15 and Lactococcus lactis ZB1 are prepared. 600 =1.0) Inoculated at 1% and 2% of the culture medium into MRS liquid medium containing 4 μg / mL tylosin. The medium was aliquoted into 1 mL sterile centrifuge tubes and incubated at 37°C. Three replicates were set up, with MRS broth medium without the strain serving as a control. Samples were taken at 0, 24, and 48 h of incubation for sample pretreatment and detection. The samples were immediately centrifuged at 10000 r / min for 10 minutes at 4°C. 600 µL of the supernatant was accurately pipetted and 1.2 mL of ethyl acetate (supernatant to extractant volume ratio 1:2) was added. The mixture was vortexed for 3 minutes to ensure thorough mixing. The mixture was centrifuged at 3000 r / min for 15 minutes. After separation, 800 µL of the upper organic phase was carefully pipetted and dried under a gentle nitrogen stream. The residue was reconstituted with 400 µL of 0.1 mol / L ammonium acetate solution and vortexed thoroughly. Finally, the solution was filtered through a 0.22 μm organic phase filter membrane to obtain the HPLC sample. The sample was analyzed under the same chromatographic conditions as the standard solution. The measured peak area of ​​tylosin was substituted into the regression equation of the standard curve to calculate its residual concentration (Ct) at each time point.

[0036] The degradation rate of tylosin is calculated using the following formula: Degradation rate (%) = (C0 - C) / ... t ) / C0×100%, where C0 is the initial antibiotic concentration; Ct Let t be the antibiotic concentration at time t.

[0037] according to Figure 4 The trend of tylosin concentration over time shows that both Lactococcus gasseri ZB15 and Lactococcus lactis ZB1 showed significant degradation ability of tylosin after 24 h and 48 h of culture, and the degradation efficiency of Lactococcus gasseri ZB15 was higher than that of Lactococcus lactis ZB1.

[0038] (5) Verification of the degradation ability of tylosin-based screening lactic acid bacteria The activated bacterial suspensions (OD) of Lactococcus gasseri ZB15 and Lactococcus lactis ZB1 were used to prepare the bacterial suspensions. 600 =1.0) At an inoculation rate of 2%, each sample was inoculated into 1 mL of MRS liquid medium containing a final concentration of 3 μg / mL of tylosin. The samples were placed in a constant temperature incubator at 37℃ for static incubation. Three replicate samples were set up for the experiment, and MRS broth medium without the strain (control group) and inactivated bacterial suspensions of the two strains after high temperature inactivation treatment (adsorption control group) were set up as controls. Samples were taken at 0 h and 24 h of incubation and the samples were processed. The sample processing and detection methods were the same as those in "(4) Detection of tylosin content in bacterial cell-free supernatant".

[0039] Inactivation process steps: Take OD 600 500 μL of bacterial culture was adjusted to pH 1.0 and centrifuged at 4 °C and 6,000 × g for 10 min. The supernatant was discarded. The culture was resuspended in 500 μL of sterile PBS and centrifuged and washed twice. Finally, the bacterial cells were resuspended in 500 μL of sterile PBS, heated in an 80 °C water bath for 30 min, cooled on ice for 3 min, and gently mixed before being used as the adsorption control group.

[0040] The results are as follows Figure 5 As shown, the removal rate of the live bacteria group was significantly higher than that of the inactivated group, and the removal rate of strain ZB15 was significantly higher than that of strain ZB1. The results indicate that within the set culture period, the removal of tylosin by strain ZB15 mainly depends on biodegradation rather than simple adsorption.

[0041] (6) Preliminary study on degradation characteristics To investigate the degradation characteristics of Lactococcus gasseri ZB15, single-factor experiments were conducted by changing key experimental conditions based on the verification of its degradation ability. The effects of different initial concentrations of tylosin (3.0, 4.0, 5.0 μg / mL), different inoculum amounts (1%, 2%, 3%, 5%, 8%), different culture temperatures (22℃, 30℃, 37℃, 40℃), and different initial pH values ​​of culture media (6.0, 8.0) on the degradation rate of tylosin at 24 h and 48 h were studied. Each experiment was replicated in parallel, and the culture, sampling, and HPLC detection were performed according to the method in "(4) Detection of tylosin content in bacterial cell-free supernatant".

[0042] The results are as follows Figures 6-9 As shown, strain ZB15 exhibited the highest degradation rate at an initial concentration of 3.0 μg / mL of tylosin; degradation was optimal at inoculum sizes of 3%-8%, with the best effect observed at 5% inoculum; degradation was best at incubation temperatures of 22℃-37℃, with the best effect observed at 37℃; and degradation was more effective at pH 8.0 than at pH 6.0. Furthermore, under different conditions, degradation was more effective at 48 h than at 24 h.

[0043] By comparing and analyzing the final degradation rates at different levels of various factors, the optimal conditions for efficient degradation of tylosin by Lactococcus gasseri ZB15 can be preliminarily determined (inoculum size of strain is 5%, culture temperature is 37℃, pH is 8.0, and degradation time is 48h), providing data support for its subsequent application.

[0044] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Lactococcus gasseri ( Lactococcus garvieae The application of ZB15 or its bacterial suspension in the degradation of tylosin, characterized in that, The preservation number of the Lactococcus gasseri ZB15 is GDMCC No: 65947.

2. The application of Lactococcus gasseri ZB15 or its bacterial suspension in the preparation of a tylosin-degrading bacterial agent, characterized in that, The preservation number of the Lactococcus gasseri ZB15 is GDMCC No: 65947.

3. The application as described in claim 1 or 2, characterized in that, The OD of the bacterial suspension 600 =1.

0.

4. A method for degrading tylosin, characterized in that, The procedure includes inoculating Lactococcus gasseri ZB15 or a suspension thereof in an environment containing tylosin; The preservation number of the Lactococcus gasseri ZB15 is GDMCC No: 65947.

5. The method as described in claim 4, characterized in that, Includes the following steps: Inoculate Lactococcus gasseri ZB15 suspension at an inoculum concentration of 3%-8% in an environment containing tylosin, and incubate at a temperature of 22℃-37℃ and a pH of 6.0-8.

0.

6. The method as described in claim 5, characterized in that, The inoculation dose is 5%.

7. The method as described in claim 5, characterized in that, The OD of the bacterial suspension 600 =1.

0.

8. The method as described in claim 5, characterized in that, The temperature is 37°C.

9. The method as described in claim 5, characterized in that, The pH is 8.

0.

10. The method as described in claim 5, characterized in that, The incubation period is 24 h to 48 h.

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