Microbial composition and application thereof in degradation of antibiotics

Abstract

The invention belongs to the technical field of microorganisms, and discloses a microbial composition and application thereof in degradation of antibiotics. The microbial composition for meat antibiotic degradation is designed according to the household use scene, spiramycin (SPM) left on the surface and deep layers of meat is rapidly removed through biodegradation of microorganisms and metabolites of the microorganisms, and the household diet safety pain point is solved. The selected microorganisms comprise bacillus safensis, bacillus tequilensis, bacillus subtilis, bacillus pumilus and lactobacillus buchneri, the microorganisms belong to probiotics, and new potential food safety hazards cannot be caused after livestock and poultry meat is treated; in addition, the selected auxiliaries are conventional additives in the food field, so that the edible safety of the treated meat is ensured. The degradation rate of the microbial composition on SPM in pork reaches 99% or above, and the effect is remarkable.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a microbial composition and application thereof in degrading antibiotics. Background Art

[0002] In recent years, the environmental and human health risks caused by overuse of antibiotics have become a global problem. Antibiotics are widely used in medicine and animal husbandry to inhibit or kill pathogenic microorganisms. However, antibiotics are not fully metabolized by organisms and ultimately enter the environment and the food chain in their original form or as metabolites. Studies have shown that long-term exposure to residual tetracycline, sulfonamides, and other antibiotics can cause allergic reactions, disrupt intestinal flora, and even contribute to the development and spread of "superbugs" by inducing drug-resistant genes.

[0003] Spiramycin (SPM) is a macrolide antibiotic used to treat ear, nose, throat, and respiratory tract infections caused by Gram-positive and certain Gram-negative bacteria. It can also be used to treat oral and otolaryngological infections such as otitis media, periodontitis, and acute sinusitis caused by susceptible bacteria. Furthermore, to improve economic efficiency, SPM has been widely used as a feed additive to promote growth and prevent disease in livestock and poultry. Consequently, SPM residues are often found in livestock and poultry meat, and these residues cannot be effectively eliminated.

[0004] In order to quickly remove antibiotic residues on the surface and deep layers of meat, solve the pain points of family food safety, and make the kitchen an "antibiotic-free line of defense", discovering microorganisms with the ability to degrade antibiotics and realizing their large-scale application has become an urgent problem that the industry needs to solve. Summary of the Invention

[0005] The technical problem addressed by the present invention is to provide a safe microbial composition for the efficient degradation of spiramycin (SPM) in food, particularly meat. This microbial composition rapidly removes residual spiramycin from the surface and deep layers of meat through the biodegradation of microorganisms and their metabolites. Specifically, the present invention provides the following detailed technical solutions.

[0006] In a first aspect, the present invention provides a microbial composition, wherein the active ingredients of the microbial composition include: Bacillus saffron, Bacillus tergillosis, Bacillus subtilis, Bacillus pumilus, and Lactobacillus buchneri;

[0007] The deposit number of the Bacillus saffron is CGMCC NO.28599;

[0008] The deposit number of the Bacillus tequila is: CGMCC NO.21317;

[0009] The deposit number of the Bacillus subtilis is: CCTCC NO.M20221487;

[0010] The deposit number of the Bacillus pumilus is: CGMCC NO.24796;

[0011] The preservation number of the Lactobacillus buchneri is: CGMCC NO.28790.

[0012] Furthermore, the components of the microbial composition include: a mixed bacterial suspension made from the active ingredient and an adjuvant; the mixed bacterial suspension components are: based on the bacterial suspension volume ratio, Bacillus saffron suspension: Bacillus tequila suspension: Bacillus subtilis suspension: Bacillus pumilus suspension: Lactobacillus buchneri suspension = 25-35:20-30:15-25:10-20:5-15; the bacterial concentration of each bacterial suspension is not less than 10 10 CFU / mL.

[0013] Furthermore, the auxiliary agents include: surfactants, penetration enhancers, buffer system regulators, and bacterial nutrient supplements.

[0014] Furthermore, the surfactant is selected from: one or two of sophorolipids and rhamnolipids;

[0015] The penetration enhancer is selected from: one or two of glycerol and food grade sorbitol;

[0016] The buffer system regulator includes: sodium bicarbonate;

[0017] The bacterial cell nutritional supplement is selected from one or two of glucose and resistant dextrin.

[0018] Furthermore, the content of the surfactant in the mixed bacterial suspension is ≤0.2% by mass;

[0019] Calculated by mass ratio, the content of the penetration enhancer in the mixed bacterial suspension is ≤3.5%;

[0020] The content of the buffer system regulator in the mixed bacterial suspension is ≤0.5% by mass;

[0021] Calculated by mass ratio, the content of the bacterial nutrient supplement in the mixed bacterial suspension is ≤2.2%.

[0022] In a second aspect, the present invention provides a use of the microbial composition, wherein the microbial composition is used to degrade antibiotics.

[0023] Furthermore, in the above application, the microbial composition is used to degrade residual antibiotics in food.

[0024] Furthermore, in the above application, the antibiotics include: spiramycin.

[0025] Compared with the prior art, the present invention "a microbial composition and its use in degrading antibiotics" has at least the following beneficial effects:

[0026] 1) Source of ingredients

[0027] This invention, targeting household use, has designed a microbial composition for degrading spiramycin. The microorganisms selected in this invention include Bacillus safensis, Bacillus equilensis, Bacillus subtilis, Bacillus pumilus, and Lactobacillus buchneri. These microorganisms are probiotics and do not pose new food safety risks after treating livestock and poultry meat. Furthermore, the selected additives are all conventional additives in the food industry, ensuring the safety of the treated meat. Conventional degraders, such as photodegraders and thermo-oxidative degraders, are mostly obtained through chemical synthesis.

[0028] 2) Degradation mechanism

[0029] The microbial composition provided by the present invention can quickly remove antibiotics remaining on the surface and deep layers of meat through the biodegradation technology of microorganisms and their metabolites. The mechanism of action of the microbial composition for degrading antibiotics includes: (1) secreting various active enzymes, such as hydrolases, oxidoreductases, etc., which react with antibiotic molecules to decompose them and remove antibiotics, for example, by opening the ring structure of the antibiotic to make it inactive, or decomposing the antibiotic molecules into small molecules, etc. (2) through the direct adsorption of residual antibiotics on the surface of the bacteria. (3) Secondary metabolites such as lactic acid produced by microbial fermentation reduce the pH value of the system and promote the inactivation of antibiotics. Therefore, the microbial composition provided by the present invention does not involve violent chemical reactions, has no safety hazards to human health and the environment, and can be used to degrade antibiotics in food. Ordinary antibiotic degraders generally produce degradation effects through chemical reactions. For example, photodegraders produce excited molecules or free radicals under the action of ultraviolet rays, which react with surrounding substances to initiate the degradation process; thermal oxidation degraders undergo oxidation reactions under heating and oxygen conditions, causing the molecular structure of surrounding substances to break and decompose.

[0030] 3) Degradation effect

[0031] The microbial strains used in the present invention have the following advantages: (1) Specificity: They have high degradation specificity for specific types of antibiotics. (2) Mildness: The degradation process of microorganisms is mild, does not produce violent chemical reactions, and has little impact on the environment and human body. (3) Sustainability: As long as these microorganisms can survive, they can exert their degradation effect and maintain the sustainability of the degradation effect. (4) Beneficiality: These microorganisms themselves are beneficial to the human body and can regulate intestinal flora and enhance human immunity. The present invention has confirmed that the microbial composition has a degradation rate of more than 99% for SPM in pork, with significant results. However, ordinary antibiotic degraders have poor specificity for antibiotics; in addition, ordinary antibiotic degraders require specific equipment and conditions, such as high temperature, strong light, etc. to play a degradation role; finally, ordinary antibiotic degraders will produce some intermediates or residual substances during the degradation process, some of which may be toxic or difficult to degrade, causing secondary pollution and harm to the human body and the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 OD of CK group and SPM group in the SPM degradation effect test 600 The horizontal axis represents the culture time, and the vertical axis represents the OD 600 value.

[0033] Figure 2 These are the liquid chromatography results for the TSB-treated group in an SPM degradation study. The horizontal axis represents retention time (minutes), which is used to determine the location of the characteristic SPM peak and ensure the qualitative accuracy of the detected substance. The vertical axis (AU) represents absorbance, which is positively correlated with substance concentration. Peak area can be used to quantify SPM content.

[0034] Figure 3 These are the liquid chromatography results for the SPM-treated group in an SPM degradation study. The horizontal axis represents retention time (minutes), which is used to determine the location of the characteristic SPM peak and ensure the qualitative accuracy of the detected substance. The vertical axis (AU) represents absorbance, which is positively correlated with substance concentration. Peak area can be used to quantify SPM content. DETAILED DESCRIPTION

[0035] The present invention is explained below with reference to the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental methods in the following examples are conventional methods and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0037] The deposit information of the microorganisms involved in the following examples is shown in Table 1.

[0038] Table 1. Microbial deposit information

[0039]

[0040]

[0041] Example 1

[0042] This example describes the degradation of spiramycin (SPM) by a bacterial suspension.

[0043] 1. Bacteria fermentation

[0044] 1) Culture medium preparation

[0045] Fermentation culture media of Bacillus saffron SF 2-2, Bacillus tequila M408F10-3F8, Bacillus subtilis BSC6ΔA8, Bacillus pumilus Hhb.018, and Lactobacillus buchneri HAC1b23.7 were prepared as shown in Table 2.

[0046] Table 2. Microbial fermentation medium components

[0047]

[0048]

[0049] 2) Inoculation and cultivation

[0050] The above strains were inoculated into the corresponding fermentation medium, and the fermentation conditions were as follows:

[0051] Bacillus saffron SF 2-2, fermentation temperature 35°C, humidity 60-70%, incubation time 24-48h;

[0052] Bacillus tequila M408F10-3F8, fermentation temperature 30-35°C, humidity 60-70%, incubation time 48h;

[0053] Bacillus subtilis BSC6ΔA8, fermentation temperature 30-35°C, humidity 60-70%, incubation time 24h;

[0054] Bacillus pumilus Hhb.018, fermentation temperature 35-37°C, humidity 60-70%, culture time 24-48h; Lactobacillus buchneri HAC1b23.7, anaerobic fermentation, fermentation temperature 37°C, humidity 60-70%, culture time 24-48h.

[0055] 3) Harvesting bacteria

[0056] The fermented bacteria were collected and centrifuged at 4°C and 5000 rpm for 15-20 min. The bacterial pellets were washed several times with sterile water and finally resuspended with sterile water. The bacterial concentration of each suspension was adjusted to 1×10 11 CFU / mL.

[0057] 2. Mixing of strains

[0058] Calculated by the volume ratio of the bacterial suspension, 30% of the Bacillus saffron SF 2-2 bacterial suspension, 25% of the Bacillus tequila M408F10-3F8 bacterial suspension, 20% of the Bacillus subtilis BSC6ΔA8 bacterial suspension, 15% of the Bacillus pumilus Hhb.018 bacterial suspension and 10% of the Lactobacillus buchneri HAC1b23.7 bacterial suspension were mixed to obtain a mixed bacterial suspension.

[0059] 3. SPM degradation test

[0060] First, prepare an appropriate amount of TSB liquid culture medium (trypticase 17.0 g / L, soybean papain hydrolyzate 3.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, sodium chloride 5.0 g / L, glucose 2.5 g / L, pH = 7.3 ± 0.2). Divide the TSB liquid culture medium into two parts: one part is not treated for the time being, recorded as the TSB treatment group; the other part is inoculated with the mixed bacterial suspension and cultured for 2 to 3 days, then adjust the OD 600 The value was raised to approximately 0.8. This portion of the fermentation broth was then divided into two groups: one group was supplemented with SPM to a final concentration of 50 mg / L, designated the SPM group; the other group was supplemented with an equal volume of sterile water as a control, designated the CK group. Simultaneously, SPM was also supplemented with the TSB-treated group to a final concentration of 50 mg / L. Specific groupings and treatments for each group are shown in Table 3.

[0061] Table 3. Experimental groups and treatments

[0062]

[0063]

[0064] After 6 days of culture, the bacterial solution OD of CK group and SPM group was measured respectively. 600 The SPM content in the TSB-treated group and the SPM-treated group was determined by liquid chromatography.

[0065] OD of CK group and SPM group 600 Value Figure 1 ,from Figure 1 It can be seen that under 50 mg / L SPM treatment, compared with the initial OD 600 The bacteria in the SPM group could continue to survive and grow, but their growth was inhibited compared with the CK treatment group. This indicates that under the condition of 50 mg / mL SPM treatment, SPM-degrading bacteria can survive and maintain a certain growth ability.

[0066] The results of liquid chromatography determination of TSB treatment group are shown in Figure 2 The results of liquid chromatography determination of SPM treatment group are shown in Figure 3 The SPM content was obtained based on the measurement results and is shown in Table 4.

[0067] Table 4. SPM test results

[0068] Trial Groups SPM content (mg / mL) TSB treatment group 49.2 SPM treatment group 0

[0069] Depend on Figure 2 、 Figure 3 As shown in Table 4, there was an obvious SPM peak in the TSB treatment group, but no SPM peak was detected in the SPM treatment group inoculated with the mixed bacterial suspension, which indicated that the mixed bacterial suspension could effectively degrade SPM with a good degradation effect.

[0070] Example 2

[0071] This example describes the preparation of a microbial composition for the degradation of an antibiotic (spiramycin).

[0072] The preparation method of the mixed bacterial suspension is referred to Example 1, and then the adjuvant is added to the mixed bacterial suspension. In terms of mass ratio, 0.05% of sophorolipid (surfactant), 0.03% of rhamnolipid (surfactant), 1.5% of glycerol (penetration enhancer), 1.0% of food-grade sorbitol (penetration enhancer), 0.3% of sodium bicarbonate (buffer system regulator), 0.8% of glucose (microorganism nutrient supplement), 0.5% of resistant dextrin (microorganism nutrient supplement) were added to the mass of the mixed bacterial suspension, stirred and mixed evenly to obtain a microbial composition, and stored at 4 ° C.

[0073] Example 3

[0074] This example describes the preparation of a microbial composition for the degradation of an antibiotic (spiramycin).

[0075] The preparation method of the mixed bacterial suspension refers to Example 1, and then the adjuvant is added to the mixed bacterial suspension. In terms of mass ratio, 0.07% of sophorolipid (surfactant), 0.05% of rhamnolipid (surfactant), 1.8% of glycerol (penetration enhancer), 1.2% of food-grade sorbitol (penetration enhancer), 0.4% of sodium bicarbonate (buffer system regulator), 1.0% of glucose (microorganism nutrient supplement), 0.7% of resistant dextrin (microorganism nutrient supplement) were added to the mass of the mixed bacterial suspension, stirred and mixed evenly to obtain a microbial composition, and stored at 4 ° C.

[0076] Example 4

[0077] This example describes the preparation of a microbial composition for the degradation of an antibiotic (spiramycin).

[0078] The preparation method of the mixed bacterial suspension is referred to Example 1, and then the adjuvant is added to the mixed bacterial suspension. In terms of mass ratio, 0.1% of sophorolipid (surfactant), 0.08% of rhamnolipid (surfactant), 2.0% of glycerol (penetration enhancer), 1.5% of food-grade sorbitol (penetration enhancer), 0.5% of sodium bicarbonate (buffer system regulator), 1.2% of glucose (microorganism nutrient supplement), 1.0% of resistant dextrin (microorganism nutrient supplement) were added to the mass of the mixed bacterial suspension, stirred and mixed evenly to obtain a microbial composition, and stored at 4 ° C.

[0079] Example 5

[0080] This example describes the degradation effect of the microbial composition on SPM residues in meat.

[0081] The microbial composition prepared in Example 3 (preferred) was used in the degradation test of SPM residues in pork. The test design was as follows:

[0082] SPM group: 100g of raw pork was rinsed with tap water to remove any dust and other impurities. The pork was then placed in 300mL of tap water (pre-mixed with 10mL of the microbial composition) and soaked for 30 minutes. The control group did not receive the microbial composition, but all other treatment conditions were the same. Three replicates were set up for both the experimental and control groups. The experimental groupings are shown in Table 5. After soaking, the SPM content in the SPM and control groups was measured using liquid chromatography, and the SPM degradation rate was calculated based on the SPM content:

[0083] SPM degradation rate = [(SPM content in the control group - SPM content in the SPM group) / SPM content in the control group] × 100%.

[0084] Table 5. SPM degradation effect test groups and experimental results

[0085] Trial Groups Treatment Degradation rate (%) SPM group Pork + microbial composition + tap water 99.1 Control group (CK) Pork + tap water /

[0086] Table 5 shows that the microbial composition achieved a 99.1% degradation rate for SPM in pork, demonstrating that it significantly degraded residual SPM in pork, reducing SPM levels. These microorganisms may precisely destroy the antibiotic's molecular structure by secreting enzymes such as natural esterases and β-lactamases. Furthermore, the microorganisms may lower the pH by producing lactic acid, synergizing with enzymatic reactions to accelerate antibiotic inactivation, thereby achieving excellent SPM degradation.

[0087] In summary, the present invention designs a spiramycin degradation product for household use scenarios. Through the biodegradation technology of microorganisms and their metabolites, it quickly removes antibiotics remaining on the surface and deep layers of meat, solves the pain points of family food safety, and makes the kitchen a "non-antibiotic defense line". The microorganisms selected by the present invention include: Bacillus safensis, Bacillus tequilensis, Bacillus subtilis, Bacillus pumilus, and Lactobacillus buchneri. These microorganisms are probiotics and will not cause new food safety hazards after processing livestock and poultry meat. In addition, the selected additives are all conventional additives in the food field to ensure the edible safety of the processed meat. The degradation rate of SPM in pork by the microbial composition reaches more than 99%, with significant results.

[0088] The embodiments described above are only some of the embodiments of the present invention, not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A microbial composition, characterized in that The active ingredients of the microbial composition are: Bacillus sabdariffa, Bacillus tequila, Bacillus subtilis, Bacillus pumilus, Lactobacillus buchneri; the deposit number of the Bacillus sabdariffa is CGMCC NO.28599; The deposit number of the Bacillus tequila is: CGMCC NO.21317; The deposit number of the Bacillus subtilis is: CCTCC NO.M20221487; The deposit number of the Bacillus pumilus is: CGMCC NO.24796; The preservation number of the Lactobacillus buchneri is: CGMCC NO.28790.

2. The microbial composition according to claim 1, characterized in that The components of the microbial composition include: a mixed bacterial suspension made from the active ingredients and an adjuvant; The mixed bacterial suspension components are as follows: based on the bacterial suspension volume ratio, Bacillus saffron suspension: Bacillus tequila suspension: Bacillus subtilis suspension: Bacillus pumilus suspension: Lactobacillus buchneri suspension = 25-35:20-30:15-25:10-20:5-15; The bacterial concentration of each bacterial suspension is not less than 10 10 CFU / mL.

3. The microbial composition according to claim 2, characterized in that The auxiliary agents include: surfactants, penetration enhancers, buffer system regulators, and bacterial nutrient supplements.

4. The microbial composition according to claim 3, characterized in that: The surfactant is selected from: one or two of sophorolipids and rhamnolipids; The penetration enhancer is selected from: one or two of glycerol and food grade sorbitol; The buffer system regulator includes: sodium bicarbonate; The bacterial cell nutritional supplement is selected from one or two of glucose and resistant dextrin.

5. The microbial composition according to claim 4, characterized in that: Calculated by mass ratio, the content of the surfactant in the mixed bacterial suspension is ≤0.2%; Calculated by mass ratio, the content of the penetration enhancer in the mixed bacterial suspension is ≤3.5%; The content of the buffer system regulator in the mixed bacterial suspension is ≤0.5% by mass; Calculated by mass ratio, the content of the bacterial nutrient supplement in the mixed bacterial suspension is ≤2.2%.

6. The use of the microbial composition according to claim 1, characterized in that: The microbial composition is used for degrading antibiotics.

7. The use according to claim 6, characterized in that The microbial composition is used for degrading residual antibiotics in food.

8. The use according to claim 7, characterized in that The antibiotics include: spiramycin.

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