Cotton bollworm source pediococcus acidilactici with gossypol degradation capability and application thereof

Abstract

The invention discloses a cotton bollworm source pediococcus acidilactici with gossypol degradation capability and application thereof, and belongs to the technical field of microorganisms, the cotton bollworm source pediococcus acidilactici with gossypol degradation capability is classified and named as JYM-1, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCCM2025009, and the preservation number is CCTCCM2025009. The 16SrDNA sequence of the cotton bollworm source pediococcus acidilactici is as shown in SEQ ID NO: 1. Probiotics capable of degrading gossypol are separated from intestinal tracts of cotton bollworms, the probiotic characteristics and safety of the probiotics are evaluated through tests of gossypol tolerance, gossypol detoxification capacity, antioxidant capacity, bacteriostatic activity, gastrointestinal stability, antibiotic sensitivity, non-hemolysis and the like, and the nutritional quality of the cottonseed meal can be remarkably improved by fermenting the cottonseed meal with the probiotics. The achievement of the invention provides a new method for improving the safety and nutritional value of the cottonseed meal as the feed, and is also beneficial to developing new probiotic resources and promoting the innovative development of the feed industry.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of *Pediococcus lactis* derived from bollworms with gossypol degradation capabilities and its applications. Background Technology

[0002] Cottonseed meal, a major byproduct of cotton processing, boasts a crude protein content exceeding 50%, making it a potential high-quality protein feed ingredient to replace soybean meal. However, the presence of anti-nutritional factors such as free gossypol (FG) in cottonseed meal severely limits its application in monogastric animal feed. Free gossypol exhibits chronic toxicity, and long-term intake can lead to growth inhibition, organ damage, and reproductive dysfunction in animals. Therefore, removing free gossypol (FG) from cottonseed meal is a crucial step in enhancing its feed value.

[0003] Currently, cottonseed meal detoxification technologies mainly include physical methods (such as heat treatment), chemical methods (such as sodium hydroxide and ferrous sulfate treatment), and microbial fermentation. While chemical methods can effectively reduce gossypol content, they suffer from problems such as high solvent consumption, high cost, easy residue of chemicals, and destruction of nutrients. In contrast, microbial detoxification methods, due to their environmental friendliness, low cost, and ability to simultaneously improve feed nutritional value, are gradually becoming a research hotspot. Currently, yeast and lactic acid bacteria are commonly used in microbial detoxification methods. Pediococcus lactis possesses probiotic properties, exhibiting antagonistic effects against pathogenic microorganisms in animals. It can competitively inhibit pathogenic microorganisms, enhance the animal's immune function, produce beneficial metabolites, activate the activity of acidic proteases, participate in the body's metabolism, and prevent the production of harmful substances. However, existing strains still have the following limitations in practical applications: Most commercially available bacterial strains have limited functionality. Most strains focus solely on gossypol degradation or nutritional improvement, lacking a synergistic effect of "detoxification-nutrition-probiotics." While some strains can degrade gossypol, their insufficient acid production leads to pH fluctuations in the fermentation system, affecting the stability of the final product. Other strains, although possessing probiotic functions, have poor tolerance to high concentrations of gossypol, making it difficult to effectively degrade gossypol in cottonseed meal.

[0004] Existing strains lack adaptability to the environment. They exhibit weak tolerance to extreme environments (such as low pH and high bile salt concentrations), resulting in low survival rates in the animal gastrointestinal tract and hindering their ability to exert long-term beneficial effects. Furthermore, some lactic acid bacteria show low survival rates in artificial gastric fluid after 2 hours, severely limiting their practical application.

[0005] Existing strains have safety deficiencies. Some strains pose a risk of hemolysis or resistance to antibiotics (such as gentamicin and streptomycin), which may be transferred to pathogens at the gene level, threatening animal and human health. Traditional strains have a narrow antibacterial spectrum and limited antagonistic effects against common pathogens (such as Escherichia coli and Salmonella), making it difficult to effectively maintain the balance of the intestinal microecology.

[0006] There are bottlenecks in process efficiency. Existing microbial detoxification processes have low gossypol degradation rates and long fermentation cycles, resulting in high production costs and limited large-scale applications.

[0007] The root cause of the above problems lies in the imperfect existing strain screening system, which lacks strains that combine efficient gossypol degradation capabilities, strong environmental adaptability, and comprehensive probiotic characteristics.

[0008] Therefore, in view of the above-mentioned technical problems, it is necessary to provide a *Pediococcus lactis* strain derived from bollworm with gossypol degradation ability and its application.

[0009] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0010] The purpose of this invention is to provide a *Pediococcus lactis* strain with gossypol degradation capability and its application, which can solve the problems of single function of strains, insufficient tolerance to extreme environments, and lack of in vitro probiotic function in existing gossypol microbial detoxification technologies.

[0011] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution: A type of Pediococcus lactis derived from bollworm with gossypol degradation ability, wherein the strain of Pediococcus lactis derived from bollworm is classified as JYM-1, the depositary institution is China Center for Type Culture Collection, and the deposit number is CCTCCM2025009; The 16S rDNA sequence of the *Pediococcus lactis* derived from *Botrytis cinerea* is shown in SEQ ID NO: 1. It can grow in MRS medium with free gossypol as the sole carbon source and is tolerant to gossypol concentrations ≤600 mg / kg.

[0012] In one or more embodiments of the present invention, the *Pediococcus lactis* derived from *Bollworm* has at least one of the following biological characteristics: (a) After culturing in MRS liquid medium containing 400 mg / kg gossypol for 24 h, the degradation rate of gossypol was ≥82.41%; (b) Maintains growth activity in an environment with pH ≥ 3.5, and has a survival rate ≥ 80.57% after 1.5 h of treatment in artificial gastric fluid; (c) It produced inhibition zones against Escherichia coli, Pseudomonas aeruginosa, Salmonella and Staphylococcus aureus, with significant differences in the diameter of the inhibition zones (p<0.05). (d) DPPH free radical scavenging rate ≥81.63%.

[0013] In one or more embodiments of the present invention, the physiological and biochemical characteristics of the *Pediococcus lactis* derived from *Bollworm* include: (a) Positive for both the aescin test and the sodium hippurate test; (b) Does not utilize raffinose, maltose, and sucrose; (c) It maintains growth activity in an environment with pH 3.5-12, and the optimal growth pH is 5.5-7.0; (d) 24-hour self-coagulation rate ≥79.97%.

[0014] In one or more embodiments of the present invention, the safety and drug resistance of the *Pediococcus lactis* derived from *Bollworm* meet the following requirements: (a) Gamma hemolytic; (b) Sensitive to penicillin, cefotaxime, clindamycin, and chloramphenicol; (c) Resistance to gentamicin, streptomycin, polymyxin B and enrofloxacin.

[0015] In one or more embodiments of the present invention, the application of a *Pediococcus lactis* strain with gossypol-degrading ability from bollworms in a microbial inoculant, wherein the microbial inoculant comprises live *Pediococcus lactis* JYM-1 cells, its metabolites, or freeze-dried bacterial powder, wherein the live count is ≥1×10⁻⁶. 8 CFU / g.

[0016] In one or more embodiments of the present invention, the application of a *Pediococcus lactis* strain with gossypol-degrading ability in the degradation of free gossypol, using *Pediococcus lactis* JYM-1 for biodegradation, includes the following steps: (1) The strain was inoculated into a culture medium containing gossypol and cultured at 37°C and 180 rpm for 24 h; (2) The concentration of free gossypol in the culture medium is 400 mg / kg; The culture medium is a glucose-free MRS medium, with gossypol as the sole carbon source.

[0017] In one or more embodiments of the present invention, the application of a cotton bollworm-derived *Pediococcus lactis* with gossypol-degrading ability in solid-state fermentation of cotton meal, using *Pediococcus lactis* JYM-1, includes the following steps: (1) Crush the cottonseed meal to a particle size of 60 mesh; (2) Inoculate with activated bacterial solution at an inoculation rate of 7% of the weight of cottonseed meal, with a material-to-water ratio of 1:0.4; (3) Ferment at 37℃ for 48 hours, and control the pH of the fermentation system to be stable at 5.25±0.01; Among them, the degradation rate of free gossypol in the fermented cottonseed meal was ≥67.51%, while the crude protein content increased by ≥45.2% and the crude fat content increased by ≥24.3%.

[0018] In one or more embodiments of the present invention, the application of a cotton bollworm-derived *Pediococcus lactis* with gossypol degradation capability in the preparation of probiotic feed additives includes: (1) Inoculate Pyrococcus lactis JYM-1 into MRS medium and culture at 37°C until the logarithmic growth phase; (2) Collect the bacterial cells by centrifugation, mix them with a protectant, and freeze-dry them to obtain freeze-dried bacterial powder; The number of live bacteria in the freeze-dried bacterial powder is ≥1×10⁻⁶ 8 CFU / g.

[0019] In one or more embodiments of the present invention, the use of a *Pediococcus lactis* strain with gossypol-degrading ability in the preparation of articles having the following functions, said articles comprising: Detoxifying agent for degrading free gossypol in cottonseed meal; Probiotic preparations that inhibit pathogenic bacteria in the intestines of livestock and poultry; Feed additives that increase crude protein in cottonseed meal through solid-state fermentation; When the product is used as a feed additive, the survival rate of the strain is ≥71.84% after treatment with artificial gastrointestinal fluid for 4.5 hours, and the surface hydrophobicity is 21.31%, with a self-aggregation rate of 79.97% after 24 hours.

[0020] In one or more embodiments of the present invention, a method for preparing *Pediococcus faecium* derived from *Bollworm* with gossypol degradation ability, wherein the *Pediococcus faecium* is isolated from the intestinal contents of 6th instar mature *Bollworm*, the specific steps of which are as follows: S1. Select the third generation of 6th instar cotton bollworms after 24 hours of starvation, sterilize their body surface with 75% ethanol and 1% sodium hypochlorite, and rinse 10 times with sterile water. S2. Dissect the worm, collect its intestinal contents, and perform serial dilutions, selecting a dilution gradient of 10⁻⁶. 5 –10- 6The bacterial suspensions of different concentrations were inoculated into the primary screening medium and cultured in a constant temperature incubator at 37℃ for 48 hours. S3. Select healthy individual plants and place them on MRS solid medium, and incubate at 37°C for 24 hours. S4. By determining the 16S rRNA gene sequence of the strain, the strain was identified as Pediococcus lactis.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention is the first to screen and isolate a strain of Pediococcus lactis JYM-1 with highly efficient gossypol degradation ability from the intestine of cotton bollworm. This strain can grow normally in MRS medium with free gossypol as the sole carbon source, and the gossypol degradation rate in the fermentation broth is ≥82.41%; 2) In the cottonseed meal fermentation experiment, the screened strain achieved a degradation rate of 67.51% for free gossypol in cottonseed meal, and the pH value stabilized at 5.52 after fermentation. This strain can also significantly improve the nutritional quality of cottonseed meal, specifically by significantly increasing the content of nutrients such as crude protein and crude fat in cottonseed meal after fermentation using this strain. 3) The screened bacteria have strong hydrophobic self-aggregation properties and strong antioxidant capacity; they have antibacterial effects against Escherichia coli, Pseudomonas aeruginosa, Salmonella and / or Staphylococcus aureus; they are resistant to gentamicin, streptomycin, polymyxin B and enrofloxacin, and sensitive to penicillin and erythromycin; they exhibit γ-hemolysis and no toxic side effects, indicating that the strain has good probiotic function. Its application in livestock production can achieve both gossypol degradation and in vitro probiotic effects. Attached Figure Description

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a colony morphology diagram of *Pediococcus lactis* JYM-1 derived from cotton bollworms in one embodiment of the present invention; Figure 2 This is a Gram-stained microscopic image of Pediococcus lactis JYM-1 from a bollworm-borne strain in one embodiment of the present invention; Figure 3 This is an phylogenetic tree diagram of the *Pediococcus lactis* JYM-1 strain derived from bollworms in one embodiment of the present invention; Figure 4 This is a diagram showing the results of the gossypol resistance test of the *Pediococcus lactis* JYM-1 strain derived from bollworms in one embodiment of the present invention; Figure 5This is a growth curve and acid production rate curve of the *Pediococcus lactis* JYM-1 strain derived from cotton bollworm in one embodiment of the present invention; Figure 6 This is a growth curve of the *Pediococcus lactis* JYM-1 strain derived from bollworms in one embodiment of the present invention at different pH values. Figure 7 This is a diagram showing the antibacterial test results of the *Pediococcus lactis* JYM-1 strain derived from cotton bollworm against *Pseudomonas aeruginosa* in one embodiment of the present invention. Figure 8 This is a diagram showing the antibacterial test results of the *Pediococcus lactis* JYM-1 strain derived from cotton bollworms against *Escherichia coli* in one embodiment of the present invention. Figure 9 This is a diagram showing the antibacterial test results of the *Pediococcus lactis* JYM-1 strain derived from cotton bollworm against *Salmonella* in one embodiment of the present invention. Figure 10 This is a diagram showing the antibacterial test results of the *Pediococcus lactis* JYM-1 strain derived from cotton bollworm against *Staphylococcus aureus* in one embodiment of the present invention. Figure 11 This is a diagram showing the hemolytic test results of the *Pediococcus lactis* JYM-1 strain derived from cotton bollworm in one embodiment of the present invention; Figure 12 This is a diagram showing the bile salt tolerance test results of the *Pediococcus lactis* JYM-1 strain derived from cotton bollworm in one embodiment of the present invention; Figure 13 This is a diagram showing the self-aggregation and surface hydrophobicity test results of the *Pediococcus lactis* JYM-1 strain derived from cotton bollworm in one embodiment of the present invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0025] This invention aims to isolate probiotics capable of degrading gossypol from the intestines of cotton bollworms. Its probiotic properties and safety are evaluated through tests such as gossypol tolerance, gossypol detoxification ability, antioxidant capacity, antibacterial activity, gastrointestinal stability, antibiotic sensitivity, and non-hemolytic activity. Fermentation of cottonseed meal with these probiotics significantly improves the nutritional quality of the cottonseed meal. This invention will provide a new method for improving the safety and nutritional value of cottonseed meal as feed, and will also contribute to the development of new probiotic resources, promoting innovation in the feed industry. Cotton bollworm-derived *Pediococcus lactis* can be used to prepare feed additives that degrade free gossypol (FG) and possess probiotic functions.

[0026] One embodiment of the present invention discloses a Pediococcus acidilactici strain with gossypol degradation ability, which is classified and named JYM-1. The depositary institution is the China Center for Type Culture Collection, with accession number CCTCCM2025009. The 16S rDNA sequence of Pediococcus acidilactici is shown in SEQ ID NO: 1.

[0027] This strain was isolated from the intestinal contents of 6th instar mature cotton bollworms. The screening criteria included, but were not limited to, characteristics such as gossypol degradation rate, cottonseed meal fermentation function, acid resistance, alkali resistance, bile salt resistance, hemolytic activity, and antibacterial activity.

[0028] When this strain was inoculated into glucose-free MRS solid medium with a free gossypol concentration of 400 mg / kg, it formed round or oval colonies that were milky white with neat edges and a smooth, moist surface, indicating that it was a Gram-positive bacterium.

[0029] Example 1: Isolation and Identification of Strains 1. Experimental materials and bacterial source Source of bacterial strain: The bacterial strain isolated in this experiment was derived from healthy sixth-instar cotton bollworms of the third generation after subculture.

[0030] Preparation of primary screening medium: MRS broth medium without glucose (purchased from Qingdao Haibo Biotechnology Co., Ltd.) was prepared into a 66.2 g / L solution, autoclaved at 121℃ for 15 min, and then gossypol acetate was added at a concentration of 400 mg / kg.

[0031] Preparation of MRS solid culture medium: MRS agar medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) was prepared into a 66.2 g / L solution and autoclaved at 121℃ for 15 min.

[0032] 2. Isolation and Identification of Pediococcus lactis Third-generation sixth-instar cotton bollworms, after 24 hours of starvation, were selected and their bodies were sterilized with 75% ethanol and 1% sodium hypochlorite, followed by rinsing 10 times with sterile water. After dissection, the intestinal contents were collected and serially diluted. Bacterial suspensions at dilution gradients of 10⁻⁵–10⁻⁶ were inoculated into primary screening media and cultured at 37°C for 48 hours. Single colonies with good growth were then picked and inoculated onto MRS solid medium and cultured at 37°C for 24 hours. Colony characteristics were observed, and single colony smears were taken with a sterile inoculation loop and Gram-stained. The morphology of the bacteria was observed under an oil immersion microscope.

[0033] The colonies are round or oval in shape, milky white in color, with neat edges and a smooth, moist surface.

[0034] The strain turned blue-purple after Gram staining, indicating that it is a Gram-positive bacterium.

[0035] 3. Molecular biological identification of Pediococcus lactis The strain was inoculated into MRS liquid medium and cultured at 37°C for 24 hours to activate and preserve the strain. The PCR amplification product of the 16S rRNA of this bacterium was sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for 16S rRNA sequencing. The sequencing results were confirmed as Pediococcus acidilactici by NCB1Blast comparison.

[0036] 4. Biochemical identification of Pediococcus lactis Physiological and biochemical tests of the strain were performed using bacterial micro-biochemical identification tubes (HBIG11, Qingdao Haibo Biotechnology Co., Ltd.), following the instructions. Results showed that the *Pediococcus lactis* JYM-1, derived from *Botrytis cinerea*, could not utilize sugars such as raffinose, maltose, and sucrose; both the aescin test and the sodium hippurate test were positive.

[0037] Table 1 Physiological and biochemical characteristics of Pediococcus lactis JYM-1 Table l Physiological and biochemical characteristics of Pediococcus acidilactici JYM-1

[0038] +: positive; : negative.

[0039] Example 2: Functional assessment of gossypol tolerance in *Pediococcus lactis* The strain was inoculated onto MRS solid medium containing 200 mg / kg, 400 mg / kg, 600 mg / kg, and 800 mg / kg gossypol acetate, respectively, and streaked onto plates for isolation. The plates were then incubated at 37°C for 48 hours. The growth status of the strain was observed during these 48 hours to assess its tolerance to gossypol.

[0040] The results are as follows Figure 4 In MRS medium with gossypol as the sole carbon source, *Pediococcus acidilactici* JYM-1 exhibited a maximum gossypol tolerance concentration of 600 mg / kg (normal growth was observed at 200 mg / kg and 400 mg / kg, but complete inhibition occurred at 800 mg / kg). This indicates that *Pediococcus acidilactici* JYM-1 possesses high gossypol tolerance.

[0041] Example 3: Evaluation of the Gossypol Degradation Function of Pediococcus lactis Single colonies of the target strain were picked and inoculated into 5 mL MRS liquid medium for amplification. The inoculated tubes were placed in a 37°C constant-temperature shaking incubator and incubated at 180 rpm for 24 h to obtain the primary seed culture in the logarithmic growth phase. JYM-1 was inoculated into MRS medium with gossypol as the sole carbon source (FG = 400 mg / kg) and incubated at 37°C and 180 rpm for 24 h. After centrifugation (10000 rpm, 4°C), the supernatant was filtered through a 0.22 μm microporous membrane for sterilization, finally obtaining sterile fermentation broth. The gossypol content in the fermentation broth before and after fermentation was determined by high-performance liquid chromatography (HPLC). The final determination showed that JYM-1 degraded 82.41% of gossypol.

[0042] Example 4: Determination of the Probiotic Properties of Pediococcus lactis 1. Growth curves and acid production curves of Pediococcus lactis The activated third-generation bacterial strain was inoculated at a rate of 2% into MRS liquid medium and cultured at 37°C in a shaker (180 rpm). Aseptic samples were taken at 0, 2, 6, 9, 12, 15, 18, 21, 24, 30, and 36 hours, and the OD was measured using a spectrophotometer. 600 The hydrogen ion concentration in the culture medium was simultaneously measured using a pH meter. Each experiment was performed in triplicate. OD values ​​were used to measure the hydrogen ion concentration in the culture medium. 600 The growth curve of *Pediococcus lactis* strain was plotted with the pH value as the ordinate and the incubation time as the abscissa. A time-pH change curve was also constructed simultaneously. Data were fitted using Origin 9.0 software.

[0043] The growth curves and acid production curves of *Pediococcus lactis* JYM-1 are shown below. Figure 4As shown, the strain was in the growth retardation stage from 0 to 2 h. After 2 hours of inoculation, the strain entered the logarithmic growth phase. When the inoculation time reached 21 hours, the growth of the strain entered the stationary phase, indicating that this Pediococcus acidilactici had good growth performance.

[0044] As Figure 5 shown, the pH of the Pediococcus acidilactici JYM-1 bacterial liquid decreased slowly within 2 h after inoculation, from the initial 5.86 to 5.54, and then the pH decreased rapidly. After 18 h of inoculation, the pH tended to be stable, and finally the pH stabilized at about 4.20. This indicated that this Pediococcus acidilactici had good acid-producing ability.

[0045] 2. Growth curve of Pediococcus acidilactici in different pH The activated third-generation strain was inoculated into MRS broth medium (pH gradient: 2.0, 2.5, 3.0, 3.5, 4.5, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0) at an inoculation amount of 2%. The culture medium was continuously cultured in a constant temperature shaker at 37 °C (180 rpm) for 48 h. Sterile samples were taken at 0, 2, 5, 8, 12, 18, 24, 30, 36 h respectively, and the change in the number of bacterial cells was measured by the spectrophotometer method (OD 600 ) Each group was set with 3 biological replicates. With the pH value as the abscissa and the OD 600 value of the total number of colonies as the ordinate, a curve was plotted.

[0046] The growth of Pediococcus acidilactici JYM-1 in different pH environments was as Figure 6 shown. When pH ≥ 3.5, the strain could still grow. Its OD 600 value was pH 5.5 < pH 6.0 < pH 7.0, indicating that this strain had good acid tolerance.

[0047] 3. Determination of the in vitro antibacterial activity of Pediococcus acidilactici The Oxford cup method was used to evaluate the antibacterial activity of the test strain. The standard strains of Gram-positive bacteria and Gram-negative bacteria were cultured in a liquid medium at 37 °C until the logarithmic growth phase. Prepare an MRS agar plate. Before the plate solidified, 4 sterile Oxford cups were placed equidistantly. After the plate solidified, it was taken out. 100 μL of the filtrate of the test strain was added into each well and cultured at 37 °C for 24 hours. A vernier caliper was used to measure the diameter of the antibacterial circle. Each group was set with three parallel replicates. An antibacterial circle difference ≥ 2 mm was judged as a significant antibacterial effect (p < 0.05).

[0048] By Figure 7-10As shown in Table 2, the bacterial suspension and cell-free supernatant of *Pediococcus lactis* JYM-1 effectively inhibited the growth and proliferation of four pathogenic bacteria. There was no significant difference in the inhibitory effects of the bacterial suspension on *Pseudomonas aeruginosa*, *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus*. The cell-free supernatant showed significantly better inhibitory effects on *Salmonella* than on *Escherichia coli*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* (P<0.05). This indicates that this strain possesses good antibacterial properties.

[0049] Table 2 Results of antibacterial test of P. lactis JYM-1

[0050] 4. Antibiotic susceptibility testing of Pediococcus lactis After activating the bacterial strain, 100 μL was evenly spread onto the surface of an MRS agar plate. Disks containing 16 antibiotics (CLSI recommended concentrations), including penicillin (PEN), cefotaxime (CF), and ampicillin (AMP), were fixed to the culture medium surface using sterile forceps and arranged in an equidistant triangular pattern. The plates were incubated at 37°C for 48 hours, and the presence and diameter of inhibition zones were accurately measured. The experiment was performed in triplicate, and the data were averaged from all three trials.

[0051] Pediococcus lactis JYM-1 showed strong sensitivity to four antibiotics: cefotaxime, clindamycin, chloramphenicol, and penicillin, with inhibition zones larger than 23 mm in diameter; inhibition zones of 15–22 mm were observed to four other antibiotics: vancomycin, rifampin, erythromycin, and ampicillin; inhibition zones of 7–14 mm were observed to one antibiotic: tetracycline; and it was not sensitive to seven other antibiotics: levofloxacin, gentamicin, streptomycin, polymyxin B, enrofloxacin, ciprofloxacin, and ofloxacin.

[0052] Table 3. Results of antibiotic susceptibility testing for Pediococcus lactis JYM-1 Table 3 Results of antibiotic susceptibility test of Pediococcusacidilactici JYM-1

[0053] +++: Inhibition zone diameter 23-30 mm; ++: Inhibition zone diameter 15-22 mm; +: Inhibition zone diameter 7-14 mm; -: No inhibition zone.

[0054] +++: inhibition circle diameter 23 to 30 mm; ++: inhibition circle diameter l5 to 22 mm; +:inhibition circle diameter 7 to14 mm; -:no inhibition circle. 5. Identification of hemolytic activity of Pediococcus lactis Hemolytic activity identification: The strain was inoculated at 1% in MRS liquid medium and kept at 37℃ for 24 hours. Then it was inoculated into blood agar medium with Bacillus tropicalis as a positive control. The culture was kept at 37℃ for 16 hours and the presence of hemolytic zones was observed.

[0055] The results are as follows Figure 11 As shown, *Pediococcus lactis* JYM-1 showed no hemolytic zone, indicating γ-hemolysis; the positive control *Bacillus tropicalis* showed a clear hemolytic zone, indicating β-hemolysis. This indicates that the strain has high safety.

[0056] 6. Gastrointestinal tolerance test of Pietrococcus lactis Bile salt tolerance of strains: After 24 hours of activation culture, the bacterial suspension was centrifuged at 10000×g for 10 min to enrich the bacterial cells. The cells were washed twice with sterile phosphate buffer, and the supernatant was discarded. The bacterial pellet was resuspended in MRS liquid medium containing gradient concentrations of ox bile salt (0.1%, 0.3%, 0.5%, 0.7%, 0.9% w / v) and incubated at 37℃ for 0 and 4 hours, respectively. After treatment, 1 mL of the bacterial suspension was serially diluted 10-fold, and suitable gradients were inoculated into MRS liquid medium. After incubation at 37℃ for 24 hours, samples were taken, and the OD of the strain was determined using a spectrophotometer. 600 Value. Perform three replicates and take the average. Plot the total bacterial count (OD) on the x-axis, with bile salt concentration as the abscissa. 600 Plot the bile salt tolerance curve with y as the vertical axis.

[0057] like Figure 12 The results showed that the growth of Pseudococcus lactis JYM-1 was relatively slow as the salt concentration increased. Although the growth of the strain was slow at high salt concentrations, it did not stop, indicating that the strain has a certain salt tolerance and can be regarded as having potential intestinal colonization ability.

[0058] Tolerance of artificial gastrointestinal models: After 24 hours of activation and culture, the bacterial culture was centrifuged at 10000×g for 10 min to enrich the bacterial cells. The cells were then washed twice with sterile phosphate-buffered saline (PBS, pH 7.4), and the supernatant was discarded. A two-stage treatment was implemented: (1) Gastric juice: The bacterial suspension was inoculated into an artificial gastric juice environment at an inoculum volume of 3% and incubated in a constant temperature shaking incubator at 37℃ at 180 r / min. Samples were collected at 0h, 2h, and 4h, and after serial dilution by 10-fold, they were evenly spread on selective solid medium and incubated at 37℃ for 48 hours. Valid plates with CFU in the range of 30-300 were selected for colony counting, with three replicates and the average of the three counts. The survival rate was calculated according to the following formula: Survival rate / % = Nt / No × 100 Where: Nt represents the number of viable bacteria at each time point, in CFU / mL; N0 represents the number of viable bacteria at 0h, in CFU / mL. (2) Intestinal fluid: The bacterial suspension was inoculated into the artificial intestinal fluid system at a 4% inoculum and cultured at 180 r / min in a 37℃ constant temperature shaking incubator. Bacterial suspension samples were collected at 0 h, 4 h, and 8 h, and diluted using a tenfold serial dilution method. The appropriately diluted bacterial suspension was evenly spread onto MRS selective solid medium and incubated at 37℃ for 48 hours. Effective culture plates with CFU counts between 30 and 300 were screened for colony counting. Three replicates were set up, and the average of the three replicates was taken. The survival rate was calculated using the following formula: Survival rate / % = Nt / No × 100 In the formula, Nt represents the viable bacterial count at each time period, in CFU / mL; N0 represents the viable bacterial count at 0h, in CFU / mL. The survival rate of the strain was 80.57% after 1.5 hours of inoculation with artificial gastric fluid, 90.54% after 3 hours of inoculation with artificial intestinal fluid, and 71.84% after 4.5 hours of inoculation with artificial gastrointestinal fluid.

[0059] Table 4. Survival rate of Pleurotus ostreatus JYM-1 in artificial gastrointestinal fluid. Table 4 Survival rates of Pediococcus acidilactici JYM-1 ingastrointestinal juices

[0060] 7. Determination of self-aggregation ability and surface hydrophobicity of Pediococcus lactis (1) Determination of self-cohesive force: The bacterial culture, activated for 24 hours, was centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded, and the bacterial pellet was washed twice with PBS. The bacterial cell resuspended in an equal volume of sterile PBS, and the OD was measured using a spectrophotometer. 630(A1). After shaking for 10 seconds, the bacterial suspension was aliquoted and incubated at 37°C for 8 h, 16 h, and 24 h. The OD of the bacterial suspension was measured at each time point. 630 (A2). The experiment was set up in three replicates, and the final data was the average of the three replicates. The self-agglomeration rate was calculated using the following formula: Self-cohesion rate (%) = (1 - A2 / A1) × 100% The autoaggregation rates of Pyotrophic Lateral Sclerosis (PFS) JYM-1 after 8, 16, and 24 hours of culture were 45.66%, 63.84%, and 79.97%, respectively.

[0061] (2) Surface hydrophobicity determination: The bacterial suspension, activated and cultured for 24 hours, was centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the bacterial pellet was washed twice with PBS. The bacterial cell resuspended in PBS, and the OD was measured using a spectrophotometer. 630 (A3). Mix an equal volume of bacterial suspension with xylene-chloroform solution, vortex for 5 minutes, then transfer to a 37°C incubator and let stand for 4 hours. After the solution separates into layers, collect the upper aqueous phase using a pipette and measure its OD. 630 Value (A4). Set three parallel values ​​and take the average of the three values. Hydrophobicity is calculated using the standard formula: Hydrophobicity (%) = [1 - (A4 / A3)] × 100% The results showed that the surface hydrophobicity of Pyorrhic Lactococcus JYM-1 was 21.31%, indicating that the strain has moderate surface hydrophobicity.

[0062] 8. Determination of antioxidant capacity of Pediococcus lactis The 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) (BC4750) radical detection kit (Beijing Solarbio Science & Technology Co., Ltd.) was used, and the experiments were performed according to the standard operating procedure in the instruction manual. Three replicates were set up, and the average value of the three experiments was taken.

[0063] The results showed that *Pediococcus lactis* JYM-1 had a high scavenging capacity for DPPH free radicals, with a DPPH free radical scavenging rate of 81.63%. This indicates that the strain has good antioxidant properties.

[0064] Example 5: Solid-state fermentation experiment of cottonseed meal with Pediococcus lactis After two generations of activation, the bacterial strain was inoculated into MRS liquid medium to obtain primary bacterial culture. This primary culture was then inoculated into cottonseed meal (which had been pulverized and sieved through a 60-mesh sieve) at a 7% inoculation rate for solid-state fermentation for 48 hours. The material-to-water ratio was 1:0.4, and fermentation was carried out at 37℃ for 48 hours. The free gossypol content in the fermented cottonseed meal was determined using the international phloroglucinol method. Three replicates were performed, and the average absorbance values ​​were collected. The gossypol content in the fermented cottonseed meal was calculated based on the gossypol standard curve.

[0065] Crude protein content was determined using a FOSS 8400 fully automatic Kjeldahl nitrogen analyzer; dry matter, crude fat, crude ash, neutral detergent fiber, and acid detergent fiber were all measured in accordance with their respective national standards.

[0066] The results of solid-state fermentation of cottonseed meal are shown in Tables 5 and 6. *Pediococcus lactis* (from bollworm) Pediococcus acidilactici JYM-1 stabilizes the pH of the system at 5.25±0.01 through acid production metabolism, achieves a degradation rate of 67.51% for free gossypol in cottonseed meal, and significantly improves nutritional indicators such as crude protein, acid-soluble protein, and crude fat.

[0067] Table 5. Effects of JYM-1 fermentation on the nutritional value of cottonseed meal substrate (%)

[0068] Table 6. Effects of JYM-1 fermentation on free gossypol content and pH in cottonseed meal.

[0069] This invention is the first to screen and isolate a strain of Pediococcus acidilactici JYM-1 with high gossypol degradation ability from the intestine of cotton bollworm. This strain can grow normally in MRS medium with free gossypol as the sole carbon source, and the gossypol degradation rate in the fermentation broth is ≥82.41%.

[0070] In cottonseed meal fermentation experiments, the screened strain achieved a 67.51% degradation rate of free gossypol in cottonseed meal, and the pH value remained stable at 5.52 after fermentation. This strain also significantly improved the nutritional quality of cottonseed meal, specifically by significantly increasing the content of crude protein, crude fat, and other nutrients in the fermented cottonseed meal.

[0071] The screened bacteria exhibit strong hydrophobic self-aggregation and antioxidant capacity; they show antibacterial effects against *Escherichia coli*, *Pseudomonas aeruginosa*, *Salmonella* and / or *Staphylococcus aureus*; they are resistant to gentamicin, streptomycin, polymyxin B, and enrofloxacin, but sensitive to penicillin and erythromycin; they exhibit γ-hemolysis without toxic side effects. This indicates that the strain possesses good probiotic functions, and its application in livestock production can achieve both gossypol degradation and probiotic effects.

[0072] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0073] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A type of *Pediococcus lactis* derived from *Botrytis cinerea* with gossypol-degrading ability, characterized in that, The strain of *Pediococcus lactis* derived from the cotton bollworm was classified and named JYM-1. The depositary institution was the China Center for Type Culture Collection, and the accession number was CCTCCM2025009. The 16S rDNA sequence of the *Pediococcus lactis* derived from *Botrytis cinerea* is shown in SEQ ID NO:

1. It can grow in MRS medium with free gossypol as the sole carbon source and is tolerant to gossypol concentrations ≤600 mg / kg.

2. The *Pediococcus lactis* strain derived from bollworms with gossypol degradation capability according to claim 1, characterized in that, The *Pediococcus lactis* derived from the cotton bollworm has at least one of the following biological characteristics: (a) After culturing in MRS liquid medium containing 400 mg / kg gossypol for 24 h, the degradation rate of gossypol was ≥82.41%; (b) Maintains growth activity in an environment with pH ≥ 3.5, and has a survival rate ≥ 80.57% after 1.5 h of treatment in artificial gastric fluid; (c) It produced inhibition zones against Escherichia coli, Pseudomonas aeruginosa, Salmonella and Staphylococcus aureus, with significant differences in the diameter of the inhibition zones (p<0.05). (d) DPPH free radical scavenging rate ≥81.63%.

3. The *Pediococcus lactis* strain derived from *Botrytis cinerea* with gossypol degradation capability according to claim 1, characterized in that, The physiological and biochemical characteristics of the *Pediococcus lactis* derived from *Bollworm* include: (a) Positive for both the aescin test and the sodium hippurate test; (b) Does not utilize raffinose, maltose, and sucrose; (c) It maintains growth activity in an environment with pH 3.5-12, and the optimal growth pH is 5.5-7.0; (d) 24-hour self-coagulation rate ≥79.97%.

4. The *Pediococcus lactis* strain derived from bollworms with gossypol degradation capability according to claim 1, characterized in that, The safety and drug resistance of the *Pediococcus lactis* derived from *Botrytis cinerea* meet the following requirements: (a) Gamma hemolytic; (b) Sensitive to penicillin, cefotaxime, clindamycin, and chloramphenicol; (c) Resistance to gentamicin, streptomycin, polymyxin B and enrofloxacin.

5. The application of *Pediococcus faecium* derived from *Botrytis cinerea* with gossypol degradation ability as described in any one of claims 1-4 in a microbial inoculant, characterized in that... The microbial agent comprises live cells of *Pediococcus lactis* JYM-1 as described in any one of claims 1-4, its metabolites, or freeze-dried bacterial powder, wherein the live cell count is ≥1×10⁻⁶. 8 CFU / g.

6. The application of *Pediococcus pyogenes*, a *Lactococcus* species with gossypol-degrading ability as described in any one of claims 1-4, in the degradation of free gossypol, characterized in that... Biodegradation using *Pediococcus lactis* JYM-1 according to any one of claims 1-4 includes the following steps: (1) The strain was inoculated into a culture medium containing gossypol and cultured at 37°C and 180 rpm for 24 h; (2) The concentration of free gossypol in the culture medium is 400 mg / kg; The culture medium is a glucose-free MRS medium, with gossypol as the sole carbon source.

7. The application of *Pediococcus faecium* derived from *Botrytis cinerea* with gossypol-degrading ability as described in any one of claims 1-4 in solid-state fermentation of cottonseed meal, characterized in that... The method of using Pediococcus lactis JYM-1 according to any one of claims 1-4 includes the following steps: (1) Crush the cottonseed meal to a particle size of 60 mesh; (2) Inoculate with activated bacterial solution at an inoculation rate of 7% of the weight of cottonseed meal, with a material-to-water ratio of 1:0.4; (3) Ferment at 37℃ for 48 hours, and control the pH of the fermentation system to be stable at 5.25±0.01; Among them, the degradation rate of free gossypol in the fermented cottonseed meal was ≥67.51%, while the crude protein content increased by ≥45.2% and the crude fat content increased by ≥24.3%.

8. The application of *Pediococcus faecium* derived from *Botrytis cinerea* with gossypol degradation ability as described in any one of claims 1-4 in the preparation of probiotic feed additives, characterized in that... include: (1) Inoculate the Pyrococcus lactis JYM-1 according to any one of claims 1-4 into MRS medium and culture at 37°C until the logarithmic growth phase; (2) Collect the bacterial cells by centrifugation, mix them with a protectant, and freeze-dry them to obtain freeze-dried bacterial powder; The number of live bacteria in the freeze-dried bacterial powder is ≥1×10⁻⁶ 8 CFU / g.

9. The use of *Pediococcus faecium* from *Botrytis cinerea* with gossypol-degrading ability as described in any one of claims 1-4 in the preparation of articles having the following functions, characterized in that, The product includes: Detoxifying agent for degrading free gossypol in cottonseed meal; Probiotic preparations that inhibit pathogenic bacteria in the intestines of livestock and poultry; When the product is used as a feed additive, the survival rate of the strain is ≥71.84% after treatment with artificial gastrointestinal fluid for 4.5 hours, and the surface hydrophobicity is 21.31%, with a self-aggregation rate of 79.97% after 24 hours.

10. A method for preparing *Pediococcus faecium* derived from *Botrytis cinerea* with gossypol degradation ability as described in any one of claims 1-4, characterized in that, The *Pediococcus lactis* derived from *Bollworm* was isolated from the intestinal contents of 6th instar mature *Bollworm*, and the specific steps are as follows: S1. Select the third generation of 6th instar cotton bollworms after 24 hours of starvation, sterilize their body surface with 75% ethanol and 1% sodium hypochlorite, and rinse 10 times with sterile water. S2. Dissect the worm, collect its intestinal contents, and perform serial dilution, selecting a dilution gradient of 10. -5 -10 -6 The bacterial suspensions of different concentrations were inoculated into the primary screening medium and cultured in a constant temperature incubator at 37℃ for 48 hours. S3. Select a single colony with good growth and inoculate it onto MRS solid medium containing 200 mg / kg gossypol, and incubate at 37°C for 24 h. S4. By determining the 16S rRNA gene sequence of the strain, the strain was identified as Pediococcus lactis.

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