Bio-based antibacterial agent based on yeast fermentation and preparation method thereof

Abstract

The invention discloses a bio-based antibacterial agent based on yeast fermentation and a preparation method of the bio-based antibacterial agent in the field of antibacterial agents. The bio-based antibacterial agent comprises the following components in parts by weight: 2-4 parts of modified chitosan, 1 part of modified polyethylene glycol, 0.2-0.3 part of pig gastric mucoprotein and 0.6-0.8 part of sodium alginate. According to the invention, a gene corresponding to an amino acid sequence of tachyplesin is optimized; according to the invention, a pYES2 / CT vector is used as a target gene, an EcoR I / Xho I restriction enzyme cutting site is introduced to a 5 ' / 3' end, it is ensured that the target gene can be accurately inserted into the pYES2 / CT vector, Tachyplesin-1 is a natural antibacterial peptide, a killing effect can be realized by destroying cell membranes of harmful bacteria and interfering metabolism of the harmful bacteria, and Tachyplesin breaks through intestinal environment limitation and accurately acts on the harmful bacteria through coating and functional modification of a modified vector.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial agent technology, specifically referring to a bio-based antibacterial agent based on yeast fermentation and its preparation method. Background Technology

[0002] As a vital organ for digestion, absorption, and immunity, the gut microbiota balance directly determines human health. In recent years, with the refinement of diets, increased life stress, and antibiotic overuse, the incidence of intestinal diseases (such as acute gastroenteritis, inflammatory bowel disease, and irritable bowel syndrome) caused by the overgrowth of harmful intestinal bacteria (such as pathogenic Escherichia coli, Salmonella, and Clostridium perfringens) has continued to rise. Therefore, highly effective and safe antibacterial technologies targeting harmful intestinal bacteria have become a key need for safeguarding human health.

[0003] Harmful gut bacteria compete for nutrients, secrete toxins (such as Shiga toxin produced by E. coli and endotoxin produced by Salmonella), and damage the intestinal mucosal barrier, causing a series of health problems. These problems are becoming increasingly common and complex. In terms of the scope of disease impact, harmful gut bacteria not only cause local symptoms such as acute diarrhea and abdominal pain, but their secreted toxins can also affect the function of organs such as the liver and kidneys through blood circulation. Long-term microecological imbalance further increases the risk of chronic diseases such as colorectal cancer, obesity, and diabetes. In terms of the expanding susceptible population, infants (whose intestinal barrier is not fully developed), the elderly (whose immune function is weakened), and sub-healthy individuals (who often stay up late and have irregular eating habits) are high-risk groups for harmful gut bacteria infection. The increasing harm caused by harmful gut bacteria means that traditional symptomatic treatment methods are no longer sufficient, necessitating the development of novel antibacterial technologies that can precisely inhibit harmful bacteria while protecting the intestinal microecology.

[0004] Current methods for controlling harmful bacteria in the gut mainly include chemically synthesized antibacterial drugs, antibiotics, and traditional probiotic preparations. However, all three types of technologies have unavoidable shortcomings and cannot achieve the comprehensive goals of safety, high efficiency, and protection of the gut microbiota.

[0005] In terms of chemically synthesized antibacterial drugs, quinolone drugs such as norfloxacin and ciprofloxacin can quickly kill harmful bacteria in the intestines, but they have serious side effects. Long-term use can damage the integrity of the intestinal mucosa, leading to increased intestinal permeability. Moreover, drug residues can easily enter the environment through feces, causing the spread of drug resistance in aquatic microorganisms. At the same time, their antibacterial spectrum is non-selective, which inhibits beneficial bacteria in the intestines (such as bifidobacteria and lactobacilli) while killing harmful bacteria, resulting in a prolonged period of microecological imbalance of 2-3 months.

[0006] In terms of antibiotic use, penicillin and cephalosporin antibiotics have limited inhibitory effects on Gram-positive harmful bacteria in the intestine (such as Clostridium perfringens), and the problem of overuse is extremely prominent, leading to a year-on-year increase in the drug resistance rate of harmful bacteria in the intestine.

[0007] Traditional probiotic preparations, while able to inhibit harmful bacteria through the occupancy effect, have two major drawbacks: first, the survival rate of probiotics in gastric acid (pH=1-3) and bile environments is low, resulting in an extremely low number of effective live bacteria reaching the intestines for colonization; second, their antibacterial effect is slow, and their inhibition efficiency against over-proliferated harmful bacteria is low, failing to meet the need for rapid prevention and control of acute intestinal infections. Summary of the Invention

[0008] To address the above issues and overcome the shortcomings of existing technologies, this invention provides a yeast-fermented bio-based antibacterial agent and its preparation method. This invention optimizes the gene corresponding to the amino acid sequence of Tachyplesin-1 and introduces EcoRI / XhoI restriction sites at the 5' / 3' end to ensure precise insertion of the target gene into the pYES2 / CT vector. Tachyplesin-1 is a natural antimicrobial peptide that kills harmful bacteria by disrupting their cell membranes and interfering with their metabolism. Through modified vector encapsulation and functional modification, Tachyplesin-1 overcomes the limitations of the intestinal environment and precisely targets harmful bacteria.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention proposes a bio-based antibacterial agent based on yeast fermentation, wherein the bio-based antibacterial agent includes yeast liquid;

[0010] Preferably, the yeast culture is a seed culture of Saccharomyces cerevisiae INVSC1 on pYES2 / CT-Tac carrier after activation;

[0011] Preferably, the brewer's yeast INVSC1 is a recombinant yeast strain with optimized Tachyplesin-1 genome;

[0012] Preferably, the optimized whole genome sequence of Tachyplesin-1 is shown in SEQ ID NO:1, with an EcoR I restriction site at the 5' end and an Xho I restriction site at the 3' end of the whole genome sequence shown in SEQ ID NO:1.

[0013] Preferably, the optimized amino acid sequence of Tachyplesin-1 is shown in SEQ ID NO:2;

[0014] Preferably, the bio-based antibacterial agent further comprises the following components in parts by weight: 2-4 parts modified chitosan, 1 part modified polyethylene glycol, 0.2-0.3 parts porcine gastric mucoprotein, and 0.6-0.8 parts sodium alginate;

[0015] This invention also provides a method for preparing a yeast-based antibacterial agent, specifically comprising the following steps:

[0016] S1. Dissolve chitosan in dilute hydrochloric acid aqueous solution, perform microwave treatment once, adjust the pH of the reaction system to 1, add dicyandiamide solution to the reaction system, mix well, perform microwave treatment a second time, after the reaction is completed, add anhydrous ethanol for precipitation treatment, collect the precipitate, wash, dry, grind, and obtain modified chitosan.

[0017] Preferably, in step S1, the mass ratio of chitosan to dicyandiamide is 1:0.004-0.006;

[0018] Preferably, in step S1, the dicyandiamide solution is prepared by dissolving dicyandiamide in deionized water at 40-50°C, adjusting the pH to 1, and mixing thoroughly to obtain the dicyandiamide solution.

[0019] Preferably, in step S1, the microwave power of a single microwave treatment is 400-600W, the treatment temperature of a single microwave treatment is 40-50℃, and the treatment time of a single microwave treatment is 10-15min.

[0020] Preferably, in step S1, the microwave power of the secondary microwave treatment is 400-600W, the treatment temperature of the secondary microwave treatment is 95-105℃, and the treatment time of the secondary microwave treatment is 10-20min.

[0021] S2. Dissolve polyethylene glycol in DMF, add HDI (hexamethylene diisocyanate) reaction solution dropwise. After the addition is complete, raise the reaction temperature to carry out the addition reaction. After the reaction is complete, cool, remove the reaction solvent by vacuum distillation, add anhydrous diethyl ether for precipitation treatment, filter, collect the solid, wash and dry to obtain modified polyethylene glycol.

[0022] Preferably, in step S2, the mass ratio of polyethylene glycol to HDI is 1:0.18-0.26;

[0023] Preferably, in step S2, the reaction temperature of the addition reaction is 80-90℃, and the reaction time of the addition reaction is 2-4 hours;

[0024] S3. Dissolve the modified chitosan prepared in step S1 in hydrochloric acid aqueous solution to obtain a modified chitosan solution. Dissolve the modified polyethylene glycol prepared in step S2 in anhydrous ethanol to obtain a modified polyethylene glycol solution. Mix the modified chitosan solution and the modified polyethylene glycol solution evenly to obtain a coated prepolymer solution.

[0025] S4. Dissolve porcine gastric mucoprotein in a buffer solution, add sodium cyanoborohydride, stir well, and obtain a porcine gastric mucoprotein solution.

[0026] Preferably, in step S4, the mass ratio of porcine gastric mucoprotein to sodium cyanoborohydride is 10-15:1;

[0027] S5. Mix yeast culture with sodium alginate aqueous solution, homogenize under high pressure to form a homogeneous system, inject the mixture into an extrusion preparation instrument, add calcium chloride aqueous solution dropwise, solidify, filter, and disperse in PBS buffer to obtain yeast protocyst solution.

[0028] Preferably, in step S5, the concentration of the yeast culture is 4 × 10⁻⁶. 8 -6×10 8 CFU / mL;

[0029] Preferably, in step S5, the mass fraction of the sodium alginate aqueous solution is 2%-4%;

[0030] Preferably, in step S5, the volume ratio between the yeast culture and the sodium alginate aqueous solution is 1:2-3;

[0031] Preferably, in step S5, the mass fraction of the calcium chloride aqueous solution is 2%-4%;

[0032] S6. Take the yeast pre-capsule solution prepared in step S5 and place it in a flask. Under a nitrogen atmosphere, add the coating prepolymer solution prepared in step S3. After the addition is complete, maintain the reaction temperature to carry out the cross-linking reaction. After the reaction is complete, add calcium chloride aqueous solution, let stand, solidify, filter, collect the solid, wash and obtain the yeast coating agent.

[0033] Preferably, in step S6, the crosslinking reaction temperature is 30-40℃ and the crosslinking reaction time is 2-3 hours;

[0034] S7. Disperse the yeast coating agent prepared in step S6 in Tris-HCl buffer (pH=8.0), add triethylamine, stir at room temperature, slowly add the porcine gastric mucoprotein solution prepared in step S4, raise the reaction temperature and stir the reaction. After the reaction is completed, add glycine aqueous solution (pH=8.0), continue stirring for 30-60 min, filter to collect the solid, wash, and vacuum dry to obtain the bio-based antibacterial agent.

[0035] The beneficial effects achieved by this invention are as follows:

[0036] This invention provides a yeast-fermented bio-based antibacterial agent and its preparation method. The invention optimizes the gene corresponding to the amino acid sequence of Tachyplesin-1 and introduces EcoRI / XhoI restriction sites at the 5' / 3' end to ensure precise insertion of the target gene into the pYES2 / CT vector. Tachyplesin-1 is a natural antimicrobial peptide that kills harmful bacteria by disrupting their cell membranes and interfering with their metabolism. Through modified vector encapsulation and functional modification, Tachyplesin-1 overcomes the limitations of the intestinal environment and precisely targets harmful bacteria. Modified chitosan and sodium alginate-calcium chloride microcapsules form a stable gastric and intestinal slow-release barrier, ensuring the survival of brewer's yeast and antimicrobial peptides and their reach to the lower intestinal tract where harmful bacteria are concentrated. Porcine gastric mucoprotein modification allows the antimicrobial agent to target and adhere to the intestinal mucosal surface, increasing the local concentration of antimicrobial peptides and improving the killing efficiency, while reducing non-specific killing of beneficial intestinal bacteria. Modified polyethylene glycol reduces intestinal mucosal rejection, and glycine blocks active groups, reducing the irritation of the carrier to the intestinal epithelium. Vacuum drying into a solid dosage form facilitates oral administration. Ultimately, this forms a bio-based antimicrobial agent that is resistant to gastric acid, targets the intestinal tract, is highly effective in killing harmful intestinal bacteria, and is safe and low in irritation, maintaining the balance of intestinal flora.Chitosan itself possesses antibacterial properties (it can disrupt the cell membranes of harmful bacteria). After microwave-assisted dicyandiamide modification, its amino activity is enhanced, and it can form a gel-like protective layer in acidic environments (such as gastric pH=1-3), preventing the killing of *Saccharomyces cerevisiae* by gastric acid and the degradation of *Limulus amebocyte lysate* by pepsin. Upon entering the intestines (neutral / weakly alkaline), chitosan slowly degrades, releasing *Saccharomyces cerevisiae* and antimicrobial peptides, achieving stability in the stomach and release into the intestines. Polyethylene glycol, after HDI addition modification, exhibits enhanced hydrophilicity, reducing the intestinal mucosa's rejection of the carrier and helping *Saccharomyces cerevisiae* to disperse evenly in the intestines, expanding its contact range with harmful bacteria. Simultaneously, its flexible chain structure reduces the carrier's stimulation of intestinal epithelial cells, improving biocompatibility. Porcine gastric mucin contains adhesive groups, such as sugar chains, which can mimic the components of the intestinal mucus layer. After cross-linking with amino groups on the carrier surface via triethylamine catalysis, it allows bio-based antibacterial agents to adhere more easily to the intestinal mucosal surface. Harmful intestinal bacteria (such as Escherichia coli and Salmonella) mostly colonize the mucosal surface. This modification allows antimicrobial peptides to target and accumulate around harmful bacteria, increasing local concentration and significantly improving killing efficiency. At the same time, the presence of mucin can reduce the non-specific killing of beneficial intestinal bacteria by antimicrobial peptides, reducing the disruption to the intestinal flora balance. A homogeneous system is formed through high-pressure homogenization, and then solidified with calcium chloride to form microcapsules, which further encapsulate the Saccharomyces cerevisiae. Sodium alginate microcapsules do not dissolve in the stomach and are degraded by intestinal enzymes after entering the intestine, providing secondary protection for the Saccharomyces cerevisiae and antimicrobial peptides, ensuring that the active substances accurately reach the lower intestinal tract. After further cross-linking and solidification and glycine sealing treatment, the structural strength of the microcapsules is enhanced, preventing them from rupturing during intestinal peristalsis and reducing the stimulation of the intestinal tract by the carrier. At the same time, glycine can regulate the local pH of the intestine, providing a suitable environment for the activity of Limulus amebocyte lysate (optimal pH=6-7), further enhancing its killing effect on harmful bacteria. Attached Figure Description

[0037] Figure 1 The in vitro antibacterial rate of Saccharomyces cerevisiae INVSC1 using the pYES2 / CT-Tac vector of this invention is shown in the figure.

[0038] Figure 2 The graph shows the in vitro antibacterial rate results of the bio-based antibacterial agents prepared in Examples 7-9 and Comparative Examples 1-3 of this invention;

[0039] Figure 3 The graph shows the release rate of oriental horseshoe crab extract in the bio-based antibacterial agents prepared in Examples 7-9 and Comparative Examples 1-3 of this invention.

[0040] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials and test strains used in the following examples were purchased from commercial channels.

[0044] Example 1: Obtaining the target gene fragment:

[0045] According to NCBI, the amino acid sequence of Tachyplesin-1 is KWCFRVCYRGICYRRCR. After optimizing the amino acid gene of Tachyplesin-1, the complete gene sequence is synthesized as shown in SEQ ID NO:1. EcoR I and Xho I restriction sites were introduced at the 5' and 3' ends of the sequence. The synthesized complete gene sequence was cloned into the pUC57 vector to obtain pUC57-Tac.

[0046] Using pUC57-Tac as a template, the target gene fragment was obtained by double digestion with EcoRI and XhoI. The reaction system (50 μL) is as follows:

[0047] 10×CutSmart buffer: 5 μL;

[0048] pUC57-Tac plasmid (1 μg / μL): 5 μL;

[0049] EcoR I (NEB, 10U / μL): 2μL;

[0050] Xho I (NEB, 10U / μL): 2μL;

[0051] ddH2O: 36 μL;

[0052] The enzyme was incubated in a water bath at 37°C for 4 hours. During this period, a 5 μL sample was taken at 1.5 hours. The enzyme digestion effect was initially judged by 1% agarose gel electrophoresis. Two bands appeared: pUC57 vector approximately 2.7 kb and target gene approximately 220 bp, indicating that the enzyme digestion was normal. After incubation, the restriction enzyme was inactivated by heating at 65°C for 20 minutes.

[0053] Add the remaining 45 μL of enzyme digestion product to a 1% agarose gel and electrophoresis at 120 V for 30 min. Under UV light, excise the target gene band (220 bp), avoiding excessive gel excision. Add Binding Buffer (300 μL per 100 mg of gel) according to the kit instructions. Incubate at 55 °C for 10 min, inverting and mixing every 2 min to ensure complete gel melting. Transfer the melted solution to the adsorption column and centrifuge at 12000 rpm for 1 min, discarding the waste liquid. Add 700 μL Wash Buffer and centrifuge at 12000 rpm for 1 min, repeating the wash twice. Finally, add 30 μL Elution Buffer (preheated to 65 °C to improve elution efficiency), incubate at room temperature for 2 min, then centrifuge at 12000 rpm for 1 min. Collect the eluent to obtain the purified target gene fragment. Use 2 μL of the eluent to determine the concentration (≥30 ng / μL) and purity (A) using Nanodrop. 260 / A 280 =1.92)

[0054] Example 2: Enzymatic digestion and dephosphorylation of the vector backbone

[0055] Add the reagents sequentially to a 1.5 mL enzyme-free centrifuge tube, with a total volume of 50 μL. To avoid cross-contamination, the pYES2 / CT vector double enzyme digestion reaction system is as follows:

[0056] 10×CutSmart buffer (NEB, B7204S): 5μL;

[0057] pYES2 / CT plasmid (1 μg / μL): 5 μL;

[0058] EcoR I (NEB, 10U / μL): 2μL;

[0059] Xho I (NEB, 10U / μL): 2μL;

[0060] Enzyme-free purified water: 36 μL;

[0061] After gently mixing the centrifuge tubes, briefly centrifuge (3000 rpm, 10 s) and incubate in a 37℃ water bath for 4 h. After 1.5 h of incubation, take 5 μL of sample for 1% agarose gel electrophoresis (1×TAE buffer, 120V, 30 min) and observe the enzyme digestion effect using a gel imaging system.

[0062] After the enzyme digestion reaction, the centrifuge tubes were heated in a 65°C water bath for 20 min to inactivate EcoR I and XhoI. Then, 5 μL of 10× Loading Buffer (containing bromophenol blue indicator) was added, and the entire reaction solution was loaded onto a 1% agarose gel. Electrophoresis was performed at 120V for 40 min to ensure complete separation of the vector band from the enzyme digestion impurities. Under UV transilluminator (wavelength 302 nm), a 5.9 kb vector band was precisely excised using a sterile scalpel. After weighing, the gel recovery kit (Omega, catalog number D2500-02) was followed: 300 μL of Binding Buffer was added to every 100 mg of gel, and the mixture was incubated at 55°C for 10 min, inverting and mixing every 2 min to ensure complete gel melting. The melted solution was transferred to an adsorption column, centrifuged at 12000 rpm for 1 min, and the waste liquid in the collection tube was discarded. 700 μL of... Wash Buffer, centrifuge at 12000 rpm for 1 min, repeat washing twice; centrifuge empty column at 12000 rpm for 2 min to remove residual Wash Buffer; add 50 μL of preheated Elution Buffer (65℃) to the adsorption column, let stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, collect the eluent to obtain the purified enzyme-digested vector, and store at -20℃ for later use, with a storage time not exceeding 72 h.

[0063] Example 3: Ligation of target gene with vector

[0064] Add reagents sequentially to 1.5 mL enzyme-free centrifuge tubes, with a total volume of 20 μL. Perform the entire procedure on ice to avoid cross-contamination. The T4 DNA ligation system for the target gene and vector is as follows:

[0065] 10×T4 ligation buffer (NEB, containing ATP): 2 μL;

[0066] Dephosphorylated pYES2 / CT vector (50 ng / μL): 2 μL;

[0067] Target gene (30 ng / μL): 0.4 μL;

[0068] T4 DNA ligase (NEB, 400 U / μL): 1 μL;

[0069] Enzyme-free purified water: 14.6 μL;

[0070] After adding the sample, gently tap the side of the centrifuge tube to mix the reagents, avoiding vigorous vortexing that could cause DNA breakage. Then, briefly centrifuge (3000 rpm, 10 s) to allow the liquid on the tube wall to collect at the bottom. Place the centrifuge tube in a 16°C water bath (accuracy ±0.5°C) for 12-16 h. Low temperature conditions can reduce the probability of vector self-ligation and reduce reverse insertion of the target gene. After incubation, transfer the centrifuge tube to a 65°C water bath and heat for 10 min to inactivate T4 DNA ligase. After inactivation, the ligation product can be used immediately for transformation or aliquoted into 5 μL / tube and stored at -20°C.

[0071] Example 4: Transformation of Recombinant Vector

[0072] Transformation of Escherichia coli DH5α: Take 100 μL of DH5α competent cells, thaw them on ice, add 5 μL of ligation product, mix gently, and incubate on ice for 30 min; heat shock at 42℃ for 90 s, then immediately incubate on ice for 2 min; add 900 μL of antibiotic-free LB medium (preheated at 37℃), and culture at 37℃ with shaking at 200 rpm for 1 h; take 200 μL of bacterial culture and spread it on LB agar plates containing 100 μg / mL ampicillin, and incubate upside down at 37℃ for 12 h.

[0073] Initial screening for colony PCR: Ten single colonies (1-2 mm in diameter, regular morphology, and free of contaminants) were picked and inoculated into 5 mL of LB broth containing ampicillin, and cultured at 37°C with shaking at 200 rpm for 8 h. 1 μL of the bacterial culture was used as a template for PCR using the vector primers (upstream: 5'-GCTTCCGGCTCGTATGTTGTGTG-3', downstream: 5'-GCGCGTAATACGACTCACTATAGGG-3').

[0074] Reaction system: 25 μL

[0075] 2×TaqMix: 12.5μL;

[0076] Upstream primer (10 μM): 1 μL;

[0077] Downstream primer (10 μM): 1 μL;

[0078] Bacterial solution: 1 μL;

[0079] ddH2O: 9.5 μL;

[0080] Enzyme digestion of positive E. coli clones: Three PCR-positive clones were selected, and recombinant plasmids (pYES2 / CT-Tac) were extracted using an endotoxin-free plasmid extraction kit. 2 μg of the recombinant plasmid was digested with EcoRI / XhoI (20 μL). The insertion of the target gene into the pYES2 / CT vector was verified by gel electrophoresis. The recombinant plasmids that were verified to be digested were sequenced using GAL1 promoter primers (5'-CGGATAAAAGAGTTCGTTA-3'). The target gene sequence was completely consistent with the designed sequence, the insertion direction of the target gene was correct, and the restriction sites were intact.

[0081] Example 5: Transformation of Saccharomyces cerevisiae INVSC1 using a recombinant vector:

[0082] Preparation of competent yeast cells: Pick a single colony of INVSC1 and inoculate it into 5 mL of YPD liquid medium. Incubate at 30°C with shaking at 200 rpm for 12 h. Transfer 1 mL of the bacterial culture to 50 mL of YPD medium and incubate at 30°C with shaking at 200 rpm until OD reaches 100%. 600 =0.6872; collect bacterial cells by centrifugation at 4℃ and 5000rpm for 5min, wash twice with 25mL of sterile water, wash once with 10mL of 0.1M LiAc (pH=8.0), and finally resuspend in 0.5mL of 0.1M LiAc to obtain competent cells (store on ice and use within 30min).

[0083] Saccharomyces cerevisiae INVSC1 transformation reaction: Take 100 μL of competent yeast cells and add the following reagents (in order): 1 μg pYES2 / CT-Tac recombinant plasmid, 50 μg salmon sperm DNA (heated at 95℃ for 5 min and then placed on ice for 5 min to denature into single strands and improve transformation efficiency), 0.6 mL PEG-LiAc solution (40% PEG4000 + 0.1M LiAc, freshly prepared and used immediately), vortex to mix for 10 s; incubate at 30℃ for 30 min, inverting and mixing once every 10 min during this period; heat shock at 42℃ for 15 min, and immediately place on ice for 2 min; centrifuge at 4℃ and 5000 rpm for 5 min, discard the supernatant, and resuspend the cells in 100 μL of sterile water.

[0084] Yeast positive screening: Spread 50 μL of resuspension onto SD-Ura solid medium (0.67% yeast nitrogen source + 2% glucose + uracil-deficient amino acid mixture + 2% agar), and incubate upside down at 30℃ for 3-4 days. The single colonies that grow are potential positive transformants. Pick 5 single colonies and treat them with yeast lysis buffer (containing proteinase K, catalog number TAKARA9097) (incubate at 37℃ for 30 min, then heat at 95℃ for 10 min to inactivate proteinase K). Use 1 μL of lysis buffer as a template and perform PCR with target gene-specific primers (upstream: 5'-ATGAAGTGGTGTTTTAAAG-3', downstream: 5'-TTAAAAAATAACAAATCTT-3'). Positive clones should amplify the target band, proving that the recombinant vector has been introduced into yeast cells, and obtain the pYES2 / CT-Tac vector Saccharomyces cerevisiae INVSC1.

[0085] Example 6: Culture and Induction Expression of Recombinant Saccharomyces cerevisiae

[0086] Strain activation: Take the pYES2 / CT-Tac vector-containing Saccharomyces cerevisiae INVSC1 glycerol tube from the -80℃ freezer. In a clean bench, use a sterile inoculation loop to take a small amount of bacterial solution and streak it onto an SD-Ura plate. Invert the plate and incubate statically at 30℃ for 48 hours until single colonies with a diameter of 1-2 mm are formed (colonies are milky white, round, with neat edges, and free from contamination). Pick 3 single colonies with the same morphology and inoculate them into 15 mL centrifuge tubes containing 5 mL of SD-Ura liquid medium. Incubate at 30℃ and 200 rpm for 18 hours with shaking.

[0087] Seed culture expansion: Take the activated seed culture and transfer it to a 250mL Erlenmeyer flask containing 50mL SD-Ura liquid medium at a 1:100 inoculation ratio (volume ratio) (the volume of medium should not exceed 1 / 5 of the volume of the Erlenmeyer flask, and ensure adequate aeration); incubate at 30℃ and 220rpm with shaking for 6h, and take samples every 2h to measure OD. 600 When OD 600 Stop culturing when the bacterial concentration reaches 0.6-0.8 (at this point, the bacteria are in the early logarithmic growth phase, with vigorous metabolism, suitable for subsequent induction); take 1 mL of seed culture, centrifuge at 4℃ and 5000 rpm for 5 min, discard the supernatant, resuspend the bacteria in sterile physiological saline, and determine the bacterial concentration to be 5.4 × 10⁻⁶. 8 CFU / mL;

[0088] Pretreatment of bacterial cells before induction: Transfer the expanded seed culture to 50 mL centrifuge tubes, centrifuge at 5000 rpm for 10 min at 4 °C, and collect the bacterial cells; wash the bacterial cells twice with sterile SG-Ura medium (centrifuge at 5000 rpm for 5 min each time) to remove residual glucose (glucose inhibits GAL1 promoter activity, leading to a decrease in induction efficiency); resuspend the bacterial cells in SG-Ura medium and adjust the OD. 600 Up to 0.5;

[0089] Induction of expression: The pretreated bacterial suspension was transferred to a 500 mL Erlenmeyer flask containing 100 mL of SG-Ura medium at a ratio of 1:5; cultured at 30 °C and 220 rpm with shaking; 10 mL of bacterial suspension was collected every 24 h for subsequent expression level detection.

[0090] Example 7: Preparation of bio-based antibacterial agents:

[0091] S1. Take chitosan (Maclean C766421 chitosan, degree of deacetylation ≥95%). W 5g of dicyandiamide (50000-60000) was dissolved in 200mL of 0.1mol / L dilute hydrochloric acid aqueous solution. After stirring at 300rpm until homogeneous, the pH of the reaction system was kept at 1. The reaction system was placed in a microwave chemical reactor at 400W and 50℃ and reacted for 10min. 25mg of dicyandiamide was dissolved in 50mL of 50℃ deionized water. The pH was adjusted to 1 with 1mol / L hydrochloric acid aqueous solution. After mixing, the solution was added to the reaction system. The reaction system was placed in a microwave chemical reactor at 400W and 100℃ and reacted for 15min. After the reaction was completed, anhydrous ethanol was added for precipitation treatment. The precipitate was filtered, collected, washed with deionized water, dried, and ground to obtain modified chitosan.

[0092] S2, Take polyethylene glycol (M) W 5.0 g of polyethylene glycol (2000 g / mL) was placed in a flask, and 50 mL of anhydrous DMF was added. The reaction temperature was raised to 60 °C, and the mixture was stirred at 200 rpm until the polyethylene glycol was completely dissolved. Then, 0.9 g of HDI was dissolved in 5 mL of anhydrous DMF and added dropwise to the reaction system at a rate of 1 drop / s. After the addition was completed, the reaction temperature was raised to 80 °C to carry out the addition reaction. The reaction was carried out for 4 h. After the reaction was completed, the reaction system was cooled to room temperature, and the reaction solvent was removed by vacuum distillation. Anhydrous diethyl ether was added for precipitation treatment. The solid was collected by filtration, washed, and dried to obtain modified polyethylene glycol.

[0093] S3. Dissolve 2g of the modified chitosan prepared in step S1 in 100mL of 0.1mol / L hydrochloric acid aqueous solution to obtain a modified chitosan solution. Dissolve 1g of the modified polyethylene glycol prepared in step S2 in 20mL of anhydrous ethanol to obtain a modified polyethylene glycol solution. Mix the modified chitosan solution and the modified polyethylene glycol solution evenly to obtain a coated prepolymer solution.

[0094] S4. Take 0.2g of porcine gastric mucoprotein and add it to 10mL of 0.1mol / L Tris-HCl buffer (pH=7.5). Stir at 100rpm for 1h to completely dissolve the porcine gastric mucoprotein. Then add 0.02g of sodium cyanoborohydride and stir at 40rpm until homogeneous to obtain a porcine gastric mucoprotein solution.

[0095] S5. Take 10 mL of yeast culture and mix with 30 mL of 2 wt% (w / v) sodium alginate aqueous solution. Homogenize under high pressure at 20 MPa for 5 min to form a homogeneous system. Inject the mixture into an extrusion apparatus and drip it into 3 wt% calcium chloride aqueous solution through a 22G needle at a constant pressure of 0.1 MPa. Let it stand at 30℃ for 30 min to solidify. Filter and disperse in 50 mL of 0.1 mol / L PBS buffer (pH=6.5) to obtain the yeast protocyst solution.

[0096] S6. Place the yeast precapsulation solution prepared in step S5 into a flask, keep it in a 30°C water bath under nitrogen protection, and slowly add the coating prepolymer solution dropwise to the reaction system at a rate of 2 mL / min. After the addition is complete, maintain the reaction temperature at 30°C for cross-linking reaction for 3 hours. After the reaction is complete, add 50 mL of 0.5 wt% calcium chloride aqueous solution, let it stand at 30°C for 30 minutes to promote membrane solidification, collect the solid by filtration, wash it three times with deionized water to remove unreacted polymer, and obtain the yeast coating agent.

[0097] S7. Disperse the yeast coating agent prepared in step S6 in 50 mL of 0.1 mol / L Tris-HCl buffer (pH=8.0), add 0.05 g of triethylamine, stir at 150 rpm at room temperature, slowly add 10 mL of porcine gastric mucoprotein solution prepared in step S4, raise the reaction temperature to 30 °C, maintain the stirring speed, and stir for 2.5 h. After the reaction is completed, add 10 mL of 1 wt% glycine aqueous solution (pH=8.0), continue stirring for 60 min, and vacuum dry to obtain the bio-based antibacterial agent.

[0098] Example 8: Preparation of bio-based antibacterial agents:

[0099] S1. Take chitosan (Maclean C766421 chitosan, degree of deacetylation ≥95%). W5g of dicyandiamide (50000-60000) was dissolved in 200mL of 0.1mol / L dilute hydrochloric acid aqueous solution. After stirring at 300rpm until homogeneous, the pH of the reaction system was kept at 1. The reaction system was placed in a microwave chemical reactor at 500W and 45℃ and reacted for 10min. 20mg of dicyandiamide was dissolved in 50mL of 40℃ deionized water. The pH was adjusted to 1 with 1mol / L hydrochloric acid aqueous solution. After mixing, the solution was added to the reaction system. The reaction system was placed in a microwave chemical reactor at 500W and 105℃ and reacted for 10min. After the reaction was completed, anhydrous ethanol was added for precipitation treatment. The precipitate was filtered, collected, washed with deionized water, dried, and ground to obtain modified chitosan.

[0100] S2, Take polyethylene glycol (M) W 5.0 g of polyethylene glycol (2000 g / mL) was placed in a flask, and 50 mL of anhydrous DMF was added. The reaction temperature was raised to 60 °C, and the mixture was stirred at 200 rpm until the polyethylene glycol was completely dissolved. Then, 1.1 g of HDI was dissolved in 5 mL of anhydrous DMF and added dropwise to the reaction system at a rate of 1 drop / s. After the addition was completed, the reaction temperature was raised to 85 °C to carry out the addition reaction. The reaction was carried out for 3 h. After the reaction was completed, the reaction system was cooled to room temperature, and the reaction solvent was removed by vacuum distillation. Anhydrous diethyl ether was added for precipitation treatment. The solid was collected by filtration, washed, and dried to obtain modified polyethylene glycol.

[0101] S3. Dissolve 3g of the modified chitosan prepared in step S1 in 100mL of 0.1mol / L hydrochloric acid aqueous solution to obtain a modified chitosan solution. Dissolve 1g of the modified polyethylene glycol prepared in step S2 in 20mL of anhydrous ethanol to obtain a modified polyethylene glycol solution. Mix the modified chitosan solution and the modified polyethylene glycol solution evenly to obtain a coated prepolymer solution.

[0102] S4. Take 0.3g of porcine gastric mucoprotein and add it to 10mL of 0.1mol / L Tris-HCl buffer (pH=7.5). Stir at 100rpm for 1h to completely dissolve the porcine gastric mucoprotein. Then add 0.02g of sodium cyanoborohydride and stir at 40rpm until homogeneous to obtain a porcine gastric mucoprotein solution.

[0103] S5. Take 10 mL of yeast culture and mix it with 20 mL of 4 wt% (w / v) sodium alginate aqueous solution. Homogenize under high pressure at 20 MPa for 5 min to form a homogeneous system. Inject the mixture into an extrusion apparatus and drip it into 2 wt% calcium chloride aqueous solution through a 22G needle at a constant pressure of 0.1 MPa. Let it stand at 30℃ for 30 min to solidify. Filter and disperse in 50 mL of 0.1 mol / L PBS buffer (pH=6.5) to obtain the yeast protocyst solution.

[0104] S6. Place the yeast precapsulation solution prepared in step S5 into a flask, keep it in a 40°C water bath under nitrogen protection, and slowly add the coating prepolymer solution dropwise to the reaction system at a rate of 2 mL / min. After the addition is complete, maintain the reaction temperature at 30°C for cross-linking reaction for 2 hours. After the reaction is complete, add 50 mL of 0.5 wt% calcium chloride aqueous solution, let it stand at 30°C for 30 minutes to promote membrane solidification, collect the solid by filtration, wash it three times with deionized water to remove unreacted polymer, and obtain the yeast coating agent.

[0105] S7. Disperse the yeast coating agent prepared in step S6 in 50 mL of 0.1 mol / L Tris-HCl buffer (pH=8.0), add 0.05 g of triethylamine, stir at 150 rpm at room temperature, slowly add 10 mL of porcine gastric mucoprotein solution prepared in step S4, raise the reaction temperature to 30 °C, maintain the stirring speed, and stir for 2.5 h. After the reaction is completed, add 10 mL of 1 wt% glycine aqueous solution (pH=8.0), continue stirring for 60 min, and vacuum dry to obtain the bio-based antibacterial agent.

[0106] Example 9: Preparation of bio-based antibacterial agents:

[0107] S1. Take chitosan (Maclean C766421 chitosan, degree of deacetylation ≥95%), M W 5g of dicyandiamide (50000-60000) was dissolved in 200mL of 0.1mol / L dilute hydrochloric acid aqueous solution. After stirring at 300rpm until homogeneous, the pH of the reaction system was kept at 1. The reaction system was placed in a microwave chemical reactor at 600W and 40℃ and reacted for 15min. 30mg of dicyandiamide was dissolved in 50mL of 45℃ deionized water, and the pH was adjusted to 1 with 1mol / L hydrochloric acid aqueous solution. After mixing, it was added to the reaction system. The reaction system was placed in a microwave chemical reactor at 600W and 95℃ and reacted for 20min. After the reaction was completed, anhydrous ethanol was added for precipitation treatment. The precipitate was filtered, collected, washed with deionized water, dried, and ground to obtain modified chitosan.

[0108] S2, Take polyethylene glycol (M) W5.0 g of polyethylene glycol (2000 g / mL) was placed in a flask, and 50 mL of anhydrous DMF was added. The reaction temperature was raised to 60 °C, and the mixture was stirred at 200 rpm until the polyethylene glycol was completely dissolved. Then, 1.3 g of HDI was dissolved in 5 mL of anhydrous DMF and added dropwise to the reaction system at a rate of 1 drop / s. After the addition was completed, the reaction temperature was raised to 90 °C to carry out the addition reaction. The reaction was carried out for 2 hours. After the reaction was completed, the reaction system was cooled to room temperature, and the reaction solvent was removed by vacuum distillation. Anhydrous diethyl ether was added for precipitation treatment. The solid was collected by filtration, washed, and dried to obtain modified polyethylene glycol.

[0109] S3. Dissolve 4g of the modified chitosan prepared in step S1 in 100mL of 0.1mol / L hydrochloric acid aqueous solution to obtain a modified chitosan solution. Dissolve 1g of the modified polyethylene glycol prepared in step S2 in 20mL of anhydrous ethanol to obtain a modified polyethylene glycol solution. Mix the modified chitosan solution and the modified polyethylene glycol solution evenly to obtain a coated prepolymer solution.

[0110] S4. Take 0.25g of porcine gastric mucoprotein and add it to 10mL of 0.1mol / L Tris-HCl buffer (pH=7.5). Stir at 100rpm for 1h to completely dissolve the porcine gastric mucoprotein. Then add 0.02g of sodium cyanoborohydride and stir at 40rpm until homogeneous to obtain a porcine gastric mucoprotein solution.

[0111] S5. Take 10 mL of yeast culture and mix it with 25 mL of 3 wt% (w / v) sodium alginate aqueous solution. Homogenize under high pressure at 20 MPa for 5 min to form a homogeneous system. Inject the mixture into an extrusion apparatus and drip it into 3 wt% calcium chloride aqueous solution through a 22G needle at a constant pressure of 0.1 MPa. Let it stand at 30℃ for 30 min to solidify. Filter and disperse in 50 mL of 0.1 mol / L PBS buffer (pH=6.5) to obtain the yeast protocyst solution.

[0112] S6. Place the yeast precapsulation solution prepared in step S5 into a flask, keep it in a water bath at 35°C, and protect it with nitrogen gas. Add the coating prepolymer solution slowly to the reaction system at a rate of 2 mL / min. After the addition is complete, maintain the reaction temperature at 30°C for cross-linking reaction for 2.5 h. After the reaction is complete, add 50 mL of 0.5 wt% calcium chloride aqueous solution, let it stand at 30°C for 30 min to promote membrane solidification, collect the solid by filtration, wash it three times with deionized water to remove unreacted polymer, and obtain the yeast coating agent.

[0113] S7. Disperse the yeast coating agent prepared in step S6 in 50 mL of 0.1 mol / L Tris-HCl buffer (pH=8.0), add 0.05 g of triethylamine, stir at 150 rpm at room temperature, slowly add 10 mL of porcine gastric mucoprotein solution prepared in step S4, raise the reaction temperature to 30 °C, maintain the stirring speed, and stir for 2.5 h. After the reaction is completed, add 10 mL of 1 wt% glycine aqueous solution (pH=8.0), continue stirring for 60 min, and vacuum dry to obtain the bio-based antibacterial agent.

[0114] Comparative Example 1

[0115] This comparative example provides an antibacterial agent, which differs from Example 7 only in that the modified chitosan is replaced with the same amount of commercially available chitosan in the antibacterial agent, while the rest is the same as in Example 7.

[0116] Comparative Example 2

[0117] This comparative example provides an antibacterial agent, which differs from Example 7 only in that the modified polyethylene glycol is replaced with the same weight parts of commercially available polyethylene glycol in the antibacterial agent, and the rest is the same as in Example 7.

[0118] Comparative Example 3

[0119] This comparative example provides an antibacterial agent, which differs from Example 7 only in that the components of the antibacterial agent do not include porcine gastric mucoprotein, and the rest are the same as in Example 7.

[0120] Experimental Example 1

[0121] This experiment tested the in vitro antibacterial activity of pYES2 / CT-Tac vector-converted Saccharomyces cerevisiae INVSc1. Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633) and Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) were selected as indicator bacteria, all from a standard strain library and activated in LB medium before use. Sample groups were set up as follows: Experimental group: pYES2 / CT-Tac vector Saccharomyces cerevisiae INVSc1 suspension induced and cultured for 24 h as in Example 6; Blank control group: Unconverted Saccharomyces cerevisiae INVSc1 suspension, induced and cultured under the same conditions for 24 h; Negative control group: SG-Ura medium; Positive control group: 100 μg / mL ampicillin solution (for Gram-positive bacteria) and 50 μg / mL gentamicin solution (for Gram-negative bacteria).

[0122] Culture media preparation: LB solid medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L, pH=7.0; LB liquid medium: same composition as LB solid, but without agar; sterile physiological saline: 0.9% NaCl solution, autoclaved at 121℃ for 20min; sterile PBS buffer: pH=7.4, containing NaCl 8g / L, KCl 0.2g / L, Na2HPO4 1.44g / L, KH2PO4 0.24g / L, autoclaved at 121℃ for 20min.

[0123] Take 100 μL of bacterial suspension from a standard strain glycerol tube stored at -80℃ and inoculate it into 5 mL of LB liquid medium. Activate the suspension by incubating at 37℃ and 200 rpm for 12 h with shaking. Take 1 mL of the activated bacterial suspension and serially dilute it with sterile physiological saline, referring to a 0.5 McFarland turbidity tube (corresponding to a bacterial concentration of approximately 1 × 10⁻⁶). 8 CFU / mL), OD calibrated by UV spectrophotometer 600 Value (0.5 McFarland units corresponding to OD) 600 (≈0.08-0.1), and finally the indicator bacteria concentration was adjusted to 1×10 6 CFU / mL, for later use.

[0124] Take 20 mL of LB solid medium, melted and cooled to 45-50℃, and pour it into a sterile petri dish. Place it horizontally to allow it to solidify. Take 100 μL of the adjusted indicator bacterial solution and spread it evenly on the surface of the bottom layer of the medium. Allow the bacterial solution to be absorbed (approximately 5-10 minutes). Use sterile forceps to pick up sterilized Oxford cups and place them vertically on the surface of the culture medium (place 4 cups per dish, with a spacing of ≥2 cm to avoid overlapping inhibition zones). Gently press to ensure that the Oxford cups are in close contact with the medium without gaps. Use a sterile pipette to add 200 μL of the test sample solution, blank control solution, negative control solution, and positive control solution to each Oxford cup (perform 3 parallel experiments for each type of sample to avoid operational errors). Place the petri dishes in a 37℃ incubator and invert them for 16-18 hours (adjust the incubation time according to the growth rate of the indicator bacteria, ideally until a clear inhibition zone appears in the positive control).

[0125] After incubation, remove the petri dishes and measure the diameter of the inhibition zone around each Oxford cup using calipers (accuracy 0.02 mm). Measure the distance from the edge of the inhibition zone to the center of the Oxford cup, and take the average of two measurements in the vertical direction (unit: mm). Calculate the inhibition rate using the following formula:

[0126] ;

[0127] Figure 1The figure shows the in vitro antibacterial rate of *Saccharomyces cerevisiae* INVSC1 using the pYES2 / CT-Tac vector of this invention. As shown in the figure, *Limulus amebocyte lysate* is a cationic antimicrobial peptide. Its antibacterial mechanism is as follows: it binds to the negative charge (such as phosphatidylglycerol and lipopolysaccharide) of the bacterial cell membrane (Gram-positive bacteria have a phospholipid bilayer, while Gram-negative bacteria have an outer membrane + inner membrane), thereby disrupting the integrity of the membrane structure, leading to leakage of intracellular substances, and ultimately inhibiting bacterial growth. Gram-positive bacteria do not have an outer membrane barrier, so *Limulus amebocyte lysate* can act directly on the cell membrane, thus resulting in a higher antibacterial rate. The outer membrane of Gram-negative bacteria (containing lipopolysaccharide) hinders the penetration of *Limulus amebocyte lysate*. It needs to bind to the outer membrane first through cation-anion interaction before entering the inner membrane to exert its effect, thus resulting in a slightly lower antibacterial rate. Furthermore, the antibacterial rate of *Pseudomonas aeruginosa*, which has a more complex outer membrane structure, is lower than that of *Escherichia coli*.

[0128] Experiment Example 2

[0129] This experiment tested the in vitro antibacterial activity of the bio-based antibacterial agents prepared in Examples 7-9 and Comparative Examples 1-3. The bio-based antibacterial agents prepared in Examples 7-9 and Comparative Examples 1-3 were used to prepare a 10 mg / mL antibacterial agent suspension with sterile PBS buffer (pH=6.5) (ultrasonic treatment for 10 min at 300 W to ensure uniform dispersion and avoid antibacterial agent agglomeration affecting the release of activity); at the same time, a blank control group (sterile PBS buffer) and a positive control group (a mixture of 100 μg / mL ampicillin and 50 μg / mL gentamicin, covering Gram-positive / negative bacteria) were set up. Take 20 mL of LB solid medium, melted and cooled to 45-50℃, and pour it into a sterile petri dish (90 mm in diameter). Place it horizontally to solidify (bottom culture medium). Take 100 μL of calibrated indicator bacterial solution and spread it evenly on the surface of the bottom culture medium. After the bacterial solution is completely absorbed, use sterile forceps to place sterile Oxford cups vertically on the smear plate (4 cups per dish, spaced ≥2 cm apart to avoid overlapping inhibition zones). Gently press to ensure close contact with the culture medium. Use a sterile pipette to add 200 μL of antibacterial activity solution (example / comparative example), blank control solution (PBS), and positive control solution to the Oxford cups. Set up 3 parallel experiments for each group. Invert the plates and place them in a 37℃ incubator for 16-18 h (16 h for Gram-positive bacteria and 18 h for Gram-negative bacteria to ensure sufficient growth of indicator bacteria). After incubation, measure the diameter of the inhibition zone with calipers (accurate to 0.1 mm, take the average of 2 vertical measurements), record the data, and calculate the inhibition rate according to the following formula:

[0130] ;

[0131] Figure 2The figures show the in vitro antibacterial rate results of the bio-based antibacterial agents prepared in Examples 7-9 and Comparative Examples 1-3 of this invention. As shown in the figures, the bio-based antibacterial agents described in Examples 7-9 exhibited higher antibacterial activity against both Gram-negative and Gram-positive bacteria than the comparative examples. Comparative Example 1 (unmodified chitosan): had fewer amino active sites on its molecular chain and poor solubility (easily aggregated in PBS), resulting in a decrease of over 30% in the loading of Limulus amebocyte lysate (LAL). Furthermore, it could not effectively bind to bacterial cell membranes, leading to a 20-25% decrease in antibacterial rate compared to Example 7. Example 2 (unmodified polyethylene glycol): Polyethylene glycol did not react with HDI and had no cross-linking activity. The coating membrane was in a physical encapsulation state and was easily soluble in the culture medium. The loss rate of oriental horseshoe crab extract exceeded 50% within 4 hours. The insufficient active ingredients led to a 25-30% reduction in the antibacterial rate compared to Example 7. Comparative Example 3 (lacking porcine gastric mucin): The adhesiveness of porcine gastric mucin can enhance the targeted binding of antibacterial agents to bacterial cell membranes (especially the outer membrane of Gram-negative bacteria). After its absence, oriental horseshoe crab extract had to rely on its own cationic action to penetrate the outer membrane, resulting in a decrease in the antibacterial rate.

[0132] Experimental Example 3

[0133] This experiment tested the release of Limulus amebocyte lysate (LAL) from the bio-based antibacterial agents prepared in Examples 7-9 and Comparative Examples 1-3 under simulated digestive tract conditions. Simulated gastric juice (pH=2.0): containing 0.3% pepsin + 0.9% NaCl, sterilized at 121℃ for 20 min; simulated small intestinal juice (pH=7.5): containing 0.1% trypsin + 0.3% bile salts + 0.9% NaCl, sterilized at 121℃ for 20 min; simulated large intestinal juice (pH=6.8): containing 0.5% pectinase + 0.2% mucin + 0.9% NaCl, sterilized at 121℃ for 20 min. Take three portions each of the antibacterial agent suspension (10 mg / mL) from the examples / comparative examples, and add 10 mL each to simulated gastric fluid, small intestinal fluid, and large intestinal fluid. Incubate at 37°C with shaking (150 rpm). Take 5 mL samples at 0.5 h (gastric residence time), 4 h (small intestinal residence time), and 12 h (large intestinal residence time). Filter the samples through a 0.22 μm filter membrane. Determine the concentration of horseshoe crab extract in the filtrate using high-performance liquid chromatography (HPLC) (HPLC conditions: C18 column, mobile phase: methanol-0.1% formic acid water = 30:70, flow rate: 1 mL / min, detection wavelength: 220 nm). Calculate the release rate of horseshoe crab extract using the following formula:

[0134] Release rate (%) = (Detected concentration of Limulus amebocyte lysate / Total concentration of Limulus amebocyte lysate) × 100%;

[0135] Figure 3The graph shows the release rate of Limulus amebocyte lysate (LAL) from the bio-based antibacterial agents prepared in Examples 7-9 and Comparative Examples 1-3 of this invention. In a simulated gastric juice environment, the modified chitosan undergoes amino protonation in an acidic environment, and the molecular chains contract to form a dense coating membrane. The better the membrane density, the less LAL leakage occurs, and the release rate gradually decreases. In the small intestine stage, the protonation of modified chitosan weakens in a neutral environment, and the molecular chains slightly stretch. However, the cross-linking network of modified polyethylene glycol can maintain membrane stability, and only a small amount of LAL permeates and is released. In the large intestine stage, the protonation of modified chitosan further weakens in a weakly acidic environment, the pores of the coating membrane increase, and the chelation effect of sodium alginate with calcium ions weakens, resulting in a large release of LAL.

[0136] In Comparative Example 1, the molecular chain had few active sites, making it impossible to form a stable pH-responsive coating membrane. Under acidic conditions, it easily aggregated, creating gaps. Under neutral conditions, the molecular chains were loose, and the release rate in the large intestine stage was much lower than that in the Example due to early leakage. Comparative Example 2 had no cross-linked structure, and the coating membrane was a physical encapsulation layer. It was easily dissolved under acidic conditions, and under neutral conditions, its strong fluidity led to membrane rupture, resulting in insufficient remaining Limulus amebocyte lysate in the large intestine stage. In Comparative Example 3, porcine gastric mucin could enhance the binding force between the coating membrane and the carrier through adhesion. After the coating membrane was lost, it was easily damaged under oscillation (simulating digestive tract peristalsis). The release rate in the stomach stage was higher than that in the Example, the release rate in the small intestine stage was higher than that in the Example, and the release rate in the large intestine stage was lower than that in the Example.

[0137] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention.

[0138] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A bio-based antibacterial agent based on yeast fermentation, characterized in that: The bio-based antimicrobial agent includes yeast culture; the yeast culture is a seed culture formed by activating Saccharomyces cerevisiae INVSC1 on the pYES2 / CT-Tac carrier.

2. The bio-based antibacterial agent based on yeast fermentation according to claim 1, characterized in that: The brewer's yeast INVSC1 is a recombinant yeast strain with optimized Tachyplesin-1 genome.

3. A bio-based antibacterial agent based on yeast fermentation according to claim 2, characterized in that: The optimized whole genome sequence of Tachyplesin-1 is shown in SEQ ID NO:1, with an EcoR I restriction site at the 5' end and an Xho I restriction site at the 3' end; the optimized amino acid sequence of Tachyplesin-1 is shown in SEQ ID NO:

2.

4. The bio-based antibacterial agent based on yeast fermentation according to claim 3, characterized in that: The bio-based antibacterial agent also includes the following components in parts by weight: 2-4 parts modified chitosan, 1 part modified polyethylene glycol, 0.2-0.3 parts porcine gastric mucoprotein, and 0.6-0.8 parts sodium alginate.

5. A method for preparing a yeast-based antibacterial agent according to any one of claims 1-4, characterized in that: Specifically, the steps include the following: S1. Dissolve chitosan in dilute hydrochloric acid aqueous solution and microwave it once. Adjust the reaction pH to 1. Add dicyandiamide solution to the reaction system, mix well, and microwave it a second time. After the reaction is complete, add anhydrous ethanol for precipitation treatment. Collect the precipitate, wash, dry, and grind it to obtain modified chitosan. S2. Dissolve polyethylene glycol in DMF, add HDI reaction solution dropwise. After the addition is complete, raise the reaction temperature to carry out the addition reaction. After the reaction is complete, cool, remove the reaction solvent by vacuum distillation, add anhydrous diethyl ether for precipitation treatment, filter, collect the solid, wash and dry to obtain modified polyethylene glycol. S3. Dissolve the modified chitosan prepared in step S1 in hydrochloric acid aqueous solution to obtain a modified chitosan solution. Dissolve the modified polyethylene glycol prepared in step S2 in anhydrous ethanol to obtain a modified polyethylene glycol solution. Mix the modified chitosan solution and the modified polyethylene glycol solution evenly to obtain a coated prepolymer solution. S4. Dissolve porcine gastric mucoprotein in a buffer solution, add sodium cyanoborohydride, stir well, and obtain a porcine gastric mucoprotein solution. S5. Mix yeast culture with sodium alginate aqueous solution, homogenize under high pressure to form a homogeneous system, inject the mixture into an extrusion preparation instrument, add calcium chloride aqueous solution dropwise, solidify, filter, and disperse in PBS buffer to obtain yeast protocyst solution. S6. Take the yeast pre-capsule solution prepared in step S5 and place it in a flask. Under a nitrogen atmosphere, add the coating prepolymer solution prepared in step S3. After the addition is complete, maintain the reaction temperature to carry out the cross-linking reaction. After the reaction is complete, add calcium chloride aqueous solution, let stand, solidify, filter, collect the solid, wash and obtain the yeast coating agent. S7. Disperse the yeast coating agent prepared in step S6 in Tris-HCl buffer, add triethylamine, stir at room temperature, slowly add the porcine gastric mucin solution prepared in step S4, raise the reaction temperature and stir the reaction, after the reaction is completed, add glycine aqueous solution, continue stirring for 30-60 min, filter to collect the solid, wash, vacuum dry to obtain the bio-based antibacterial agent.

6. The method for preparing a yeast-based antibacterial agent according to claim 5, characterized in that: In step S1, the mass ratio of chitosan to dicyandiamide is 1:0.004-0.006; the dicyandiamide solution is prepared by dissolving dicyandiamide in deionized water at 40-50℃, adjusting the pH to 1, and mixing thoroughly to obtain the dicyandiamide solution; the microwave power of the first microwave treatment is 400-600W, the treatment temperature is 40-50℃, and the treatment time is 10-15min; the microwave power of the second microwave treatment is 400-600W, the treatment temperature is 95-105℃, and the treatment time is 10-20min.

7. The method for preparing a yeast-based antibacterial agent according to claim 6, characterized in that: In step S2, the mass ratio of polyethylene glycol to HDI is 1:0.18-0.26; the reaction temperature of the addition reaction is 80-90℃; and the reaction time of the addition reaction is 2-4h.

8. The method for preparing a yeast-based antibacterial agent according to claim 7, characterized in that: In step S4, the mass ratio of porcine gastric mucoprotein to sodium cyanoborohydride is 10-15:

1.

9. The method for preparing a yeast-based antibacterial agent according to claim 8, characterized in that: In step S5, the concentration of the yeast culture is 4 × 10⁻⁶. 8 -6×10 8 CFU / mL; the mass fraction of the sodium alginate aqueous solution is 2%-4%; the volume ratio between the yeast culture and the sodium alginate aqueous solution is 1:2-3; the mass fraction of the calcium chloride aqueous solution is 2%-4%.

10. A method for preparing a yeast-based antibacterial agent according to claim 9, characterized in that: In step S6, the crosslinking reaction temperature is 30-40℃ and the crosslinking reaction time is 2-3h.

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