A high-antibacterial edible packaging film based on lactobacillus plantarum nanoemulsion and a preparation method thereof

Abstract

This invention belongs to the field of food packaging film technology, and relates to a highly antibacterial edible packaging film based on *Lactobacillus plantarum* nanoemulsion and its preparation method. This invention creatively combines three modifiers—L-ascorbic acid, Tween 20, and trehalose—to solve the problem of activity loss and significantly improve the survival rate of probiotics. It also uses glyceryl monostearate as a co-surfactant to optimize the pH value and droplet acceleration of the aqueous phase. A combination of high-pressure homogenization and intermittent ultrasonication solves the problems of poor stability and uneven particle size in the original emulsion, achieving a particle size variation coefficient (CV) ≤15% and particle size control between 50 and 100 nm. Furthermore, a gradient drying process solves the problems of easy cracking and numerous surface pores during film drying, improving the integrity and stability of the film. The packaging film maintains >75% antibacterial efficiency within 28 days; the tensile strength of the packaging film is ≥12 MPa, the elongation at break is ≥35%, and the film remains intact without cracks.

Description

Technical Field

[0001] This invention belongs to the field of food packaging film technology, specifically relating to a highly antibacterial edible packaging film based on Lactobacillus plantarum nanoemulsion and its preparation method. Background Technology

[0002] With increasing consumer demand for food safety, nutrition, and green packaging, edible packaging films, as a new type of biodegradable and environmentally friendly material, have received widespread attention in recent years. However, microbial contamination is one of the main factors leading to food spoilage during storage. Edible packaging films can not only act as a physical barrier for food but also, by introducing functional ingredients, impart antibacterial and antioxidant activities, thereby delaying food spoilage and extending product shelf life. Therefore, developing edible packaging materials with good antibacterial properties is of great significance for ensuring food safety.

[0003] Currently, researchers have attempted to add various natural antibacterial active substances, such as plant essential oils, polyphenols, and inorganic nanomaterials, to edible matrices to improve their antibacterial properties. However, these substances still face challenges in application, including poor stability, volatility, and safety controversies. Probiotics, as highly safe microorganisms with physiological functions, are widely used in the food industry. Among them, *Lactobacillus plantarum* (… Lactobacillus plantarum Because it can produce antibacterial substances such as organic acids and bacteriocins, and inhibit the growth of spoilage bacteria through nutrient competition, probiotics have promising applications in the food preservation field. Existing research shows that introducing probiotics into edible films can, to some extent, endow the material with antibacterial properties. However, current probiotic edible films generally suffer from the following technical defects: First, probiotics are easily affected by environmental factors (temperature, humidity, matrix constraints) during film preparation (such as drying) and storage, leading to reduced activity or even inactivation, resulting in a significant reduction in the film's antibacterial performance; second, probiotics have poor dispersion in the film matrix, making it difficult to fully contact harmful microorganisms on the food surface, thus limiting antibacterial efficiency; third, it is difficult to simultaneously achieve both mechanical and antibacterial properties of the film, and the addition of probiotics can easily lead to problems such as film embrittlement and breakage, affecting practical applications.

[0004] To address the aforementioned issues, while existing technologies have attempted to improve the dispersibility of probiotics using nanoemulsion technology, problems remain, including insufficient nanoemulsion stability, limited probiotic activity retention, and poor antibacterial durability. Furthermore, a systematic, industrially applicable preparation process has not yet been established, and the poor mechanical properties make it difficult to meet the practical needs of food packaging. For example, some literature discloses edible antibacterial films containing Lactobacillus probiotics, which directly introduce probiotics into an edible matrix, endowing the film with certain antibacterial properties and probiotic functions. However, in this method, the probiotics exist in a free state within the matrix, making them susceptible to external environmental factors such as temperature, ultraviolet radiation, and pH changes, resulting in a low survival rate of the probiotics and consequently affecting the stability and durability of their antibacterial effect. Therefore, developing an edible antibacterial film based on Lactobacillus plantarum nanoemulsion is of great significance for improving food preservation and expanding the application of probiotics in the packaging field. Summary of the Invention

[0005] The purpose of this invention is to address the problem of insufficient antibacterial properties of probiotic films by providing a method for preparing an edible film with enhanced antibacterial activity. This method involves adding *Lactobacillus plantarum* to a polyvinyl alcohol matrix after nanoemulsification to prepare an edible probiotic nanoemulsion film with high antibacterial activity.

[0006] The technical solution of the present invention includes the following steps: (1) Pour the Lactobacillus plantarum powder into sterilized MRS liquid medium at an inoculation rate of 1~2 g / L, add L-ascorbic acid and Tween-20, shake well and culture to obtain Lactobacillus plantarum culture medium; wherein the mass fraction of L-ascorbic acid is 0.1~0.3% and the mass fraction of Tween-20 is 0.05~0.1%; Centrifuge the *Lactobacillus plantarum* culture medium, discard the supernatant (to avoid disturbing the bacterial precipitate), then wash with sterile physiological saline to remove residual culture medium components; finally, collect the washed precipitate, suspend the precipitate in sterile distilled water, add trehalose (a preservative), and stir magnetically until completely dissolved to obtain a modified probiotic suspension; the mass fraction of trehalose is 0.2-0.4%; The formula for calculating probiotic concentration is as follows: CFU / mL = (Average colony count × Dilution factor) / Inoculation volume (mL) (2) Preparation of probiotic nanoemulsion: At room temperature, hydrophobic corn germ oil and surfactant Tween-80 were mixed, and glyceryl monostearate (a co-surfactant) was added. The mixture was magnetically stirred for 30-40 min at a stirring speed of 300-400 r / min to prepare a homogeneous and stable oil phase, wherein the mass fraction of Tween-80 was 8-12% and the mass fraction of glyceryl monostearate was 0.5-1.0%. Then, the aqueous phase is slowly added dropwise to the oil phase at a rate of 2-3 mL / min, while the mixture is continuously stirred at a speed of 500-600 r / min until the addition is complete. The aqueous phase consists of 45-55% (v / v) of the modified probiotic suspension obtained in step (1), 25-35% (v / v) of glycerol, and the remainder of sterile distilled water. The pH of the aqueous phase is adjusted to 6.5-7.0 (using 0.1 mol / L NaOH or HCl solution to avoid the pH being too high or too low and affecting the activity of the probiotics). After being mixed evenly, the mixture was placed in a high-pressure homogenizer and homogenized under a pressure of 20-25 MPa to further refine the emulsion particles. After homogenization, the mixture was ultrasonically treated with an ultrasonic cell disruptor with an ultrasonic power of 200-250W and an ultrasonic time of 10-15 min to obtain a stable probiotic nanoemulsion with a particle size distribution of 50-100 nm and uniform dispersion. Among them, the particle size of the nanoemulsion was determined by using a laser particle size analyzer. Each sample was measured three times, and the average value was taken to ensure that the particle size was within the range of 50~100 nm and the coefficient of variation (CV) was ≤15%. The calculation formula is as follows: CV = (Standard deviation of particle size / Average particle size) × 100% (3) Add polyvinyl alcohol (PVA, degree of polymerization 1700~1800) to sterilized distilled water, place it in a constant temperature water bath at 70~80℃, stir for 30~40 min until completely dissolved, and obtain a transparent polyvinyl alcohol solution with a concentration of 8~10 g / 100 mL; after cooling to room temperature, add chitosan (synergistic antibacterial agent), and stir magnetically for 0.5~1 h to fully combine chitosan with polyvinyl alcohol to obtain a mixed solution, thereby improving the antibacterial and mechanical properties of the film; wherein the mass fraction of chitosan is 0.3~0.5%; The mixed solution was then kept warm in a constant temperature water bath at 30-37℃. The probiotic nanoemulsion prepared in step (2) was slowly added. The amount of nanoemulsion added was 15-20% of the mass of the polyvinyl alcohol solution. While adding, the mixture was magnetically stirred (200-300 r / min, stirring time 2h). After stirring, the mixture was placed in an ultrasonic degasser and ultrasonically degassed for 15-20 min at a power of 100-150 W and a temperature of 30-35℃ to remove air bubbles from the solution (to avoid pores on the film surface, which would affect the antibacterial performance and appearance). A uniform, bubble-free edible probiotic nanoemulsion film was obtained. (4) Pour the edible probiotic nanoemulsion liquid prepared in step (3) into a pre-sterilized glass plate and coat it evenly with a coater. The coating thickness is controlled to be 0.3~0.5 mm (to ensure uniform film thickness and avoid uneven antibacterial performance caused by excessive thickness or thinness in some areas). Place the coated glass plate in a constant temperature and humidity drying oven and set a gradient drying mode: temperature 30~32℃ and humidity 58~60% for the first 4 hours, temperature 33~34℃ and humidity 55~56% for the middle 4 hours, and temperature 35~36℃ and humidity 48~50% for the last 4~8 hours to avoid rapid drying that could cause the film to crack. After drying, peel the film off the glass plate to obtain an edible probiotic nanoemulsion film with enhanced antibacterial activity.

[0007] Preferably, the culturing step (1) is as follows: culturing in a constant temperature shaker at 32-35℃ and 180-200 r / min for 20-24 h; centrifuging the *Lactobacillus plantarum* culture medium under the following conditions: centrifugation at 6000 rpm and 4℃ for 5-10 min; the concentration of the modified probiotic suspension is 1×10⁻⁶. 7 CFU / mL; Specific procedure: Take 1 mL of the modified probiotic suspension and perform serial dilutions with sterile physiological saline (10 CFU / mL); -1 ~10 -7 (times), take 10 -6 10 -7 Two dilution gradients of bacterial culture, 0.1 mL each, were spread onto MRS solid medium, with three replicates for each gradient. After incubation at 35°C for 24 h, the colony count on each plate was counted (plates with colony counts between 30 and 300 were selected for counting). The counts were then substituted into the formula above to ensure that the probiotic concentration remained stable at 1 × 10⁻⁶. 7 CFU / mL.

[0008] Preferably, in step (1), the mass fraction of L-ascorbic acid is 0.2%, the mass fraction of Tween-20 is 0.08%, and the mass fraction of trehalose is 0.3%.

[0009] Preferably, in step (2), the corn germ oil is filtered through a 0.22 μm filter membrane before use, and the mass fraction of corn germ oil in the oil phase is 10%, the mass fraction of Tween-80 is 10%, and the mass fraction of glyceryl stearate is 0.8%. Preferably, in step (2), the pH value of the aqueous phase is 6.8; the homogenization time is 15 min, the homogenization pressure is 22 MPa, and the homogenization is divided into 3 times, each time for 5 min, with an interval of 2 min; the ultrasonic power is 220 W, the ultrasonic time is 12 min, the ultrasonic time is 3 s, and the interval is 5 s to avoid local high temperature damage to probiotics; the nanoemulsion particle size is controlled at 60~80 nm, and CV≤12%.

[0010] Preferably, in step (3), the concentration of polyvinyl alcohol is 9 g / 100 mL, the mass fraction of chitosan is 0.4%, the amount of nanoemulsion added is 18% of the mass of the polyvinyl alcohol solution, the ultrasonic degassing power is 120 W, and the degassing time is 18 min.

[0011] Preferably, in step (4), the coating thickness is 0.4 mm, the thickness after film peeling is 0.1~0.15 mm, and the moisture content is ≤10% (moisture content calculation formula: moisture content = (wet film mass - dry film mass) / wet film mass × 100%).

[0012] This invention prepares a highly antibacterial, edible packaging film based on Lactobacillus plantarum nanoemulsion by adding a nanoemulsion of probiotic solution to a polyvinyl alcohol matrix. The film has the following advantages: (1) This invention firstly adds three modifiers—L-ascorbic acid (antioxidant), Tween-20 (dispersant), and trehalose (protectant)—to the preparation of probiotic suspensions, solving the problem of probiotic activity loss during cultivation, centrifugation, and suspension, and significantly improving the survival rate of probiotics. Simultaneously, it clarifies key parameters such as inoculum size and culture speed to ensure stable probiotic concentration. Finally, by nanoemulsifying the probiotic suspension, the survival rate of probiotics during film drying and storage is significantly improved. Furthermore, controlling the glycerol content in the nanoemulsion system within the range of 25-35% helps maintain structural stability, thereby further enhancing the probiotic activity retention effect.

[0013] (2) In addition, in the preparation of nanoemulsion, the present invention adds glyceryl monostearate as a co-surfactant, optimizes the pH value and drop rate of the aqueous phase, and adopts a combination of high pressure homogenization and intermittent ultrasound to solve the problems of poor stability and uneven particle size of the original nanoemulsion, so that the particle size of the nanoemulsion is controlled at 50~100 nm, the dispersibility is better, and the activity of probiotics can be effectively protected.

[0014] (3) In this invention, chitosan is added to the film matrix as a synergistic antibacterial agent to form an antibacterial synergistic effect with probiotics, which solves the problem of insufficient antibacterial performance of single probiotics; at the same time, the concentration of polyvinyl alcohol and the amount of nanoemulsion added are optimized to take into account the antibacterial performance and mechanical properties of the film, and avoid the film from becoming brittle and broken.

[0015] (4) During the drying process, the present invention adopts a gradient drying process to replace the original single drying conditions, which solves the problems of easy cracking and many surface pores during the film drying process, and improves the integrity and stability of the film. At the same time, it supplements key detection indicators and calculation formulas such as moisture content and particle size variation coefficient, making the scheme more operable and repeatable.

[0016] In summary, this invention significantly improves the mechanical and antibacterial properties of probiotic films and significantly maintains the effective antibacterial efficiency of probiotic films; wherein the packaging film maintains an antibacterial efficiency of >75% within 28 days; the packaging film has a tensile strength ≥12 MPa, an elongation at break ≥35%, and the film is intact without cracks. Attached Figure Description

[0017] Figure 1 Comparison of antibacterial efficiency (%) of different samples after 28 days; Figure 2 The survival rate curves of probiotics at different storage times (survival rate, %, stored at 4℃). Figure 3 The graph shows the mechanical properties of the thin film in different embodiments and comparative examples. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the listed embodiments. Without departing from the core concept of the present invention, appropriate modifications or improvements can be made by those skilled in the art, and all such modifications or improvements should be considered to fall within the scope of protection of the present invention.

[0019] Example 1: (1) Preparation of modified probiotic suspension: Lactobacillus plantarum powder (active 1×10 11 The inoculum (CFU / g) was added to sterilized MRS liquid medium at an inoculation rate of 1.5 g / L. L-ascorbic acid and Tween-20 were added, and the mixture was shaken well and incubated in a constant temperature shaker at 35℃ and 180 r / min for 24 h to obtain Lactobacillus plantarum culture medium. The mass fraction of L-ascorbic acid was 0.2%, and the mass fraction of Tween-20 was 0.08%.

[0020] The culture medium was centrifuged at 6000 rpm and 4℃ for 15 min, the supernatant was discarded, and the precipitate was washed twice with sterile physiological saline, centrifuged at 4000 rpm for 5 min after each wash. The precipitate was then resuspended in sterile distilled water, trehalose was added, and the mixture was magnetically stirred for 15 min until completely dissolved, resulting in a trehalose mass fraction of 0.3% in the bacterial suspension. Finally, a serial dilution plating method was used to determine the concentration, yielding a concentration of 1×10⁻⁶. 7 Modified probiotic suspension at CFU / mL.

[0021] (2) Preparation of stable probiotic nanoemulsion: At room temperature of 25°C, corn germ oil (sterilized by 0.22 μm filter membrane) and Tween-80 were mixed and glyceryl monostearate was added. The mixture was magnetically stirred for 35 min (350 r / min) to prepare the oil phase. The mass fraction of Tween-80 was 10% and the mass fraction of glyceryl monostearate was 0.8%.

[0022] The aqueous phase (composed of 50% (v / v) modified probiotic suspension, 30% (v / v) glycerol, and 20% (v / v) sterile distilled water) was adjusted to pH 6.8 with 0.1 mol / L NaOH solution and added dropwise to the oil phase at a rate of 2.5 mL / min, while continuously stirring with a magnetic stirrer (550 r / min). After homogenization, the mixture was homogenized under high pressure at 22 MPa for 15 min (divided into 3 intervals of 5 min each, with 2 min intervals), and then sonicated for 12 min using an ultrasonic cell disruptor (220 W power) (3 s of sonication followed by 5 s of intermittent sonication). The particle size was measured using a laser particle size analyzer to obtain a stable probiotic nanoemulsion with a particle size of 60–80 nm and a CV of 11%.

[0023] (3) Preparation of edible probiotic nanoemulsion solution: Polyvinyl alcohol (degree of polymerization 1700~1800) was added to sterilized distilled water at a concentration of 9 g / 100 mL, and placed in a constant temperature water bath at 80℃. The mixture was stirred for 35 min until completely dissolved. After cooling to room temperature, chitosan (pre-dissolved and sterilized with 1% acetic acid solution) was added and magnetically stirred for 1 h to obtain a mixed solution with a chitosan mass fraction of 0.4%. The mixed solution was kept warm in a constant temperature water bath at 35℃, and 18% (relative to the mass of the polyvinyl alcohol solution) of probiotic nanoemulsion was added while magnetically stirring (250 r / min). After stirring for 2 h, the mixture was ultrasonically degassed for 18 min at a power of 120 W and a temperature of 32℃ to obtain a uniform, bubble-free edible probiotic nanoemulsion solution.

[0024] (4) Preparation of edible antibacterial film: The above nanoemulsion film solution was poured into a pre-sterilized glass plate and coated evenly with a coater to a thickness of 0.4 mm. The glass plate was placed in a constant temperature and humidity drying oven and subjected to gradient drying: 32℃ and 60% humidity for the first 4 hours, 34℃ and 55% humidity for the middle 4 hours, and 36℃ and 50% humidity for the last 6 hours, for a total drying time of 14 hours. After drying, the film was peeled off, placed in a sterile sealed bag, and stored at 4℃ and 50% relative humidity for later use, thus obtaining an edible antibacterial film.

[0025] Mechanical test results: tensile strength 14 MPa, elongation at break 42%; moisture content 9.2%, good stability, and edibility meets national standards.

[0026] Example 2: (1) Preparation of modified probiotic suspension: Lactobacillus plantarum powder (active 1×10 11The inoculum of Lactobacillus plantarum (CFU / g) was added to sterilized MRS liquid medium at an inoculation rate of 1 g / L. L-ascorbic acid and Tween-20 were added, and the mixture was shaken well and incubated in a constant temperature shaker at 35℃ and 180 r / min for 24 h to obtain the Lactobacillus plantarum culture medium. The mass fraction of L-ascorbic acid was 0.1%, and the mass fraction of Tween-20 was 0.05%.

[0027] The culture medium was centrifuged at 5800 rpm and 4℃ for 14 min, the supernatant was discarded, and the precipitate was washed twice with sterile physiological saline, centrifuged at 4000 rpm for 5 min after each wash. The precipitate was then resuspended in sterile distilled water, trehalose was added, and the mixture was magnetically stirred for 15 min until completely dissolved, resulting in a trehalose mass fraction of 0.2% in the bacterial suspension. Finally, a serial dilution plating method was used to determine the concentration, yielding a concentration of 1×10⁻⁶. 7 Modified probiotic suspension at CFU / mL.

[0028] (2) Preparation of stable probiotic nanoemulsion: At room temperature (25°C), corn germ oil (sterilized by a 0.22 μm filter membrane) and Tween-80 were mixed, and glyceryl monostearate was added. The mixture was magnetically stirred for 35 min (350 r / min) to prepare the oil phase. The mass fraction of Tween-80 was 10%, and the mass fraction of glyceryl monostearate was 0.5%. The aqueous phase (composed of 45% (v / v) modified probiotic suspension, 35% (v / v) glycerol and 20% (v / v) sterile distilled water) was adjusted to pH 6.5 with 0.1 mol / L NaOH solution and added dropwise to the oil phase at a rate of 2 mL / min, while continuously stirring with a magnetic stirrer (500 r / min). After being mixed evenly, the mixture was homogenized under high pressure at 20 MPa for 15 min (divided into 3 times, 5 min each time, with 2 min intervals). Then, it was ultrasonically treated with an ultrasonic cell disruptor (power 200 W) for 10 min (3 s of ultrasonic treatment, 5 s of intermittent treatment). The particle size was measured by a laser particle size analyzer to obtain a stable probiotic nanoemulsion with a particle size of 50~70 nm and CV=13%.

[0029] (3) Preparation of edible probiotic nanoemulsion solution: Polyvinyl alcohol (degree of polymerization 1700~1800) was added to sterilized distilled water at a concentration of 8 g / 100 mL, and placed in a constant temperature water bath at 78℃. The mixture was stirred for 30 min until completely dissolved. After cooling to room temperature, chitosan (pre-dissolved and sterilized with 1% acetic acid solution) was added and magnetically stirred for 1 h to obtain a mixed solution with a chitosan mass fraction of 0.3%. The mixed solution was kept warm in a constant temperature water bath at 30℃. 15% (relative to the mass of the polyvinyl alcohol solution) of probiotic nanoemulsion was added while magnetically stirring (200 r / min). After stirring for 2 h, the mixture was ultrasonically degassed for 15 min at a power of 100 W and a temperature of 30℃ to obtain a uniform, bubble-free edible probiotic nanoemulsion solution.

[0030] (4) Preparation of edible antibacterial film: The above nanoemulsion film solution was poured into a pre-sterilized glass plate and coated evenly with a coater to a thickness of 0.3 mm. The glass plate was placed in a constant temperature and humidity drying oven and dried using a gradient: 32℃ and 60% humidity for the first 4 hours, 34℃ and 55% humidity for the middle 4 hours, and 36℃ and 50% humidity for the last 4 hours, for a total of 12 hours. After drying, the film was peeled off, placed in a sterile sealed bag, and stored at 4℃ and 50% relative humidity for later use, thus obtaining an edible antibacterial film.

[0031] Mechanical test results: tensile strength 12 MPa, elongation at break 35%; moisture content 8.8%, good stability, and edibility meets national standards.

[0032] Example 3: (1) Preparation of modified probiotic suspension: Lactobacillus plantarum powder (active 1×10 11 Inoculation (CFU / g) was added to sterilized MRS liquid medium at a rate of 2 g / L. L-ascorbic acid and Tween-20 were added, and the mixture was shaken well and incubated in a constant temperature shaker at 35℃ and 180 rpm for 24 h to obtain a *Lactobacillus plantarum* culture broth. The mass fraction of L-ascorbic acid was 0.3%, and the mass fraction of Tween-20 was 0.1%. The culture broth was centrifuged at 6200 rpm and 4℃ for 16 min, the supernatant was discarded, and the precipitate was washed twice with sterile physiological saline, centrifuged at 4000 rpm for 5 min after each wash. Finally, the precipitate was resuspended in sterile distilled water, trehalose was added, and the mixture was magnetically stirred for 15 min until completely dissolved. The mass fraction of trehalose in the resulting bacterial suspension was 0.4%. Finally, the concentration was obtained by serial dilution plating to determine the bacterial count, resulting in a concentration of 1×10⁻⁶. 7 Modified probiotic suspension at CFU / mL.

[0033] (2) Preparation of stable probiotic nanoemulsion: At room temperature (25°C), corn germ oil (sterilized by a 0.22 μm filter membrane) and Tween-80 were mixed, and glyceryl monostearate was added. The mixture was magnetically stirred for 35 min (350 r / min) to prepare the oil phase. The mass fraction of Tween-80 was 10%, and the mass fraction of glyceryl monostearate was 1%. The aqueous phase (composed of 55% (v / v) modified probiotic suspension, 25% (v / v) glycerol and 20% (v / v) sterile distilled water) was adjusted to pH 7.0 with 0.1 mol / L HCl solution and added dropwise to the oil phase at a rate of 3 mL / min, while continuously stirring with a magnetic stirrer (600 r / min). After being mixed evenly, the mixture was homogenized under high pressure at 25 MPa for 15 min (divided into 3 times, 5 min each time, with an interval of 2 min). Then, it was ultrasonically treated with an ultrasonic cell disruptor (power 250 W) for 15 min (3 s of ultrasonic treatment, 5 s of interval). The particle size was measured by a laser particle size analyzer to obtain a stable probiotic nanoemulsion with a particle size of 70~100 nm and CV=14%.

[0034] (3) Preparation of edible probiotic nanoemulsion solution: Polyvinyl alcohol (degree of polymerization 1700~1800) was added to sterilized distilled water at a concentration of 10 g / 100 mL, and placed in a constant temperature water bath at 82℃. The mixture was stirred for 40 min until completely dissolved. After cooling to room temperature, chitosan (pre-dissolved and sterilized with 1% acetic acid solution) was added and magnetically stirred for 1 h to obtain a mixed solution with a chitosan mass fraction of 0.5%. The mixed solution was kept warm in a constant temperature water bath at 37℃. 20% (relative to the mass of the polyvinyl alcohol solution) of probiotic nanoemulsion was added while magnetically stirring (300 r / min). After stirring for 2 h, the mixture was ultrasonically degassed for 20 min at a power of 150 W and a temperature of 35℃ to obtain a uniform, bubble-free edible probiotic nanoemulsion solution.

[0035] (4) Preparation of edible antibacterial film: Pour the nanoemulsion solution from step (3) into a pre-sterilized glass plate and coat it evenly with a coater to a thickness of 0.5 mm. Place the glass plate in a constant temperature and humidity drying oven and use gradient drying: 32℃ and 60% humidity for the first 4 hours, 34℃ and 55% humidity for the middle 4 hours, and 36℃ and 50% humidity for the last 8 hours, for a total drying time of 16 hours. After drying, peel off the film, place it in a sterile sealed bag, and store it at 4℃ and 50% relative humidity for later use to obtain an edible antibacterial film.

[0036] Mechanical test results: tensile strength 15 MPa, elongation at break 45%; moisture content 9.5%, good stability, and edibility meets national standards.

[0037] Comparative Example 1: Without L-ascorbic acid, Tween-20, and trehalose modifier. (1) Preparation of probiotic suspension Lactobacillus plantarum powder (active 1×10) 11 Inoculation (CFU / g) was added to sterilized MRS liquid medium at a rate of 1.5 g / L, without the addition of L-ascorbic acid, Tween-20, or trehalose. The medium was shaken well and incubated at 35°C and 180 r / min for 24 h to obtain a *Lactobacillus plantarum* culture. The culture was centrifuged at 6000 rpm and 4°C for 15 min, the supernatant was discarded, and the precipitate was washed twice with sterile physiological saline, centrifuged at 4000 rpm for 5 min after each wash. Finally, the precipitate was resuspended in sterile distilled water, magnetically stirred for 18 min, and counted using a serial dilution plating method.

[0038] (2) Preparation of probiotic nanoemulsion The operation is exactly the same as that in Example 1.

[0039] (3) Preparation of edible probiotic nanoemulsion film liquid The operation is exactly the same as that in Example 1.

[0040] (4) Preparation of edible antibacterial film The operation is exactly the same as that in Example 1.

[0041] Test results The initial concentration of probiotics was 5.2 × 10⁻⁶. 6 The CFU / mL concentration significantly reduced the survival rate; the nanoemulsion exhibited poor dispersibility and was prone to stratification; the antibacterial efficiency of the film after 28 days was only 42%, far lower than that of the examples.

[0042] Comparative Example 2: No nanoemulsification treatment (ordinary stirred emulsion) (1) Preparation of modified probiotic suspension The formulation, culture, centrifugation, suspension, and modifier addition were exactly the same as in Example 1.

[0043] (2) Preparation of probiotic emulsion After mixing the oil and water phases, only magnetic stirring was performed, without high-pressure homogenization and intermittent ultrasonic treatment. The other conditions were the same as in Example 1.

[0044] (3) Preparation of edible probiotic film liquid It is exactly the same as Example 1.

[0045] (4) Preparation of edible antibacterial film It is exactly the same as Example 1.

[0046] Test results: The emulsion particle size was 350~500 nm, the particle size variation coefficient CV>35%, and the stability was poor; the activity of probiotics was severely lost during the drying and storage process, and the number of viable bacteria decreased by more than 65% after 28 days of storage at 4℃; the film surface had many pores and poor integrity, and the antibacterial efficiency was only 51% after 28 days.

[0047] Comparative Example 3: No chitosan added to the film matrix (1) Preparation of modified probiotic suspension The operation is exactly the same as that in Example 1.

[0048] (2) Preparation of stable probiotic nanoemulsion The operation is exactly the same as that in Example 1.

[0049] (3) Preparation of edible film liquid The polyvinyl alcohol dissolution, heat preservation, and stirring process is the same as in Example 1, except that chitosan is not added, and the amount of nanoemulsion added and the subsequent degassing steps remain unchanged.

[0050] (4) Preparation of edible antibacterial film The operation is exactly the same as that in Example 1.

[0051] Test results: No synergistic antibacterial effect, and the antibacterial rate against pathogenic bacteria decreased by 30%~35%; the antibacterial efficiency of the film was only 48% after 28 days, the antibacterial efficiency decayed rapidly, and there was no long-lasting antibacterial ability.

[0052] Comparative Example 4: Single-temperature drying (non-gradient drying) (1) Preparation of modified probiotic suspension The operation is exactly the same as that in Example 1.

[0053] (2) Preparation of stable probiotic nanoemulsion The operation is exactly the same as that in Example 1.

[0054] (3) Preparation of edible probiotic nanoemulsion film liquid The operation is exactly the same as that in Example 1.

[0055] (4) Preparation of edible antibacterial film The coating thickness was the same as in Example 1, except that a single drying condition was used: 36°C, 50% relative humidity, continuous drying for 14 hours, without gradient temperature increase and humidity reduction, and the other storage conditions were the same.

[0056] Test results: The film cracked during drying, had many surface pores, and poor integrity; the tensile strength was 9.5 MPa, the elongation at break was 22%, and it was easily brittle and damaged; the number of live probiotics decreased by 40% during the drying process, and the stability and antibacterial long-term effect of the film were significantly reduced.

[0057] The key properties of the edible films involved in the above embodiments and comparative examples were measured: 1. Determination of the survival rate of probiotics in the membrane during drying and storage: The probiotic content in the membrane is expressed as LogCFU / g. The initial bacterial count before drying was calculated based on the amount of bacterial solution added and the membrane mass. The bacterial membrane was stored at 25℃ and 75% relative humidity for 28 days, and bacterial activity was measured every 4 days. The specific method was as follows: 1 g of membrane sample was weighed and 9 mL of sterile physiological saline was added under aseptic conditions. The mixture was shaken thoroughly to completely release the bacteria. Subsequently, serial dilutions were performed, and the appropriate dilutions were spread on MRS medium. After incubation, colony counting was performed.

[0058] Antibacterial activity assay of the membrane: The antibacterial properties of the membrane were evaluated using the inhibition zone method. Nutrient agar medium (10 mL) was poured into a petri dish and allowed to cool and solidify. Then, 100 μL of a 1×10⁻⁶ m³ solution was added to the medium. 7 CFU / mL of Escherichia coli culture was evenly spread on the surface of the culture medium. Then, thin film samples cut to a diameter of 6 mm were placed in the center of the plate, and the diameter of the inhibition zone was measured after incubation. Three replicates were set up for each experiment.

[0059] Determination of the antibacterial efficiency of the membrane during storage: The membrane was stored at 25℃ and 75% relative humidity for 28 days, and its antibacterial activity was determined according to the method described above. The antibacterial efficiency of the membrane during storage was calculated using the following formula: Antibacterial efficiency: I n / I0×100%, I n Id is the diameter of the inhibition zone of the nd film, and I0 is the diameter of the inhibition zone of the 0d film. Figure 1 This is a bar chart comparing the 28-day antibacterial efficiency of the edible antibacterial films obtained in Examples 1-3 and Comparative Examples 1-4 of the present invention. As can be seen from the chart, the antibacterial films of Examples 1, 2, and 3 of the present invention, after storage at 4°C for 28 days, have antibacterial efficiencies of 78%, 76%, and 80%, respectively, all remaining above 75%. In contrast, the 28-day antibacterial efficiencies of Comparative Example 1 (without modifier), Comparative Example 2 (without nano-emulsification), Comparative Example 3 (without chitosan), and Comparative Example 4 (using a single drying process) are only 42%, 51%, 48%, and 53%, respectively, significantly lower than those of the examples of the present invention. This indicates that the present invention, through a combination of modifier addition, nano-emulsification, chitosan synergy, and gradient drying, can significantly improve the long-lasting antibacterial performance of the probiotic film.

[0060] Figure 2The graph shows the survival rate of probiotics in the antibacterial films obtained in Example 1, Comparative Example 1 (without modifier), and Comparative Example 2 (without nanoemulsification) of this invention under storage conditions of 4°C, as a function of storage time. As can be seen from the graph, the survival rate of probiotics in Example 1 remained at 78% after 28 days of storage; in Comparative Example 1, due to the lack of modifier protection, the activity of probiotics rapidly declined, with a survival rate of only 42% after 28 days; and in Comparative Example 2, due to the lack of nanoemulsification, the probiotics suffered severe losses during drying and storage, with a survival rate of only 51% after 28 days. The results indicate that the modifier and nanoemulsification process can significantly improve the survival rate of probiotics during preparation and storage.

[0061] 2. Particle size distribution: The laser particle size analyzer (dynamic light scattering method, DLS) was used according to the national standard GB / T 29022-2012 "Dynamic Light Scattering Method for Particle Size Distribution of Nanopowders in Nanotechnology". Before testing, the sample was appropriately diluted with sterile distilled water, the detection temperature was set to 25℃, water was used as the dispersion medium, the detection angle was 90°, and three parallel measurements were performed. The average particle size, particle size distribution range and coefficient of variation (CV) were recorded, and the average value of the results was taken.

[0062] The test results show that the probiotic nanoemulsion prepared by the present invention through a combination of high-pressure homogenization and intermittent ultrasonication has a concentrated particle size distribution of 60–80 nm, with a narrow particle size distribution and uniform dispersion. In contrast, the ordinary emulsion obtained by magnetic stirring only in Comparative Example 2 has a particle size distribution of 350–500 nm, with a large and wide particle size distribution and poor stability. This indicates that the nanoemulsification process of the present invention can effectively reduce the emulsion particle size, improve the dispersibility and stability of the system, and thus protect the activity of probiotics.

[0063] 3. Antibacterial performance testing and effects Detection method: The inhibition zone method and colony counting method were combined. *Escherichia coli* (ATCC 25922) and *Staphylococcus aureus* (ATCC 25923) were selected as indicator bacteria, and a concentration of 1×10⁻⁶ was prepared. 6 Indicator bacterial suspension at CFU / mL. The film sample was cut into 6 mm diameter discs and placed on LB solid medium inoculated with indicator bacterial suspension. The discs were incubated at 37°C for 24 h, and the diameter of the inhibition zone was measured. The antibacterial efficiency was calculated by colony counting.

[0064] Formula for calculating antibacterial efficiency: Antibacterial efficiency % = (Number of colonies in the control group - Number of colonies in the experimental group) / Number of colonies in the control group × 100% (Note: The blank group was a polyvinyl alcohol film without added probiotics and chitosan, and the experimental group was a probiotic nanoemulsion film prepared in this invention. Three parallel samples were set up for each group, and the average value was taken.) The inhibition zone diameter against Escherichia coli is ≥18 mm, and the inhibition zone diameter against Staphylococcus aureus is ≥16 mm. After 28 days of storage, the antibacterial efficiency against the two indicator bacteria is still >75%, which is much higher than the original technology (the original technology has an antibacterial efficiency of ≤60% after 24 days), and the antibacterial range is wider and the effect is more lasting.

[0065] 4. Mechanical property testing and results Test method: According to GB / T 1040.3-2006 standard, the film sample was cut into strips of 100 mm × 15 mm. The tensile strength and elongation at break were tested using a universal testing machine at a tensile speed of 5 mm / min. Five parallel samples were set up for each group, and the average value was taken.

[0066] Figure 3 Table 1 compares the mechanical properties and appearance of the antibacterial films obtained in Examples 1-3 and Comparative Example 4 (single-temperature drying). As shown in the table, the tensile strengths of the films in Examples 1, 2, and 3 are 14 MPa, 12 MPa, and 15 MPa, respectively, and the elongation at break is 42%, 35%, and 45%, respectively; the films are intact and without cracks. Comparative Example 4, using single-temperature drying, has a tensile strength of only 9.5 MPa and an elongation at break of only 22%, exhibiting defects such as cracking and numerous pores. This indicates that the gradient drying process of the present invention can significantly improve the mechanical properties and integrity of the film, avoiding film embrittlement and damage.

[0067] Table 1

[0068]

[0069] 5. Film stability and edibility Stability testing: The film was stored at 4℃ and 50% relative humidity for 28 days. The appearance, integrity, and dispersibility of the nanoemulsion were observed. The moisture content and probiotic activity of the film were also tested. The results showed that after 28 days of storage, the film showed no cracking, deformation, or delamination. The nanoemulsion was uniformly dispersed, the moisture content was ≤10%, and the probiotic activity remained good, demonstrating excellent stability.

[0070] In summary, this invention successfully prepared an edible probiotic nanoemulsion film with enhanced antibacterial activity through modification of probiotic suspension, preparation of stable nanoemulsions, synergistic regulation of chitosan and polyvinyl alcohol, and optimization of gradient drying process. Compared with existing technologies, this film significantly improves probiotic survival rate, antibacterial performance, mechanical properties, and stability. Furthermore, the process is simple, industrially feasible, and can be widely applied in food preservation packaging, addressing the technical limitations of existing edible probiotic films and possessing significant practical value and promising prospects for widespread application.

[0071] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a highly antibacterial edible packaging film based on Lactobacillus plantarum nanoemulsion, characterized in that, Includes the following steps: (1) Pour the Lactobacillus plantarum powder into sterilized MRS liquid medium at an inoculation rate of 1~2 g / L, add L-ascorbic acid and Tween-20, shake well and culture to obtain Lactobacillus plantarum culture medium; wherein the mass fraction of L-ascorbic acid is 0.1~0.3% and the mass fraction of Tween-20 is 0.05~0.1%; Centrifuge the Lactobacillus plantarum culture medium, discard the supernatant, and then wash with sterile physiological saline to remove residual culture medium components. Finally, collect the washed precipitate, suspend the precipitate in sterile distilled water, add trehalose, and stir magnetically until completely dissolved to obtain a modified probiotic suspension, wherein the mass fraction of trehalose is 0.2-0.4%. (2) At room temperature, the hydrophobic substance corn germ oil and the surfactant Tween-80 are mixed and glyceryl monostearate is added. The mixture is magnetically stirred for 30-40 min at a stirring speed of 300-400 r / min to prepare a uniform and stable oil phase, wherein the mass fraction of Tween-80 is 8-12% and the mass fraction of glyceryl monostearate is 0.5-1.0%. Then the aqueous phase is slowly added dropwise to the oil phase at a dropping rate of 2-3 mL / min, while the mixture is continuously stirred at a speed of 500-600 r / min until the addition is complete. The aqueous phase consists of 45-55% (v / v) of the modified probiotic suspension obtained in step (1), 25-35% (v / v) of glycerol and the remainder of sterile distilled water, and the pH value of the aqueous phase is adjusted to 6.5-7.

0. After being mixed evenly, the mixture was placed in a high-pressure homogenizer and homogenized under a pressure of 20-25 MPa to further refine the emulsion particles. After homogenization, the mixture was ultrasonically treated with an ultrasonic cell disruptor with an ultrasonic power of 200-250 W and an ultrasonic time of 10-15 min to obtain a stable probiotic nanoemulsion with a particle size distribution of 50-100 nm and uniform dispersion. (3) Add polyvinyl alcohol to sterilized distilled water, place it in a constant temperature water bath at 70~80℃, stir for 30~40min until completely dissolved, and obtain a transparent polyvinyl alcohol solution with a concentration of 8~10 g / 100 mL; after cooling to room temperature, add chitosan, stir magnetically for 0.5~1 h, so that chitosan and polyvinyl alcohol are fully combined to obtain a mixed solution, which improves the antibacterial and mechanical properties of the film; wherein the mass fraction of chitosan is 0.3~0.5%; Then place the mixed solution in a constant temperature water bath at 30-37℃ and slowly add the probiotic nanoemulsion prepared in step (2). The amount of nanoemulsion added is 15-20% of the mass of the polyvinyl alcohol solution. While adding, stir magnetically. After stirring, place the mixed solution in an ultrasonic degasser and degas it for 15-20 minutes at a power of 100-150 W and a temperature of 30-35℃ to remove bubbles in the solution and obtain a uniform, bubble-free edible probiotic nanoemulsion film solution. (4) Pour the edible probiotic nanoemulsion liquid prepared in step (3) into a pre-sterilized glass plate and coat it evenly with a coater. The coating thickness is controlled to be 0.3~0.5 mm. Place the coated glass plate in a constant temperature and humidity drying oven and set a gradient drying mode: temperature 30~32℃ and humidity 58~60% for the first 4 hours, temperature 33~34℃ and humidity 55~56% for the middle 4 hours, and temperature 35~36℃ and humidity 48~50% for the last 4~8 hours to avoid rapid drying that could cause the film to crack. After drying, peel the film off the glass plate to obtain an edible probiotic nanoemulsion film with enhanced antibacterial activity.

2. The method for preparing a highly antibacterial edible packaging film based on *Lactobacillus plantarum* nanoemulsion according to claim 1, characterized in that, The culturing steps in step (1) are as follows: place the culture in a constant temperature shaker at 32~35℃ and 180~200 r / min for 20~24 h; the centrifugation conditions for the Lactobacillus plantarum culture medium are: centrifuge at 6000 rpm and 4℃ for 5-10 min; The concentration of the modified probiotic suspension is 1×10⁻⁶. 7 CFU / mL, specific procedure: Take 1 mL of modified probiotic suspension, perform serial dilution with sterile physiological saline, then take 10... -6 10 -7 Two dilution gradients of bacterial culture, 0.1 mL each, were spread onto MRS solid medium, with three replicates for each gradient. After incubation at 35°C for 24 h, the colony count was counted on each plate. Plates with colony counts between 30 and 300 were selected for counting. The counts were then calculated using the formula to ensure the probiotic concentration remained stable at 1 × 10⁻⁶. 7 CFU / mL.

3. The method for preparing a highly antibacterial edible packaging film based on *Lactobacillus plantarum* nanoemulsion according to claim 1, characterized in that, In step (1), the mass fraction of L-ascorbic acid is 0.2%, the mass fraction of Tween-20 is 0.08%, and the mass fraction of trehalose is 0.3%.

4. The method for preparing a highly antibacterial edible packaging film based on *Lactobacillus plantarum* nanoemulsion according to claim 1, characterized in that, In step (2), the corn germ oil is filtered through a 0.22 μm filter membrane before use, and the oil phase contains 10% Tween-80 and 0.8% glyceryl stearate.

5. The method for preparing a highly antibacterial edible packaging film based on *Lactobacillus plantarum* nanoemulsion according to claim 1, characterized in that, In step (2), the pH of the aqueous phase is 6.8; the homogenization time is 15 min, the homogenization pressure is 22 MPa, and the homogenization is performed in 3 sessions, each lasting 5 min with a 2 min interval; the ultrasonic power is 220 W, the ultrasonic time is 12 min, with 3 s ultrasonic sessions followed by a 5 s interval to avoid excessive local temperature damage to the probiotics; the nanoemulsion particle size is controlled at 60~80 nm, and the CV is ≤12%.

6. The method for preparing a highly antibacterial edible packaging film based on *Lactobacillus plantarum* nanoemulsion according to claim 1, characterized in that, In step (3), the concentration of polyvinyl alcohol is 9 g / 100 mL, the mass fraction of chitosan is 0.4%, the amount of nanoemulsion added is 18% of the mass of the polyvinyl alcohol solution, the ultrasonic degassing power is 120 W, and the degassing time is 18 min.

7. The method for preparing a highly antibacterial edible packaging film based on *Lactobacillus plantarum* nanoemulsion according to claim 1, characterized in that, In step (4), the coating thickness is 0.4 mm, the thickness of the film after peeling is 0.1~0.15 mm, and the moisture content is ≤10%.

8. The packaging film prepared by the method according to any one of claims 1-7, characterized in that, The packaging film has a tensile strength ≥12 MPa, an elongation at break ≥35%, and is intact without cracks.

9. The packaging film according to claim 8 is used in the preparation of food preservation and antibacterial packaging films.

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