Cross-electrospun composite nanofiber membrane, preparation method and application thereof

Abstract

The application discloses a kind of cross electrospinning composite nanofiber membrane and its preparation method and application.The membrane is with food-grade polymer ethyl cellulose and pullulan as base material, is prepared using cross electrospinning technology, and can load Nisin, oregano oil or probiotic bacteria.Ethyl cellulose provides hydrophobic support, and pullulan strengthens mechanical properties.The membrane has significant antibacterial activity on staphylococcus aureus after loading Nisin, and can be applied to strawberry preservation and preservation;On this basis, the introduction of oregano oil and Nisin compatibility can synergistically enhance the antibacterial and antioxidant properties of cross electrospinning membrane, which can effectively prolong the shelf life of chicken.In addition, when the membrane is used to load probiotic bacteria, it can improve the survival rate of bacterial cells in storage and simulated gastrointestinal fluid, achieve colon-targeted delivery, and affect intestinal flora metabolism.The application has antibacterial, preservation and probiotic protection functions, and has application prospect in the field of active food packaging and functional carrier.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and food engineering technology, specifically relating to a cross-electrospinned composite nanofiber membrane, its preparation method, and its application. Background Technology

[0002] In recent years, with the continuous deepening of nanotechnology research, nanomaterials have gained increasing popularity in the fields of food nutrition and safety due to their unique structural characteristics and nanoeffects. Electrospinning is a method for preparing nanofibers with mild conditions, simple processes, and continuous fabrication capabilities. The resulting nanofiber membranes have a large specific surface area, high porosity, high loading capacity for active substances, and good controlled-release performance. However, currently, these carriers mainly use spinnable synthetic polymers as substrates, and their safety remains controversial. Furthermore, the hydrophilic swelling problem of the carriers limits their application effectiveness. Currently, over a hundred spinnable polymer substrates have been discovered. However, with the increasing awareness of consumer safety and health and the growing demand for sustainable development, natural polymer materials, represented by polysaccharides, have gradually become a research hotspot in the field of electrospinning due to their excellent biocompatibility, superior water solubility, and environmentally friendly degradation characteristics. Therefore, seeking food-grade spinning substrates and constructing hydrophilic- and hydrophobic tunable nanocarriers using specific spinning strategies is an important strategy to solve the aforementioned problems.

[0003] Cross-spinning technology can simultaneously spin two or more polymer solutions into interwoven network composite fiber membranes, effectively solving the problem of immiscibility of spinning solutions. It also allows for flexible adjustments to achieve functional blending and structural reinforcement of the fiber membranes, providing new technical support for their application in food active packaging and nutrient delivery. Pullulan (PUL) has good film-forming properties, resulting in fiber membranes with good mechanical properties, but its strong hydrophilicity makes it prone to disintegration upon contact with water. Ethyl cellulose (EC) fiber membranes are hydrophobic and chemically stable, but they are brittle and lack toughness, making them unsuitable for meeting the mechanical and barrier requirements of packaging materials when used alone. Simple blending or uniaxial electrospinning cannot achieve a balance between hydrophilicity / hydrophobicity and mechanical properties, limiting their application in complex environments.

[0004] In terms of antibacterial and antioxidant packaging, the commonly used antimicrobial peptide Nisin has poor inhibitory effects on Gram-negative bacteria, while plant essential oils, although broad-spectrum antibacterial, are volatile and unstable, resulting in excessively rapid release of active ingredients or insufficient antioxidant capacity when added directly. Furthermore, existing food preservation films have limited ability to control food spoilage caused by microbial infection and oxidative decay, making it difficult to meet the needs of long shelf-life preservation. Currently, there is a lack of fiber membrane systems that synergistically combine Nisin with essential oils and utilize cross-electrospinning to construct composite antibacterial and antioxidant activities.

[0005] In the field of probiotic delivery, traditional probiotic preparations suffer from low survival rates and poor intestinal colonization efficiency under conditions of gastric acid, bile salts, and heat treatment. Furthermore, existing carrier materials are mostly monopolysaccharides, which cannot achieve the organic combination of colon-targeted delivery and gut microbiota regulation. Therefore, developing a composite nanofiber membrane that combines hydrophobic support and hydrophilic biodegradability with antibacterial / antioxidant properties, as well as probiotic protection and delivery functions, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] This invention relates to a cross-electrospinned composite nanofiber membrane, its preparation method, and its applications. Using food-grade EC and PUL as substrates, an interwoven, stacked network structure is formed through cross-electrospinning, and it can be loaded with Nisin, oregano oil (OEO), or probiotics. This membrane combines hydrophobic support with hydrophilic biodegradability, exhibiting antibacterial / antioxidant properties, probiotic protection, and colonic delivery functions, making it suitable for active food packaging and functional carrier applications.

[0007] On the one hand, the present invention provides a cross-electrospun composite nanofiber membrane, which adopts the following technical solution: A cross-electrospun composite nanofiber membrane is formed by cross-electrospun electrospinning of food-grade polymer ethyl cellulose (EC) and pullulan (PUL) to create an interwoven stacked network structure. Ethyl cellulose fibers provide hydrophobic support, while pullulan fibers enhance mechanical properties. The combination of the two can obtain a composite nanofiber membrane with adjustable hydrophilicity and hydrophobicity and good mechanical properties. The composite nanofiber membrane is loaded with active ingredients, which are selected from one or more of Nisin, oregano essential oil, and probiotics.

[0008] Preferably, the active ingredient is Nisin, and the loading of Nisin in pullulan polysaccharide fiber is 3% to 7%.

[0009] Preferably, the active ingredient is oregano essential oil, which is loaded in pullulan polysaccharide fiber in the form of β-cyclodextrin inclusion complex, and the volume fraction of oregano essential oil in the spinning solution is 1.59%~5.14%.

[0010] Preferably, the active ingredient is probiotics, which are encapsulated within pullulan polysaccharide fibers; the probiotics are Lactobacillus plantarum.

[0011] On the other hand, the present invention also provides a method for preparing a cross-electrospun composite nanofiber membrane, which adopts the following technical solution: 5. A method for preparing a cross-spun composite nanofiber membrane, comprising the following steps: S1: Prepare ethyl cellulose spinning solution and pullulan polysaccharide spinning solution; wherein, the solvent of ethyl cellulose spinning solution is anhydrous ethanol or glacial acetic acid, and the solvent of pullulan polysaccharide spinning solution is deionized water; as needed, add Nisin, β-cyclodextrin-encapsulated oregano essential oil and / or probiotics to pullulan polysaccharide spinning solution. S2: Using a cross electrospinning device, ethyl cellulose spinning solution and pullulan polysaccharide spinning solution are loaded into two syringes respectively, which are connected to the positive terminal of a high-voltage power supply. A roller is used as a collector, and electrospinning is performed simultaneously. Under the action of electric field force, the two jets are alternately covered on the surface of the collector to form a composite fiber membrane with an interpenetrating network structure.

[0012] Preferably, in step S2, the propulsion flow rate of the ethyl cellulose spinning solution is 0.15~1.0 mL / h, the propulsion flow rate of the pullulan polysaccharide spinning solution is 0.15~0.4 mL / h, the spinning voltage is 15~25 kV, and the receiving distance is 10~15 cm.

[0013] Preferably, when loading oregano essential oil, the preparation method of pullulan polysaccharide spinning solution is as follows: first, β-cyclodextrin is dissolved in water, then an ethanol solution of oregano essential oil is added for inclusion, and then Nisin and pullulan polysaccharide are added and stirred to dissolve.

[0014] Preferably, when probiotics are loaded, the pullulan polysaccharide spinning solution is mixed with the probiotic solution just before spinning to ensure that the probiotics are evenly dispersed in the spinning solution.

[0015] This invention also provides the application of cross-electrospun composite nanofiber membrane in the preparation of food active packaging materials or probiotic colon-targeted delivery carriers; it can be used to inhibit Gram-positive bacteria or to synergistically inhibit Gram-negative bacteria.

[0016] Preferably, the composite nanofiber membrane is used to improve the survival rate of probiotics under heat treatment, room temperature storage and simulated gastrointestinal fluid environment, and to achieve the release of probiotics in the colon.

[0017] In summary, the beneficial effects of the present invention are as follows: This invention provides a cross-electrospinned composite nanofiber membrane, its preparation method, and its applications. The EC / PUL composite nanofiber membrane (P / E-CNF) prepared using cross-electrospinning technology forms a hydrophobic-hydrophilic interpenetrating network structure, exhibiting good mechanical strength, controllable wettability, and biodegradability. Loading Nisin yields a cross-electrospinned membrane (N@P / E-CNF), which shows significant and concentration-dependent antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus. Using this composite membrane for strawberry preservation effectively delays fruit spoilage, reduces weight loss, and extends shelf life. Further compounding with OEO yields a Nisin and OEO-loaded cross-electrospinned membrane (OEO@NP / E-CNF). Nisin and OEO exhibit synergistic antibacterial activity against Staphylococcus aureus, while also enhancing the antibacterial effect against Gram-negative bacteria and significantly improving the DPPH free radical scavenging capacity of the composite membrane, giving the membrane material excellent antibacterial and antioxidant properties. Using this composite film for chicken preservation can significantly slow down the deterioration of chicken's color and texture characteristics (hardness, elasticity, chewiness), effectively maintain the chicken's good aroma and overall acceptability, and extend its shelf life to 7 days.

[0018] As a probiotic delivery carrier, this composite fiber membrane utilizes the barrier effect of hydrophobic EC (external microbiota) and the phased degradation characteristics of hydrophilic PUL (partially fibrous) fibers to achieve efficient encapsulation and colon-targeted delivery of probiotics (such as *Lactobacillus plantarum*). Experiments show that the survival rate of the encapsulated probiotics exceeds 84% ​​after heat treatment at 60℃, reaches 74.66% after storage at 25℃ for 28 days, and maintains a high survival rate even after continuous digestion by simulated gastric and intestinal juices, releasing them into the colonic environment with intact viability. In vitro fecal fermentation experiments further confirm that this composite membrane can be degraded and utilized by intestinal flora, regulating the abundance of beneficial bacteria such as *Bifidobacterium* and *Lactobacillus*, and significantly increasing the production of short-chain fatty acids such as acetic acid and butyric acid, demonstrating its potential for regulating the intestinal microecology. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of cross electrospinning; Figure 2 Laser confocal microscopy (CLSM) image of RhB-labeled PUL / EC cross-spun composite fiber membrane (P / E-CNF); Where a is a dark-field fluorescence image (showing that the RhB-labeled PUL fibers exhibit red fluorescence); b is a bright-field image (showing that PUL and EC fibers are evenly distributed and the surface is flat and smooth); c is a superimposed fluorescence image (bright field and fluorescence superimposed to confirm the distribution of the two fibers in the composite membrane). Figure 3 (Comparison of tensile properties of different electrospun fiber membranes). Figure 4 The effect of different EC concentrations and Nisin addition on the water contact angle of fiber membranes; Figure 5 Comparison of water solubility (WS) and swelling degree (SD) of different fiber membranes; Figure 6 The antibacterial activity of N@P / E-CNF membranes loaded with different Nisin concentrations against Staphylococcus aureus was investigated. Figure 7 Comparison of macroscopic morphological changes of different films in soil after 7 days of degradation; Figure 8 The effect of different OEO addition amounts on the water contact angle of N@P / E-CNF; Figure 9 Comparison of inhibition zone diameters of OEO@NP / E-CNF against Staphylococcus aureus and Escherichia coli; Figure 10 The effect of different OEO addition amounts on the DPPH free radical scavenging rate of composite membranes; Figure 11 This represents the survival state of probiotics after cross-electrospinning. Figure 12 The effect of heat treatment on the activity of free and embedded bacteria; Figure 13 The storage stability of free and encapsulated bacteria at different temperatures; Figure 14 Survival rate of probiotics encapsulated in PUL / EC composite fiber membranes during various stages of simulated digestion in vitro; Figure 15 CLSM image of PUL / EC nanofibers loaded with Rh123-labeled probiotics; Where a is a bright field image (showing the morphology of probiotic cells released from the fibrous membrane); b is a dark-field fluorescence image (showing the green fluorescence signal of Rh123-labeled probiotics); c represents a superimposed image of light and dark areas; Figure 16 Scanning electron microscope images of PUL / EC composite fiber membrane during in vitro simulated digestion; Figure 17 The changes in species abundance at the phylum level of the gut microbiota after in vitro fecal fermentation of composite fiber membranes; Figure 18 The changes in species abundance at the genus level of gut microbiota after in vitro fecal fermentation of composite fiber membranes; Figure 19 The change in short-chain fatty acid concentration of composite fiber membrane after in vitro fecal fermentation; Figure 20 Images showing the changes in strawberry quality; Figure 21The effect of different membrane treatments on the total bacterial count of chicken during storage at 4 ℃; Figure 22 The effects of different membrane treatments on the appearance of chicken. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example Example 1 The specific steps for preparing a cross-electrospun membrane are as follows: S1. Weigh EC powder, dissolve it in acetic acid, stir magnetically for 4 hours until completely dissolved, allow to stand to remove bubbles, and obtain an EC solution with a concentration of 30% (w / v). Weigh PUL powder, dissolve it in deionized water, stir to dissolve, and allow to stand to remove bubbles, and obtain a PUL solution with a concentration of 30% (w / v).

[0022] S2. Draw EC solution into a 5 mL syringe (needle inner diameter 0.8 mm) and attach it to the left syringe pump; draw PUL solution into another syringe and attach it to the right syringe pump. The distance between the two needles should be approximately 15 cm, with the needle tips aligned with the center of the collector. Turn on the high-voltage power supply to 25 kV and simultaneously start both syringe pumps (EC flow rate 1.0 mL / h, PUL flow rate 0.4 mL / h). Spin to obtain a PUL / EC cross-electrospun membrane (P / E-CNF) for later use. Figure 1 The diagram shown is a schematic of cross electrospinning.

[0023] Example 2 The specific steps for preparing a cross-electrospun membrane are as follows: S1. Prepare a 30% EC solution and a 20% PUL solution according to Example 1. Then add Nisin to the PUL solution to make the final concentration 3% (w / v), stir to dissolve, and let stand to remove bubbles.

[0024] S2. Draw EC solution into a 5 mL syringe (needle inner diameter 0.8 mm) and attach it to the left syringe pump; draw PUL solution into another syringe and attach it to the right syringe pump. The distance between the two needles is approximately 15 cm, and the needle tips are aligned with the center of the collector. Turn on the high voltage power supply to 25 kV and start both syringe pumps simultaneously (EC flow rate 1.0 mL / h, PUL flow rate 0.4 mL / h). Electrospinning yields a Nisin-loaded PUL / EC cross-electrospun membrane (N@P / E-CNF).

[0025] Example 3 The preparation of a cross-electrospun membrane differs from Example 2 in that the Nisin concentration is changed from 3% (w / v) to 5% (w / v), while the other steps are the same as in Example 2.

[0026] Example 4 The preparation of a cross-electrospun membrane differs from that in Example 2 in that Nisin is changed from 3% (w / v) to 7% (w / v), while the other steps are the same as in Example 2.

[0027] Example 5 A method for preparing a cross-spun membrane (N@OEO-P / E-CNF) loaded with Nisin and OEO, the specific steps of which are as follows: S1. Dissolve 1.0 g of β-CD powder in 10 mL of distilled water and stir in a 55 ℃ water bath for 3 h until completely dissolved to obtain a β-CD solution with a final concentration of 10.0% (w / v). Subsequently, different volumes of OEO were added to this solution, each OEO sample having been pre-dissolved in 5 mL of anhydrous ethanol. The OEO-ethanol mixture was added dropwise to the β-CD solution, and stirring was continued in a 55 ℃ water bath for 3 h until completely dissolved, resulting in a final OEO concentration of 1% (v / v) based on the volume of distilled water.

[0028] S2. Add 5% (w / v) Nisin and 20% (w / v) PUL sequentially to the above mixture and stir overnight until completely dissolved. Separately, dissolve 3.0 g of EC powder in 10 mL of acetic acid and stir overnight until completely dissolved.

[0029] S3. Load the spinning solution into a 20 mL syringe.

[0030] The electrospinning parameters were set as follows: positive voltage: 15 kV, negative voltage: 4 kV, feed speed: 0.50 mL / h, receiving distance: 15 cm, ambient temperature: 25℃, relative humidity: 40%. Cross-electrospinning was performed separately to obtain a composite membrane denoted as N@OEO1-PUL / EC. The electrospun membrane was stored in a desiccator for later use.

[0031] Example 6 A method for preparing an N@OEO-PUL / EC electrospun membrane differs from Example 5 in that the OEO concentration in step S1 is changed from 1% (v / v) to 3% (v / v), resulting in a composite membrane denoted as N@OEO2-PUL / EC. The remaining steps are the same as in Example 5.

[0032] Example 7 A method for preparing an N@OEO-PUL / EC electrospun membrane differs from Example 5 in that the OEO in step S1 is changed from 1% (v / v) to 5% (v / v), resulting in a composite membrane denoted as N@OEO3-PUL / EC. The remaining steps are the same as in Example 5.

[0033] Example 8 The specific steps for preparing a PUL / EC cross-spun fiber membrane loaded with probiotics are as follows: S1, Lactobacillus plantarum stored at -80℃ ( L. plantarum Remove from the refrigerator. Inoculate the bacterial culture into MRS broth medium at a 2% (v / v) inoculum. Incubate statically at 37°C for 16 h. Repeat the above steps (2% inoculum, 37°C, 16 h static incubation) for a second activation.

[0034] S2. Centrifuge the activated culture medium at 4℃ and 4000 rpm for 10 min, and collect the cell pellet. Discard the supernatant, and wash the cell pellet three times with 1% peptone buffer (pH 5.6). After washing, resuspend the cell pellet in sterile water for later use.

[0035] S3. Adjust the concentration of the bacterial suspension to make... L. plantarum The final concentration was 9–10 log CFU / mL. This bacterial suspension was added to a PUL solution prepared with sterile water. Under sterile conditions, the mixture was magnetically stirred at 100 rpm for 30 minutes to ensure uniform dispersion of the bacteria. After stirring, the mixture was allowed to stand to eliminate air bubbles, resulting in a homogeneous bacterial PUL spinning solution.

[0036] S4. The EC solution and the sterile PUL solution were separately loaded into two 10 mL syringes equipped with 21-gauge stainless steel needles. These syringes were then placed on two constant flow pumps, and a 15 kV voltage was applied to each. A roller rotating at 100 rpm was used as the collector. The flow rate of the EC solution was set to 0.15 mL / h, with a needle-to-collector distance of 15 cm; the flow rate of the PUL solution was set to 0.15 mL / h, with a receiving distance of 10 cm. The spinning machine was sterilized with UV light before spinning. Aseptic operation was maintained during solution preparation and spinning to avoid contamination. The temperature was controlled at approximately 25 ± 1 ℃, and the humidity was maintained at approximately 50 ± 2%. The prepared fiber membrane was stored in a sterile bag for later use.

[0037] Comparative Example Comparative Example 1 A method for preparing a PUL electrospun film (P-NF) includes the following steps: PUL powder was weighed and dissolved in deionized water. The solution was magnetically stirred for 4 hours until completely dissolved, and then ultrasonically degassed for 10 minutes to obtain a 10% (w / v) PUL solution. The PUL solution was drawn into a 5 mL syringe, which was then attached to a syringe pump with a needle inner diameter of 0.8 mm. A cylindrical collector was placed over an aluminum foil container at a distance of 12 cm. The voltage was set to 25 kV, and the PUL solution injection rate was set to 0.4 mL / h. The high-voltage power supply was turned on, and P-NF was obtained through electrospinning.

[0038] Comparative Example 2 A method for preparing P-NF differs from the comparative example in that the concentration of the PUL solution is changed from 10% to 15%, while the remaining steps are the same as in Comparative Example 1.

[0039] Comparative Example 3 A method for preparing P-NF differs from the comparative example in that the concentration of the PUL solution is changed from 10% to 20%, while the remaining steps are the same as in Comparative Example 1.

[0040] Comparative Example 4 A method for preparing P-NF differs from the comparative example in that the concentration of the PUL solution is changed from 10% to 25%, while the remaining steps are the same as in Comparative Example 1.

[0041] Comparative Example 5 A method for preparing an EC electrospun membrane (E-NF) includes the following steps: EC powder was weighed and added to acetic acid, and magnetically stirred for 4 h until completely dissolved. After standing to remove bubbles, a 20% (w / v) EC solution was prepared. The EC solution was drawn into a 5 mL syringe, which was then attached to a syringe pump with a needle inner diameter of 0.8 mm. A cylindrical collector was placed over an aluminum foil 12 cm away. The voltage was set to 25 kV, and the EC solution injection rate was set to 1.0 mL / h. The high-voltage power supply was turned on, and E-NF was obtained through electrospinning.

[0042] Comparative Example 6 The preparation of E-NF differs from Comparative Example 5 in that the concentration of the EC solution is changed from 20% (w / v) to 25% (w / v), while the other steps are the same as in Comparative Example 5.

[0043] Comparative Example 7 The preparation of an E-NF differs from Comparative Example 5 in that the solution concentration is changed from 20% (w / v) to 30% (w / v), while the remaining steps are the same as in Comparative Example 5.

[0044] Comparative Example 8 The preparation of E-NF differs from Comparative Example 5 in that the concentration of EC powder in the solution is changed from 20% (w / v) to 35% (w / v), while the remaining steps are the same as in Comparative Example 5.

[0045] Comparative Example 9 A polyethylene (PE) film, purchased from Anhui Qiling Plastic Products Co., Ltd.

[0046] Test case Test Example 1 The fiber distribution of the cross-electrospun membrane prepared in Example 1 was observed using a laser confocal microscope (CLSM). The excitation wavelength was 552 nm, the emission wavelength was 570 nm, the laser transmittance was 20%, and the objective lens magnification was 100x. To avoid RhB fluorescence quenching, all the above operations were performed in a dark environment.

[0047] Figure 2 CLSM images of RhB-labeled PUL fibers are shown under different fields of view. Bright-field images ( Figure 2 b) shows that PUL and EC fibers are uniformly distributed in the electrospun membrane, exhibiting a typical fine fibrous structure. In dark-field fluorescence images (…),… Figure 2 In (a), the RhB-labeled PUL fibers exhibited significant red fluorescence, while the unlabeled EC fibers showed no fluorescence signal. This result indicates that RhB successfully labeled the PUL fibers, which can be used to distinguish and visualize the distribution of the two components in the composite fiber membrane. Specifically, Figure 2 b shows that the surface of the cross-spun fibers is smooth and even, consistent with the observations in the SEM images. Figure 2 The fluorescence superposition image in c further demonstrates that the cross-electrospinning technique successfully integrates both PUL and EC spinning solutions into the same fiber membrane. This proves that this technique can achieve a good composite of PUL and EC, and the fiber membrane has the potential to possess both hydrophilic and hydrophobic properties.

[0048] Test Example 2 Mechanical strength tests were conducted on the electrospun films prepared in Examples 1, 4, Comparative Examples 1-4, and Comparative Example 7 using a computer-controlled universal testing machine according to standardized protocols. Rectangular specimens (50 mm × 10 mm) were precisely cut from the electrospun film and mounted between test handles with a constant width of 10 mm. Tests were performed under controlled conditions with an initial gauge length of 30 mm and a crosshead speed of 50 mm / min to ensure repeatable measurement of the film's mechanical properties.

[0049] From the stretching process of E-NF ( Figure 3 It is evident that its tensile strength is weaker than other fiber membranes. However, the P / E-CNF prepared by cross-electrospinning exhibits significantly higher TS and EAB than E-NF. p The value was <0.05, mainly due to the introduction of PUL fibers and the cross-linked structure formed between the two-phase fibers. Furthermore, the addition of Nisin led to a decrease in TS and EAB, possibly due to the salt precipitant carried by Nisin and the resulting roughening of the fiber morphology, which negatively impacted the mechanical strength of the material. Nevertheless, the composite membrane maintained its structural integrity under a static load of 175 g.

[0050] Test Example 3 The hydrophilicity and hydrophobicity properties of the electrospun membranes prepared in Comparative Examples 3, 5-8, and Examples 1 and 4 were tested, and the specific steps are as follows: (1) Contact angle The water contact angle of the samples was measured using a handleless drop method with a DSA25 contact angle goniometer. A rectangular specimen (3×3cm) was horizontally mounted on the sample stage, and 4 μL of deionized water was carefully dropped onto the surface using a microsyringe. The droplet image was captured immediately after deposition, and the contact angle was determined using ImageJ software.

[0051] like Figure 4 As shown, the water contact angle of the fiber membrane increases significantly with increasing EC concentration from 20% to 35%. p <0.05). This may be because the EC molecular backbone is rich in hydrophobic groups such as ethyl and ether bonds. As the polymer concentration increases, these hydrophobic segments are more likely to accumulate at the membrane-air interface, effectively reducing the surface free energy and thus enhancing the hydrophobicity of the membrane material. Due to the inherent hydrophilicity of PUL, P-NF has a small contact angle (<20°) and quickly decomposes upon contact with water. After cross-electrospinning, the strong hydrophobicity of EC fibers significantly reduces the hydrophilicity of P / E-CNF, increasing the contact angle to 88.7° ( p <0.05), which can effectively solve the swelling and decay of fiber membranes, providing a guarantee for their use as active food packaging.

[0052] (2) Water solubility (WS) and swelling degree (SD) The water solubility (WS) and swelling degree (SD) of P-NF, E-NF, P / E-CNF, and N@P / E-CNF membranes were determined. First, fiber membrane samples (1 cm × 2 cm) were dried to constant weight (m1) at 50 °C. Then, the samples were immersed in distilled water at 25 °C for 24 h before weighing. After removal from the water, the surface moisture of the membrane was gently blotted with filter paper, and the samples were weighed immediately (m2). Subsequently, the samples were re-dried at 50 °C to obtain their final constant weight (m3).

[0053] The formulas for calculating WS and SD are as follows:

[0054]

[0055] like Figure 5As shown, the PUL nanofiber membrane rapidly disintegrates in water, becoming difficult to handle even with tweezers after immersion for 10 minutes. This is attributed to the large number of hydroxyl groups in the PUL fibers, which readily form hydrogen bonds with water molecules. In contrast, the EC fiber membrane only undergoes surface wetting at the water-air interface and remains intact, demonstrating its significant hydrophobicity. Both types of cross-spun fiber membranes exhibit slight shrinkage after immersion in water, but still maintain structural integrity. The interpenetrating network formed during cross-electrospinning creates a physical barrier between the hydrophobic EC fibers and the underlying hydrophilic PUL fibers, effectively protecting them from moisture intrusion. This further confirms the promising application potential of N@P / E-CNF in the field of active food packaging.

[0056] Test Example 4 The antibacterial activity of the cross-electrospun membranes prepared in Examples 1-4 was analyzed, and the specific steps are as follows: The antibacterial activity of cross-spun nanofiber membranes (NE / P CEF) containing different Nisin concentrations (0%, 3%, 5%, 7%) was qualitatively evaluated using the disc diffusion method. The membranes were cut into 6 mm diameter discs, sterilized with UV light (30 min per side, 1 h total) for later use. Suspensions of Staphylococcus aureus and Escherichia coli (approximately 10 μL) were collected... 8 100 μL of each of the CFU / mL inoculum was evenly spread on the surface of tryptic soybean agar (TSA) plates. Then, sterile fiber membrane discs were aseptically placed on the inoculated agar. After incubation at 37°C for 48 h, the diameter of the inhibition zone was measured using the vernier caliper cross-section method. Each sample was measured three times, and the average diameter was used for quantitative comparison.

[0057] The results are as follows Figure 6 As shown, N@P / E-CNF exhibits significant antibacterial activity against Staphylococcus aureus. p <0.05), while no obvious inhibition zone was observed in the blank P / E-CNF, indicating that its antibacterial activity mainly comes from Nisin. As the Nisin concentration increases, the diameter of the inhibition zone gradually increases ( p The concentration-dependent antibacterial effect (<0.05) indicates that the antibacterial effect is concentration-dependent. Therefore, N@P / E-CNF has significant application potential as a highly efficient antibacterial packaging material in the field of food preservation.

[0058] Test Example 5 The electrospun membranes prepared in Examples 1 and 4, and the commercial polyethylene (PE) film of Comparative Example 9, were placed in plastic boxes containing equal amounts of soil. After adding an appropriate amount of water, degradation experiments were conducted at room temperature. The biodegradation process of the materials was visually monitored and recorded by periodically taking digital images (on a weekly basis).

[0059] The degradation process of different films was recorded using digital imaging over a period of 7 days under ambient temperature and humidity conditions (25-35℃, 50%-95% RH). Figure 7 The results showed that, except for polyethylene (PE) film (control), all cross-spun fiber membranes exhibited significant shrinkage and degradation from day 1. By day 3, the nanofiber membranes had essentially lost their structural integrity; after one week, the PE film's mass loss was only 19.35%, while the nanofiber membrane's degradation rate exceeded 70%. The faster degradation rate of N@E / P-CNF was mainly attributed to the hydrophilic domains introduced by Nisin, which promoted water penetration and microbial colonization. PUL, as a linear polysaccharide, can be depolymerized into glucose monomers by exoenzymes in the soil (such as α-amylase, glucosidase, and isoamylase), and ultimately mineralized into CO2 and H2O. Therefore, N@P / E-CNF possesses good biodegradability and environmental compatibility, and can serve as a potential green alternative to traditional plastic packaging materials.

[0060] Test Example 7 The contact angle of the electrospun films prepared in Examples 4-7 was tested using the same method as in Test Example 3.

[0061] like Figure 8 As shown, the contact angle of the N@P / E-CNF without OEO was 84.75°, and the hydrophobicity of the membrane was significantly improved after the introduction of OEO. p <0.05). When the OEO addition ratio increased from 1% to 5% (v / v), the contact angle of the composite membrane gradually increased from 89.14° to 113.39°, effectively improving the swelling and disintegration problem caused by hydrophilicity. This change may be due to the enrichment of OEO hydrophobic functional groups on the membrane surface, altering the distribution of hydrophilic active groups on the composite membrane surface. Therefore, the OEO addition ratio can effectively regulate the surface wetting properties of nanofiber membranes, a characteristic that gives it good application potential in the field of food preservation packaging.

[0062] Test Example 8 The antibacterial effects of the electrospun membranes prepared in Examples 4-7 were tested, and the specific methods were the same as those in Test Example 4.

[0063] like Figure 9 As shown, compared to N@P / E-CNF, the antibacterial effect of OEO-loaded N@OEO-P / E-CNF is significantly enhanced. p <0.05), and the antibacterial activity showed a significant increasing trend with increasing OEO content. Specifically, when the OEO concentration was 5%, N@OEO-P / E-CNF showed [results in] [the effect on...]. S. aureus and E. coliO157:H7 exhibited the most significant antibacterial activity, with inhibition zone diameters of 21.3 mm and 18.9 mm, respectively. Therefore, introducing OEO into N@P / E-CNF can effectively broaden the antibacterial spectrum of the fiber membrane, which has important application value and significance for addressing food spoilage problems caused by diverse microbial systems.

[0064] Test Example 9 The antioxidant properties of the electrospun membranes prepared in Examples 4 and 5-7 were tested using the following methods: Accurately weigh 0.00394 g of DPPH (2,2-diphenyl-1-trinitrophenylhydrazine), dissolve it in anhydrous ethanol and dilute to 100 mL to prepare a DPPH stock solution with a concentration of 0.1 mmol / L. Prepare Nisin ethanol solution and OEO ethanol solution with concentrations of 0.07 g / mL and 0.039 μL / mL, respectively. Weigh 0.05 g of cross-electrospun membrane sample, add 3 mL of anhydrous ethanol and soak for 24 h to prepare sample soaking solution. Mix 2 mL of Nisin and OEO ethanol solutions and sample soaking solution with 2 mL of DPPH stock solution, and react in the dark for 30 min. Using anhydrous ethanol as a blank control, measure the absorbance at a wavelength of 517 nm and calculate the free radical scavenging rate according to the following formula:

[0065] In the formula: A C A represents the absorbance value of the sample after the reaction. S This is the absorbance value for the blank control.

[0066] like Figure 10 As shown, Nisin has limited antioxidant capacity, and the antioxidant effect of N@P / E-CNF with Nisin alone is poor, with a DPPH free radical scavenging rate of 18.6%. OEO is rich in active ingredients such as carvacrol and terpenes. Its mechanism of action is to capture free radicals as a hydrogen atom or electron donor, blocking the lipid peroxidation chain reaction, and can directly scavenge reactive oxygen species such as hydroxyl radicals, peroxides, and superoxides. With the increase of OEO addition, the antioxidant activity of the membrane is significantly improved. p <0.05). When the OEO concentration increased from 1% to 5%, the DPPH scavenging rate increased from 61.2% to 70.5%. These results indicate that the addition of OEO significantly improves the antioxidant performance of the cross-electrospun membrane in a concentration-dependent manner. In summary, the N@OEO-P / E-CNF prepared in this study possesses both good antibacterial and antioxidant properties, providing an important foundation for its application in the preservation of meat products susceptible to multi-microbial contamination and oxidative spoilage.

[0067] Test Case 10 The electrospinning stability of the probiotics in the composite fiber membrane prepared in Example 8 was tested, and the specific steps are as follows: The blank P / E-CNF prepared in Example 1 and the bacterial-loaded PUL / EC fiber membrane (L / P / E) prepared in Example 8 were thoroughly dissolved in PBS. Samples were taken from the liquid every 4 hours for a fixed interval of 28 hours. The absorbance was measured immediately after each sample using a UV spectrophotometer, and the absorbance OD was recorded. 600 .

[0068] like Figure 11 As shown, in contrast, the bacterial-carrying composite fiber membrane (PUL / EC / L. plantarum OD 600 The value shows a gradual increasing trend over time. OD values ​​within 0~12 h... 600 The slow increase in OD value may be attributed to the hydrophobic effect of the outer EC layer, which delays the inward penetration of the culture medium and temporarily hinders cell release. With prolonged culture time, OD... 600 The value increased rapidly within 12–28 h, likely due to the gradual swelling of the PUL after water absorption, forming microchannels that allow probiotics to be released and proliferate rapidly. This is consistent with the survival rate of probiotics in the PUL fiber membrane (92.59%), indicating that the cross-electrospinning process did not significantly damage probiotic activity and that metabolic activity could be rapidly restored and proliferated using nutrients in the culture medium. These results confirm that cross-electrospinning technology achieves sustained-release regulation of probiotics through the PUL / EC composite structure, demonstrating its potential as a stable delivery carrier for probiotics.

[0069] Test Example 11 The thermal stability of the composite fiber membrane prepared in Example 8, loaded with probiotics and free probiotics, was tested. The specific steps are as follows: Free bacteria and electrospun fiber membranes containing encapsulated probiotics were placed separately into sealed test tubes and incubated in a 60°C water bath for 10 min to evaluate the effect of fiber membrane encapsulation on the thermal stability of probiotics. After heat treatment, the samples were transferred to test tubes containing sterile water, and the survival rate was calculated according to the following formula: Survival rate (%) = N / N0 × 100 Where N is the number of viable bacteria after heat treatment (log CFU / mL); N0 is the number of viable bacteria before heat treatment (log CFU / mL).

[0070] The results are as follows Figure 12As shown, the survival rate of probiotics in the composite fiber membrane after heat treatment was 84.53%, significantly better than that of free bacteria. This indicates that the composite fiber membrane also has a significant heat protection effect on probiotics; even at a high temperature of 60℃, more than 80% of the probiotics remained viable. The survival rate of probiotics in the composite fiber membrane after treatment at 60℃ was lower than that of probiotics in the PUL membrane under the same conditions (92.59%). This suggests that the EC component in the composite fiber membrane did not significantly enhance the heat protection ability of the carrier for probiotics; on the contrary, it may have weakened its thermal stability to some extent. This phenomenon may be related to changes in the distribution and loading density of probiotics. During the cross-electrospinning process, the EC and PUL fibers are spun independently, forming a physical separation structure. This results in probiotics being loaded only inside the PUL fibers, and the spatial density of bacteria per unit weight of the fiber membrane is lower than that of the uniaxial PUL fiber membrane, thus weakening the synergistic protective effect between bacteria.

[0071] Test Example 12 The storage stability of the composite fiber membrane prepared in Example 8, loaded with probiotics and free probiotics, was tested. The specific steps are as follows: Free bacterial suspensions and electrospun fiber membranes encapsulating probiotics were stored in sterile, sealed containers at 4℃ and 25℃ for 28 days, respectively. Samples were taken at 0, 1, 4, 7, 14, and 28 days to determine the probiotic survival rate. Specifically, an appropriate weight of the electrospun fiber membrane encapsulating probiotics was weighed, dissolved thoroughly in sterile water, serially diluted, and then plated on MRS agar medium. After incubation at 37℃ for 24 hours, the bacteria were counted, and the results were expressed as CFU / g. For free probiotics, serial dilutions were directly performed followed by plate counting. The probiotic survival rate was calculated using the following formula:

[0072] In the above formula, N is the number of viable bacteria after different storage times (log CFU / mL); N0 is the initial number of viable bacteria (log CFU / mL).

[0073] like Figure 13 As shown, under refrigeration at 4℃, the viable bacterial count decreased from an initial 9.43 log CFU / g to 8.50 log CFU / g after 28 days, with a survival rate of 90.14%. The rate of decrease was relatively rapid in the first 7 days, and then stabilized after 14 days. However, under room temperature conditions at 25℃, the viable bacterial count showed a continuous decreasing trend, decreasing from 9.43 log CFU / g to 7.04 log CFU / g, with a survival rate of 74.66%. The decrease was particularly significant between 21 and 28 days, indicating that higher temperatures accelerated the inactivation process.

[0074] Test Example 13 The composite fiber membrane prepared in Example 8 was subjected to simulated digestion analysis. The specific steps are as follows: S1. Preparation of in vitro digestion simulation solution (1) Preparation of simulated gastric juice (SGF): 1 g of pepsin was added to 100 mL of sterilized 0.85% physiological saline (w / v, pH 2.0) to obtain simulated gastric juice (SGF).

[0075] (2) Preparation of simulated small intestinal fluid (SIF): 300 mg of bile salts were added to 100 mL of physiological saline (0.85%, w / v), the pH of the solution was adjusted to 6.8 and then sterilized; 1 g of trypsin was added to the 100 mL sterile bile salt solution to obtain simulated small intestinal fluid (SIF).

[0076] (3) Preparation of simulated colonic fluid (SCF): Fecal samples were obtained from three volunteers (aged 20-25 years) from Zhengzhou University of Light Industry with normal BMI (18.5 kg / m²). 2 <BMI<23.9 kg / m 2 (Applicants must) have no digestive tract diseases, have not used antibiotics for at least 3 months, and have not taken probiotic products within the past 3 weeks. On the day of the experiment, fresh stool samples were provided, quickly placed in sterile tubes, and immediately transferred to the anaerobic chamber. Three stool samples were mixed with phosphate carbonate buffer at a ratio of 1:3 (w / v) to prepare a stool slurry, which was then filtered through four layers of filter cloth. One mL of the filtered stool slurry and four mL of phosphate carbonate buffer were added to separate anaerobic bottles, the tubes were sealed, and the bottles were incubated at 37°C with precise timing.

[0077] S2, Gastrointestinal Tolerance of Probiotics The bacterial-loaded PUL / EC fiber membrane was subjected to three stages of in vitro digestion simulation: SGF (2 h), SIF (4 h), and SCF (16 h). After each stage, samples were taken, surface moisture was aspirated, and the samples were dissolved in PBS. After complete dissolution, the sample solutions were serially diluted and plated on MRS agar medium. After incubation at 37°C for 24 h, single colony counting was performed. Using the bacterial load of the PUL / EC composite fiber membrane before in vitro digestion simulation as a control, the survival rate of probiotics in the composite fiber membrane at each digestion stage was calculated according to the formula in Test Example 11. In addition, to further verify the survival of the bacterial-loaded composite fiber membrane after treatment with the gastrointestinal simulation solution, fluorescently labeled probiotics were used to prepare a composite fiber membrane containing fluorescently labeled probiotics. The bacterial-loaded composite fiber membranes treated with SGF, SIF, and SCF were dissolved in PBS solution under light-protected conditions, and the release of bacteria in the solution was observed using CLSM.

[0078] Microstructure of S3, PUL / EC composite fiber membrane In vitro digestion simulations were performed using sterile PUL / EC composite fiber membranes in three phases: SGF (2 h), SIF (4 h), and SCF. Samples were taken after each phase, surface moisture was absorbed, and the samples were allowed to air dry in a clean bench before being observed under a scanning electron microscope. Samples for the SCF phase were taken at 4 h, 8 h, 16 h, and 24 h. The SCF digestive fluid used in this experiment was a simulated colonic fluid produced from fermented feces.

[0079] First, the surface morphology of the cross-spun nanofibers was characterized using field emission scanning electron microscopy. For example... Figure 14 As shown, the initial viable count of the undigested bacterial-laden composite fiber membrane was 8.04 log CFU / g, which decreased to 5.24 log CFU / g after 2 h of SGF treatment. This decrease in survival rate was mainly attributed to the combined effects of the highly acidic environment of gastric juice and pepsin. Although EC fibers, with their hydrophobic properties and chemical stability, delayed the erosion of the membrane structure by gastric acid, the hydrophilicity of PUL fibers caused them to partially swell under acidic conditions, resulting in the exposure of some probiotics embedded in PUL fibers to gastric juice and causing cell death. However, compared to a single polysaccharide carrier, the interwoven structure of this composite carrier provided a physical barrier for probiotics, effectively improving their survival rate under gastric environmental conditions. Furthermore, after SIF treatment, the viable count further decreased to 4.92 log CFU / g. The neutral environment of SIF (pH 6.8) and the action of trypsin accelerated the degradation of PUL fibers, releasing more embedded probiotics. However, the hydrophobic barrier of EC fibers maintained partial structural integrity during the intestinal fluid stage. Its low swelling properties reduced the erosion of the membrane by the intestinal fluid, effectively preventing large-scale exposure of bacteria to digestive fluids in a short period, thus mitigating trypsin damage. These results indicate that the sustained protective effect of EC fibers and the phased degradation of PUL fibers achieved a protective effect on probiotics, preventing large-scale exposure of bacteria to digestive fluids in a short period. After SCF treatment, the viable bacterial count remained at 4.88 log CFU / g. It is noteworthy that the neutral environment of colonic fluid (pH 7.4) did not present a direct threat from strong acids or digestive enzymes; therefore, the bacterial-loaded composite fiber membrane did not show a significant loss of probiotic survival rate in the colonic simulated fluid. p >0.05). The composite membrane maintained a survival rate of over 60% after continuous erosion by gastric and intestinal fluids, indicating that the PUL / EC composite fiber membrane can provide good protection for the loaded probiotics throughout the entire gastrointestinal digestion process.

[0080] The results are as follows Figure 15As shown, the survival status of probiotics in the simulated digestion solution after 16 hours of colonic digestion under in vitro conditions using a biofilm-loaded composite fiber membrane is observed. The presence of numerous scattered green fluorescent signal spots in the dark-field fluorescence image indicates that the Rh123-labeled probiotics maintained their intact cell membrane potential and metabolic activity after successive digestion by SGF, SIF, and SCF. The uniform distribution and high density of the fluorescence signal further confirm that the composite fiber membrane effectively protects the probiotics from gastric acid, proteases, and bile salts through the synergistic effect of hydrophobic EC fibers and hydrophilic PUL fibers, ensuring their high survival rate delivery to the colonic environment. Furthermore, only discrete cellular particles were observed in the bright-field image, and their spatial distribution highly overlapped with the fluorescence signal, indicating that the fiber membrane partially dissolved during simulated colonic digestion, releasing probiotics in a free state dispersed in the digestive fluid. This phenomenon is closely related to the staged degradation behavior of the composite fiber membrane. In the upper gastrointestinal tract, EC fibers, with their hydrophobic properties and the interwoven network structure of the composite fiber membrane, delay the erosion of the membrane structure by factors such as gastric acid and proteases, thereby reducing damage to the viability of probiotics. The precise matching of fluorescence signals and cell morphology in the superimposed bright and dark images not only verified the specificity of the fluorescent labeling but also indicated that the probiotics did not undergo significant aggregation or structural damage during delivery, and their morphological integrity and viability were preserved.

[0081] Changes in the state of the carrier during gastrointestinal digestion, such as Figure 16 As shown, after 2 h of SGF treatment, only the network structure formed by EC fibers was observed on the surface of the fibrous membrane, while the surface components of hydrophilic PUL fibers were no longer visible. Combined with previous studies on the activity changes of probiotics during simulated digestion, it is speculated that the SGF stage may have only dissolved the PUL components on the surface of the fibrous membrane, while the internal PUL fibers remained partially retained due to the physical barrier of the hydrophobic EC fiber network. This stratified degradation characteristic allows EC fibers to form a hydrophobic barrier during the gastric juice stage, delaying the direct erosion of residual PUL by gastric acid, thus providing a structural basis for subsequent intestinal delivery. After 4 h of SIF treatment, the morphology of the fibrous membrane was basically consistent with that of the SGF stage, and the EC fiber network remained intact, with no significant structural damage observed. Upon entering the SCF treatment stage, the fibrous membrane exhibited dynamic degradation characteristics driven by the intestinal flora. After 4 h of SCF treatment, a small amount of adhesion began to appear in the EC fiber network, possibly due to the sustained release of deep PUL fibers and their binding with colonic flora secretions. As the reaction progressed to 16 h, the gaps in the EC fiber network were largely filled. After 24 h, the surface of the fiber membrane was completely covered, and local magnification revealed that the adhesions were enriched with a large number of intestinal microorganisms from SCF.

[0082] Test Example 14 The composition of the microbial community in the simulated colon fermentation broth prepared in Test Example 13 was analyzed, and the specific steps are as follows: (1) DNA extraction and purification: Take 1 mL of colonic simulated fermentation broth and extract total genomic DNA according to the Fast DNA® SPIN Kit for Soil instructions. The DNA concentration and purity were determined by NanoDrop 2000 and the DNA integrity was detected by 1% agarose gel electrophoresis.

[0083] (2) PCR amplification and library construction: Primers 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were selected for amplification of the V3-V4 region of the bacterial 16S gene. All PCR mixtures were amplified using 15 µL of Phusion High-Fidelity PCR Master Mix, 0.2 µM primers, and 10 ng of genomic DNA template. The amplification program consisted of a first denaturation at 98 °C for 1 min, followed by 30 cycles at 98 °C (10 s), 50 °C (30 s), and 72 °C (30 s), and finally a hold at 72 °C for 5 min. The amplified products were purified using the AxyPrep DNA gel extraction kit, and sequencing libraries were constructed using The NEBNext Ultra II DNA Library Prep Kit.

[0084] (3) High-throughput sequencing: NovaSeq 6000 was used for sequencing. The sequencing data were quantified by Qubit 2.0 Fluorometer before being sent to the sequencing machine. The raw data were processed by Cutadapt (v1.9.1) to remove primer sequences. The DADA2 algorithm in QIIME2 software was used for quality control, noise reduction and ASV (Amplicon Sequence Variant) clustering. Finally, species annotation was performed based on the SILVA database (v138.1).

[0085] Changes in phylum-level species abundance in each treatment group are as follows: Figure 17As shown, the human gut microbiota is mainly composed of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Actinobacteria have an absolutely dominant relative abundance, accounting for over 99% of the total microbiota along with Firmicutes and Bacteroidetes, a result consistent with the typical characteristics of the gut microbiota in healthy adults. Compared to the control group, the composite fiber membrane promoted an increase in the relative abundance of Bacteroidetes, but a slight decrease in the relative abundance of Actinobacteria and Firmicutes. This may be because the physical presence of the composite fiber membrane indirectly affects the competitive relationships among the microbiota by altering the local microenvironment (such as pH and redox potential). In the group treated with the bacterial-loaded composite fiber membrane, the relative abundance of Firmicutes significantly increased, which is related to the release of *Lactobacillus plantarum* from the bacterial-loaded fiber membrane. Furthermore, in the sugar-free fermentation group (L / P / E / G), no exogenous carbon sources such as glucose were added to the culture medium; the PUL / EC in the composite carrier was used as the sole carbon source for microbial metabolism to verify whether the carrier itself could be utilized by the colonic microbiota and drive metabolic activities. The results showed that the microbial structure in the sugar-free fermentation treatment group with the bacterial-loaded composite fiber membrane underwent significant changes. The relative abundance of Actinobacteria decreased, while Firmicutes and Bacteroidetes became the dominant phyla. Since human feces contain microorganisms capable of decomposing cellulose, the sugar-free conditions may have intensified competition among these microorganisms for the degradation products of the PUL / EC fiber membrane, further reshaping the microbial structure.

[0086] Figure 18 This shows the abundance changes of the bacterial community at the genus level. At the genus level, *Bifidobacterium* (…) Bifidobacterium ) held an absolute advantage; compared to the blank control, the composite fiber membrane promoted the growth of Bacteroides spp. after 24 h of in vitro fermentation. Bacteroides ) and Parabacterium genus ( Parabacteroides The relative abundance of Lactobacillus (L / P / E) decreased, thus reducing the relative abundance of Bifidobacterium. With the introduction of probiotics into the L / P / E composite fiber membrane, the relative abundance of Lactobacillus (L / P / E) increased. Lactiplantibacillus The abundance of *Bifidobacterium* increased to 3.82%, becoming the third most dominant genus. This is consistent with the abundance trend of Firmicutes in the previous section, further indicating that the bacterial-loaded composite fiber membrane can achieve colonic delivery and release of probiotics. Meanwhile, the abundance of *Bifidobacterium* decreased, suggesting that the introduction of probiotics may have a competitive inhibitory effect on *Bifidobacterium*. Furthermore, in the composite fiber membrane group after sugar-free fermentation, the structure of bacterial abundance changed significantly. The abundance of *Bifidobacterium* decreased to 6.09%, while the abundance of *Bacteroides* and *Lactobacillus* increased. Lachnoclostridium ) and Parasatella spp. Parasutterella They become the dominant bacterial group. Studies have shown that Bacteroides are influenced by polysaccharide binding sites and recruit glycosidic hydrolases to work together to break down polysaccharides.

[0087] Test Example 15 SCFAs in the colon fermentation broth of Example 13 were quantitatively analyzed using liquid chromatography-mass spectrometry (LC-MS). The specific method is as follows: (1) Establishment of standard curve: Accurately measure 11 short-chain fatty acid standards and add ultrapure water to prepare standard solutions with concentrations of 0.1 mL / L, 0.5 mL / L, 1.0 mL / L, 2.0 mL / L, 3.2 mL / L, 4.0 mL / L and 5.0 mL / L respectively. Establish the standard curve by using the ratio of standard concentration to internal standard concentration as the abscissa and the ratio of standard peak area to internal standard peak area as the ordinate.

[0088] (2) Metabolite extraction: Take 1 mL of fermentation broth, add 1 mL of 80% methanol aqueous solution, mix well, centrifuge at 12000 rpm for 10 min at 4 ℃, take 50 μL of supernatant, add 150 μL of derivatization reagent, derivatize at 40 ℃ for 40 min, and then dilute the derivatized sample with 80% methanol aqueous solution; take 95 μL of supernatant, add 5 μL of mixed internal standard 80% methanol aqueous solution, mix well, and then perform LC-MS analysis.

[0089] (3) Chromatographic and mass spectrometry conditions: Chromatographic separation was performed using a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm), with the column temperature maintained at 40 ℃. The mobile phase consisted of 10 mM ammonium acetate aqueous solution (mobile phase A) and an acetonitrile-isopropanol mixture (1:1, mobile phase B), with an injection volume of 2 μL and a flow rate of 0.30 mL / min. Mass spectrometry was performed in multiple reaction monitoring (MRM) negative ion mode, with the following parameters set: ion spray voltage (-4500 V), sheath gas pressure of 35 psi, ion source temperature of 550 ℃, auxiliary gas pressure of 50 psi, and collision gas pressure of 55 psi.

[0090] Figure 19This study shows the changes in the content of major SCFAs (acetic acid, propionic acid, and butyric acid) such as acetic acid, propionic acid, and butyric acid in the composite fiber membrane during in vitro fermentation. It can be seen that the fatty acid composition of each group is dominated by acetic acid, propionic acid, and butyric acid. In particular, the acetic acid content was highest in the control group, the P / E composite fiber membrane group, and the L / P / E composite fiber membrane group, which is consistent with the highest abundance of Bifidobacterium in the previous section's bacterial community structure analysis. Bifidobacterium mainly produces acetic acid through metabolism, and studies have shown that resistant starch can promote the rapid proliferation of Bifidobacterium to produce acetic acid. Compared with the control group and the P / E composite fiber membrane group, the total SCFAs in the L / P / E composite fiber membrane treatment group increased to 3.66 mg / mL, and the butyric acid content increased by 38.9% compared with the control group. Butyric acid is the main SCFA produced by Firmicutes metabolism, while acetate and propionate are the main SCFAs produced by Bacteroidetes metabolism. Therefore, the increased butyric acid content in the L / P / E composite fiber membrane group is related to the increased abundance of Firmicutes in this group. Furthermore, the butyric acid concentration in the L / P / EG group significantly increased to 0.47 mg / mL. This may be because, under carbon-free conditions, certain bacterial groups (Bacteroides) can obtain carbon sources for proliferation by degrading the fibrous membrane, further confirming the bacterial responsive degradation characteristics of the composite carrier.

[0091] Test Example 16 The electrospun films prepared in Examples 1 and 4 were compared with the commercial polyethylene (PE) film of Comparative Example 9 in a strawberry preservation experiment. The specific steps are as follows: S1. Fresh strawberries of uniform quality were purchased from a local supermarket for preservation research. After removing damaged and unripe fruits, strawberries of similar size and maturity were selected for storage experiments. Whole strawberries were immersed in a 0.02% sodium hypochlorite solution for 5 minutes, followed by rinsing with distilled water to remove residual solution. All strawberries were then air-dried and wrapped in P / E-CNF, N@P / E-CNF, and commercially available polyethylene (PE) film, respectively. Unwrapped strawberries served as a blank control group. All packaged samples were stored at room temperature (25℃), and indicators were measured at predetermined time intervals.

[0092] S2. Quantitative analysis was conducted on the apparent changes of strawberries during storage by evaluating their color and degree of decay. Color parameters during strawberry storage were measured using an SC-80C fully automatic colorimeter. Three different surface sites were selected for measurement on each sample, and the lightness value was determined. L * Red-green value a * Yellow-blue value b * and calculate the total color difference Δ according to formula (3). E The degree of decay was assessed using existing methods.

[0093]

[0094] In the formula:L 0*, a 0* and b 0* represents the initial colorimetric parameters of the strawberry sample at day 0; L *, a *and b *These represent the color parameters of strawberry samples after storage for different periods.

[0095] S3. Fruit firmness was determined using a texture analyzer with an 8 mm probe and a constant testing rate of 20 mm / min. Results are expressed in Newtons (N). The initial and post-storage weights of the strawberries were recorded, and the weight loss rate was calculated using the following formula:

[0096] In the formula: W 0 represents the mass of strawberries at 0 days; Wt represents the mass of strawberries at different storage times t.

[0097] S4. The total soluble solids content was determined using an Abbe refractometer. The vitamin C content was determined by titration. 5g of sample was added to 20 mL of 70% ethanol and homogenized. After centrifugation at 8000 rpm for 10 min, the supernatant was collected. 0.2 mL of the supernatant was taken and added sequentially to 1.8 mL of 50 g / L trichloroacetic acid (TCA), 1 mL of ethanol, 0.5 mL of 0.4% phosphoric acid-ethanol solution, 1 mL of 5 g / L BP-ethanol solution, and 0.5 mL of 0.3 g / L ferric chloride-ethanol solution. The mixture was incubated in the dark for 30 min, and the absorbance was measured at 534 nm.

[0098] The quality changes of strawberries during storage are manifested by weight loss and color difference. ΔE ) was characterized. For example, Figure 20 As shown, the overall color of strawberries ΔE On day 7, the strawberry color in the control group reached 21.5, with a significant color difference, indicating that the strawberries had already severely rotted and turned brown. In the PE and 0% N@P / E-CNF experimental groups, on day 3... ΔE It rises rapidly, and by day 7, ΔE reaches over 10. 7% NE / P CEF within 7 days of storage. ΔE Always keep at a low level ( ΔE <10), indicating that 7% N@P / E-CNF effectively slows down strawberry spoilage and maintains freshness, with 7% N@P / E-CNF showing the best preservation effect. Since the antibacterial film effectively inhibits microbial infection, thereby reducing strawberry spoilage and weight loss, NE / P CEF exhibits good food preservation potential and can be used as an active packaging material.

[0099] Test Example 17 The specific steps for testing chicken-wrapped chicken in Examples 1, 4, 7 and Comparative Example 9 are as follows: S1. All operating utensils, including knives and cutting boards, that came into contact with the samples were thoroughly sterilized with 75% alcohol to ensure hygienic conditions during the experiment and to avoid interference from exogenous microbial contamination. Subsequently, the chicken, repeatedly rinsed with sterile water, was cut into uniformly sized pieces (3×3×1 cm) to ensure consistent sample size and facilitate subsequent measurements. Five treatment groups were set up. Unwrapped chicken served as a blank control group. Four different films (all 8×8 cm in size) were used to wrap the chicken pieces: PE film from Comparative Example 9, P / E-CNF from Example 1, N@P / E-CNF from Example 4, and N@OEO-P / E-CNF from Example 7. All treated samples were stored at 4 ℃ for 7 days. During this period, samples were taken daily at regular intervals, and relevant quality indicators were measured to systematically evaluate the effects of different treatments on the chicken.

[0100] S2. Determination of TVC in chicken meat according to GB 4789.2-2022. Weigh the chicken meat sample, place it in a homogenizer bag containing 100 mL of physiological saline, and shake for 2 min. After the homogenate is serially diluted with physiological saline at a ratio of 1:9, perform the coating and counting.

[0101] Depend on Figure 22 It can be seen that initially (day 0), the total bacterial count of all samples was less than 4 log₂. 10 The CFU / g level met the standards for Grade 1 fresh meat. During storage, the total bacterial count in the control group, PE group, and P / E-CNF treatment group all increased significantly with prolonged storage time. The control group reached 6.13 log [value missing] on day 2. 10 CFU / g indicates the group has entered a deteriorated state; the PE group reached 6.15 log on day 3. 10 CFU / g, both were also spoiled; by day 5, the total bacterial counts of the two increased to 8.17 and 6.79 log, respectively. 10 CFU / g. In the PUL / EC treatment group, the increase in total bacterial count was relatively slower due to the oxygen barrier effect of the packaging, but it still reached 5.78 log on day 5. 10 CFU / g, close to the deterioration standard. In contrast, the total bacterial count growth in the N@P / E-CNF and N@OEO-P / E-CNF cross-electrospun membrane treatment groups was relatively slow. The NP / E-CNF group showed slow growth in the first 4 days, attributed to the physical barrier of the membrane material and the antibacterial effect of Nisin; by day 6, it had increased to 6.53 log. 10 CFU / g, entering the deterioration range. The total colony count in the N@OEO-PUL / EC treatment group was lower than that in the N-PUL / EC group throughout the entire storage period, and the difference was significant.p The value <0.05 indicates that the combination of Nisin and OEO has a synergistic antibacterial effect. The total bacterial count in this treatment group did not exceed 6 log on day 7. 10 CFU / g, compared with the control group at the same time (9.92 log). 10 Compared to CFU / g, it significantly decreased by 3.37 log. 10 CFU / g ( p <0.05).

[0102] exist Figure 22 In the initial stage of storage, all samples in each treatment group were uniformly pale white, with glossy muscle fiber surfaces and a compact tissue structure, indicating that all samples were fresh at the start of the experiment. As storage time increased, the chicken samples in the control group and the P / E-CNF group gradually changed from pale white to yellowish-brown, with a looser tissue structure, accompanied by significant moisture loss and surface wrinkling, exhibiting typical spoilage characteristics. In contrast, while the chicken samples in the PE group maintained a relatively pale white appearance during storage, their tissue structure also gradually became looser, a trend consistent with the aforementioned color and texture characteristics. Due to the relatively limited antioxidant capacity of Nisin, the chicken in the N@P / E-CNF group still showed relatively obvious oxidative discoloration. In contrast, N@OEO-P / E-CNF contained OEO with good antioxidant activity, effectively slowing down the oxidation process; therefore, the muscle fiber structure of this group remained relatively intact, and the surface color was brighter. In conclusion, N@OEO-P / E-CNF has a good preservation effect on chicken.

[0103] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A cross-spun composite nanofiber membrane, characterized in that, The composite nanofiber membrane is formed by cross-electrospinning of food-grade polymer ethyl cellulose and pullulan into an interwoven stacked network structure. The ethyl cellulose fibers provide hydrophobic support, and the pullulan fibers enhance mechanical properties. The combination of the two can obtain a composite nanofiber with adjustable hydrophilicity and hydrophobicity and good mechanical strength. The composite nanofiber membrane is loaded with active ingredients, which are selected from one or more of Nisin, oregano essential oil, and probiotics.

2. The cross-electrospun composite nanofiber membrane according to claim 1, characterized in that, The active ingredient is Nisin, and the loading of Nisin in pullulan polysaccharide fiber is 3%~7%.

3. The cross-electrospun composite nanofiber membrane according to claim 1, characterized in that, The active ingredient is oregano essential oil, which is loaded in pullulan polysaccharide fiber in the form of β-cyclodextrin inclusion complex. The volume fraction of oregano essential oil in the spinning solution is 1.59%~5.14%.

4. The cross-electrospun composite nanofiber membrane according to claim 1, characterized in that, The active ingredient is probiotics, which are encapsulated within pullulan polysaccharide fibers; the probiotics are Lactobacillus plantarum.

5. A method for preparing a cross-electrospun composite nanofiber membrane as described in any one of claims 1-4, characterized in that, Includes the following steps: S1: Prepare ethyl cellulose spinning solution and pullulan polysaccharide spinning solution; wherein, the solvent of ethyl cellulose spinning solution is anhydrous ethanol or glacial acetic acid, and the solvent of pullulan polysaccharide spinning solution is deionized water; as needed, add Nisin, β-cyclodextrin-encapsulated oregano essential oil and / or probiotics to pullulan polysaccharide spinning solution. S2: Using a cross electrospinning device, ethyl cellulose spinning solution and pullulan polysaccharide spinning solution are loaded into two syringes respectively, which are connected to the positive terminal of a high-voltage power supply. A roller is used as a collector, and electrospinning is performed simultaneously. Under the action of electric field force, the two jets are alternately covered on the surface of the collector to form a composite fiber membrane with an interpenetrating network structure.

6. The preparation method according to claim 5, characterized in that, In step S2, the propulsion flow rate of the ethyl cellulose spinning solution is 0.15~1.0 mL / h, the propulsion flow rate of the pullulan polysaccharide spinning solution is 0.15~0.4 mL / h, the spinning voltage is 15~25 kV, and the receiving distance is 10~15 cm.

7. The preparation method according to claim 5, characterized in that, When loading oregano essential oil, the preparation method of the pullulan polysaccharide spinning solution is as follows: first, β-cyclodextrin is dissolved in water, then an ethanol solution of oregano essential oil is added for inclusion, and then Nisin and pullulan polysaccharide are added and stirred to dissolve.

8. The preparation method according to claim 5, characterized in that, When probiotics are loaded, the pullulan polysaccharide spinning solution is mixed with the probiotic solution just before spinning to ensure that the probiotics are evenly dispersed in the spinning solution.

9. The application of a cross-electrospinned composite nanofiber membrane as described in any one of claims 1-4 in the preparation of food active packaging materials or probiotic colon-targeted delivery carriers; wherein the composite nanofiber membrane is used to inhibit Gram-positive bacteria or to synergistically inhibit Gram-negative bacteria.

10. The application according to claim 9, characterized in that, The composite nanofiber membrane is used to improve the survival rate of probiotics under heat treatment, room temperature storage and simulated gastrointestinal fluid environment, and to realize the release of probiotics in the colon.

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