Ultrasonic preparation method of polysaccharide fruit and vegetable fresh-keeping film loaded with nano-essential oil emulsion and application thereof

Abstract

The application discloses an ultrasonic preparation method and application of a polysaccharide fruit and vegetable fresh-keeping film loaded with nano essential oil emulsion, and relates to the technical field of food active packaging material preparation. The application takes pullulan as a matrix, and prepares a fruit and vegetable fresh-keeping film by loading soybean protein isolate / turmeric essential oil nano-emulsion and soybean protein isolate / orange essential oil nano-emulsion prepared by ultrasonic regulation control, and studies the mechanical properties, barrier properties, structural characteristics and antibacterial activity of the emulsion prepared by ultrasonic compared with the emulsion prepared without ultrasonic. Meanwhile, the application takes strawberries as a research object, studies the influence of the fresh-keeping film on the fruit fresh-keeping effect, and can meet the requirements of food active packaging, is beneficial to the development of green and environment-friendly food packaging materials, and has a potential application prospect.

Description

Technical Field

[0001] This invention relates to the field of food packaging material preparation technology, specifically to a soy protein isolate / turmeric essential oil nanoemulsion (Sp / CEO / US) and a soy protein isolate / orange essential oil nanoemulsion (Sp / OEO / US) prepared by adding ultrasound (US) with pullulan (Pul) as a matrix, and to the preparation of P / CEO / US and P / OEO / US fruit and vegetable preservation films by casting method. Background Technology

[0002] In recent years, the widespread use and non-biodegradability of petroleum-based polymer packaging materials have posed a serious threat to the environment. Edible composite films made from natural polymers, possessing biodegradability, have attracted widespread attention from researchers. Pullulan, an extracellular water-soluble mucopolysaccharide similar to dextran and xanthan gum, produced by the fermentation of *Brachystomata*, exhibits excellent film-forming ability among renewable biopolymer sources. Compared to other polysaccharide and protein-based composite films, pullulan-based composite films are colorless, odorless, and thermally stable, and can effectively delay the penetration of oil and oxygen. Therefore, pullulan-based composite films can effectively delay the oxidative rancidity of food.

[0003] Active films are edible composite films containing natural antibacterial substances (such as essential oils). They can be used to extend the shelf life of different food systems (such as fruits, vegetables, and meats) and reduce the potential risks posed by foodborne pathogens (Preparation and characterization of clove essential oil loaded nanoemulsion and pickering emulsion activated pullulan-gelatin based edible film. International Journal of Biological Macromolecules, 2021, 181, 528-539). Turmeric and orange essential oils mainly consist of curcuminoids, curcuminones, and limonene, exhibiting excellent antibacterial activity against many pathogens associated with foodborne diseases. However, due to the high volatility and strong hydrophobicity of essential oils, free essential oils are difficult to directly integrate into the composite film matrix, and the rapid loss of active compounds in the essential oils limits the active function of the composite film. Currently, an effective measure to address these issues is to encapsulate the essential oils in an oil-in-water emulsion to improve their compatibility with the composite film matrix and increase the retention rate of the essential oils.

[0004] The particle size and stability of emulsions have a significant impact on the performance of composite membranes. Nanoemulsions, referring to emulsions with droplet sizes of 20-200 nm, exhibit higher resistance to aggregation and gravity separation than traditional emulsions. Currently, to adapt to a wide range of industrial applications, the methods for preparing nanoemulsions must emphasize high energy. High-intensity ultrasound can homogenize dispersion systems and reduce the size of emulsion droplets. Generally, ultrasonic devices generate high-intensity forces through the acoustic cavitation effect, defined as the formation, expansion, and implosion collapse of cavitation bubbles, inducing strong shock waves and shear forces in liquid media, leading to droplet disintegration. Research on the ultrasonic preparation of nanoemulsions with small droplet sizes and higher kinetic stability has been reported. However, a literature review revealed no reports on the ultrasonic preparation of soy protein isolate / turmeric oil nanoemulsions and soy protein isolate / orange oil nanoemulsions, and their use in preparing pullulan-based active packaging films.

[0005] In this invention, pullulan polysaccharide is used as the film-forming matrix, and a nanoemulsion is prepared by loading ultrasound. The aim is to further enhance the mechanical properties, barrier properties, structural characteristics and antibacterial activity of the composite film by improving the physicochemical properties of the emulsion, thereby meeting the requirements of food preservation. Summary of the Invention

[0006] To address the aforementioned issues, this invention aims to provide a method for preparing fruit and vegetable preservation films using pullulan (Pul) as a matrix and loading ultrasonically controlled soybean protein isolate / turmeric essential oil nanoemulsions (Sp / CEO / US) and soybean protein isolate / orange essential oil nanoemulsions (Sp / OEO / US). The invention also investigates the effects of ultrasonically prepared emulsions compared to non-ultrasonic prepared emulsions on the mechanical properties, barrier properties, structural characteristics, antibacterial activity, and preservation effect of the composite films.

[0007] The ultrasonic preparation method of the polysaccharide and vegetable preservation film loaded with nano-essential oil emulsion of the present invention is carried out according to the following steps:

[0008] (1) Deionized water, soy protein isolate, and turmeric oil were stirred at room temperature for 30 minutes until the soy protein isolate was completely dissolved. The solution was then homogenized at 12,000 rpm for 4 minutes to prepare a crude emulsion of soy protein isolate / turmeric oil.

[0009] (2) The crude emulsion of soy protein isolate / turmeric essential oil in step (1) is subjected to ultrasonic treatment to obtain soy protein isolate / turmeric essential oil nanoemulsion prepared by ultrasound.

[0010] (3) Stir pullulan at room temperature for 1 hour until completely dissolved to prepare a polysaccharide solution.

[0011] (4) Add a certain mass concentration of glycerol to the polysaccharide solution in step (3), place the blend in a water bath constant temperature magnetic stirrer, stir for 30 min at 50℃ to obtain pullulan polysaccharide film-forming solution.

[0012] (5) Add a certain proportion of the soy protein isolate / turmeric essential oil nanoemulsion prepared by ultrasound in step (2) to the film-forming solution in step (4), and stir the mixture at room temperature for 30 min to obtain the film-forming solution.

[0013] (6) Take the film-forming solution in step (5) and form a film by casting. Dry it at 60°C for 8 hours. After the film is formed, take it out and cool it to room temperature to obtain a polysaccharide vegetable and fruit preservation film loaded with turmeric essential oil nanoemulsion.

[0014] In step (1), the ratio of deionized water, soy protein isolate, and turmeric essential oil is (89-85):(1-5):10, with the preferred ratio being 85:5:10.

[0015] In step (2), the ultrasonic power is 1200W / L, the intermittent ratio is 3s / 3s, and the ultrasonic treatment time is 2.5-10min, preferably 10min.

[0016] The mass concentration of pullulan polysaccharide solution in step (3) is 4 g / 100 mL.

[0017] In step (4), the amount of glycerol added is 15% of the total mass of pullulan.

[0018] In step (5), the amount of soybean protein isolate / turmeric oil nanoemulsion prepared by ultrasound is 4%-8% of the volume ratio of pullulan polysaccharide film-forming solution, preferably 6%.

[0019] The ultrasonic preparation method of the polysaccharide and vegetable preservation film loaded with nano-essential oil emulsion described in this invention can be carried out according to the following steps:

[0020] (1) Stir deionized water, soy protein isolate, and orange essential oil at room temperature for 30 minutes until the soy protein isolate is completely dissolved. Then homogenize the solution at 12,000 rpm for 4 minutes to prepare a crude emulsion of soy protein isolate / orange essential oil.

[0021] (2) The crude emulsion of soy protein isolate / orange essential oil in step (1) is subjected to ultrasonic treatment to obtain soy protein isolate / orange essential oil nanoemulsion prepared by ultrasound.

[0022] (3) Stir pullulan at room temperature for 1 hour until completely dissolved to prepare a polysaccharide solution.

[0023] (4) Add a certain mass concentration of glycerol to the polysaccharide solution in step (3), place the blend in a water bath constant temperature magnetic stirrer, stir for 30 min at 50℃ to obtain pullulan polysaccharide film-forming solution.

[0024] (5) Add a certain proportion of the soybean protein isolate / orange essential oil nanoemulsion prepared by ultrasound in step (2) to the film-forming solution in step (4), and stir the mixture at room temperature for 30 min to obtain the film-forming solution.

[0025] (6) Take the film-forming solution in step (5) and form a film by casting. Dry it at 60°C for 8 hours. After the film is formed, take it out and cool it to room temperature to obtain a polysaccharide vegetable and fruit preservation film loaded with orange essential oil nanoemulsion.

[0026] In step (1), the ratio of deionized water, soy protein isolate, and orange essential oil is (89-85):(1-5):10, with the preferred ratio being 85:5:10.

[0027] In step (2), the ultrasonic power is 1200W / L, the intermittent ratio is 3s / 3s, and the ultrasonic treatment time is 2.5-10min, preferably 10min.

[0028] The mass concentration of pullulan polysaccharide solution in step (3) is 4 g / 100 mL.

[0029] In step (4), the amount of glycerol added is 15% of the total mass of pullulan.

[0030] In step (5), the amount of soy protein isolate / orange essential oil nanoemulsion added during ultrasonic preparation is 4%-8% of the volume ratio of pullulan polysaccharide film-forming solution, with a preferred addition amount of 6%.

[0031] The above-mentioned polysaccharide and vegetable preservation film loaded with nano-essential oil emulsion can be used for the preservation packaging of fruits and vegetables such as strawberries.

[0032] The beneficial effects of this invention are as follows:

[0033] (1) The ultrasonic treatment technology used in the preparation of the composite film in this invention is a physical method. Ultrasonic waves are a green and environmentally friendly physical processing method that is widely used in the food industry and is a new physical processing method for preparing food packaging materials.

[0034] (2) The particle sizes of the ultrasonically prepared soy protein isolate / turmeric oil nanoemulsion and the ultrasonically prepared soy protein isolate / orange oil nanoemulsion were reduced from 214.6 nm and 222.5 nm to 116.3 nm and 120.6 nm, respectively, and both showed better dynamic storage stability during the 60-day storage period.

[0035] (3) When Sp / CEO / US nanoemulsion and Sp / OEO / US nanoemulsion prepared by ultrasound were added to the composite membrane, the tensile strength of the composite membrane increased by 120.9% and 108.2%, respectively; the elongation at break increased by 99.2% and 122.3%, respectively; the water vapor permeability decreased by 212.5% ​​and 179.2%, respectively; and the essential oil loss rate decreased by 24.3% and 20.5%, respectively.

[0036] (4) In the preservation of strawberries, P / CEO / US and P / OEO / US composite films can effectively inhibit the spoilage of strawberries and effectively delay the loss of moisture. After seven days of storage, the weight loss rate was reduced by 24.9% and 26.8%, respectively. P / CEO / US and P / OEO / US composite films are green and edible food packaging materials with potential application prospects in food packaging.

[0037] (5) This invention provides a theoretical basis for the application of ultrasonic technology in the preparation of composite films. The resulting composite film can meet the requirements of active food packaging, which is conducive to the development of green and environmentally friendly food packaging materials. It has important academic value and application prospects. Attached Figure Description

[0038] Figure 1 Particle size histograms of Sp / CEO crude emulsions prepared with different ratios of deionized water, soy protein, and turmeric oil, and Sp / OEO crude emulsions prepared with different ratios of deionized water, soy protein, and orange oil.

[0039] Figure 2 Bar graphs showing the mechanical properties of composite membranes prepared with different amounts of soy protein isolate / turmeric oil emulsion (Sp / CEO / US) added using ultrasonic methods.

[0040] Figure 3 The effects of different ultrasonic times on the particle size, zeta potential, and PDI of soy protein isolate / turmeric oil emulsion (Sp / CEO).

[0041] Figure 4 The effects of different ultrasonic times on the particle size, zeta potential, and PDI of soy protein isolate / orange essential oil emulsion (Sp / OEO).

[0042] Figure 5 The morphology of (A) soy protein isolate / turmeric oil emulsion (Sp / CEO) and (B) soy protein isolate / orange oil emulsion (Sp / OEO) prepared under laser confocal microscopy with different ultrasonic times.

[0043] Figure 6 Storage stability results of soy protein isolate / turmeric oil emulsions (Sp / CEO) prepared for different ultrasonic times.

[0044] Figure 7 Storage stability results of soy protein isolate / orange oil emulsion (Sp / OEO) prepared for different ultrasonic times.

[0045] Figure 8 The graph shows the thickness of different composite membranes. From left to right, the membranes are: pullulan composite membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric oil emulsion (P / CEO), pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / turmeric oil emulsion (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange oil emulsion (P / OEO), and pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / orange oil emulsion (P / OEO / US).

[0046] Figure 9 The figures show bar charts of moisture content and water vapor transmission rate for different composite membranes. From left to right, the membranes are: pullulan composite membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric oil emulsion (P / CEO), pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / turmeric oil emulsion (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange oil emulsion (P / OEO), and pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / orange oil emulsion (P / OEO / US).

[0047] Figure 10 The bar chart shows the mechanical properties of different composite membranes. From left to right, the membranes are: pullulan composite membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric oil emulsion (P / CEO), pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / turmeric oil emulsion (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange oil emulsion (P / OEO), and pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / orange oil emulsion (P / OEO / US).

[0048] Figure 11The figures show the Fourier transform attenuated total reflectance infrared spectra of different composite membranes. From top to bottom, the membranes are: pullulan composite membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric oil emulsion (P / CEO), pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / turmeric oil emulsion (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange oil emulsion (P / OEO), and pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / orange oil emulsion (P / OEO / US).

[0049] Figure 12 The figures show the XRD diffraction patterns of different composite membranes. From top to bottom, they are: pullulan membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric oil emulsion (P / CEO), pullulan composite membrane loaded with soy protein isolate / turmeric oil emulsion prepared by ultrasound (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange oil emulsion (P / OEO), and pullulan composite membrane loaded with soy protein isolate / orange oil emulsion prepared by ultrasound (P / OEO / US).

[0050] Figure 13 Thermogravimetric analyses (TGAs) of different composite membranes are shown. From top to bottom, the membranes are: pullulan composite membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric oil emulsion (P / CEO), pullulan composite membrane loaded with soy protein isolate / turmeric oil emulsion prepared by ultrasound (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange oil emulsion (P / OEO), and pullulan composite membrane loaded with soy protein isolate / orange oil emulsion prepared by ultrasound (P / OEO / US).

[0051] Figure 14 The figures show the first derivative curves of the thermogravimetric analysis (TGA) of different composite membranes. From top to bottom, the membranes are: pullulan composite membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric oil emulsion (P / CEO), pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / turmeric oil emulsion (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange oil emulsion (P / OEO), and pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / orange oil emulsion (P / OEO / US).

[0052] Figure 15The figures represent the retention times of essential oils in different composite membranes. From top to bottom, the membranes are: pullulan composite membrane loaded with soy protein isolate / turmeric essential oil emulsion (P / CEO), pullulan composite membrane loaded with ultrasound-prepared soy protein isolate / turmeric essential oil emulsion (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange essential oil emulsion (P / OEO), and pullulan composite membrane loaded with ultrasound-prepared soy protein isolate / orange essential oil emulsion (P / OEO / US).

[0053] Figure 16 The antibacterial effects of different composite membranes on *Escherichia coli* (as of time) are shown in the figure from top to bottom: pullulan membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric essential oil emulsion (P / CEO), pullulan composite membrane loaded with soy protein isolate / turmeric essential oil emulsion prepared by ultrasound (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange essential oil emulsion (P / OEO), and pullulan composite membrane loaded with soy protein isolate / orange essential oil emulsion prepared by ultrasound (P / OEO / US).

[0054] Figure 17 The antibacterial effects of different composite membranes on Staphylococcus aureus (as of time) are shown in the figure. From top to bottom, the membranes are: pullulan composite membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric essential oil emulsion (P / CEO), pullulan composite membrane loaded with soy protein isolate / turmeric essential oil emulsion prepared by ultrasound (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange essential oil emulsion (P / OEO), and pullulan composite membrane loaded with soy protein isolate / orange essential oil emulsion prepared by ultrasound (P / OEO / US).

[0055] Figure 18 This diagram illustrates the preservation effects of different composite film coverings on strawberries. From left to right, the diagram shows: no film covering (Control), pullulan film covering (Pul), pullulan composite film covering with soy protein isolate / turmeric oil emulsion (P / CEO), pullulan composite film covering with soy protein isolate / turmeric oil emulsion prepared by ultrasound (P / CEO / US), pullulan composite film covering with soy protein isolate / orange oil emulsion (P / OEO) front view, pullulan composite film covering with soy protein isolate / orange oil emulsion (P / OEO) bottom view, and pullulan composite film covering with soy protein isolate / orange oil emulsion prepared by ultrasound (P / OEO / US).

[0056] Figure 19The graph shows the weight loss rate of strawberries under different composite film coverings during storage. From top to bottom, the graph represents: no film covering (Control), pullulan film covering (Pul), pullulan composite film covering with soy protein isolate / turmeric oil emulsion (P / CEO), pullulan composite film covering with soy protein isolate / turmeric oil emulsion prepared by ultrasound (P / CEO / US), pullulan composite film covering with soy protein isolate / orange oil emulsion (P / OEO), and pullulan composite film covering with soy protein isolate / orange oil emulsion prepared by ultrasound (P / OEO / US).

[0057] Figure 20 The images show samples of different composite membranes. From left to right, they are: pullulan membrane (Pul), pullulan composite membrane loaded with soy protein isolate / turmeric essential oil emulsion (P / CEO), pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / turmeric essential oil emulsion (P / CEO / US), pullulan composite membrane loaded with soy protein isolate / orange essential oil emulsion (P / OEO), and pullulan composite membrane loaded with ultrasonically prepared soy protein isolate / orange essential oil emulsion (P / OEO / US). Detailed Implementation

[0058] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is described in further detail below with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0059] In the following embodiments of the present invention, the Chinese names, full English names, or abbreviations of the following terms may be used. Regardless of whether the Chinese name, full English name, or abbreviation is used, it represents a compound, drug, or reagent. See Table 1 for details:

[0060] Table 1. English-Chinese Abbreviation Table

[0061]

[0062]

[0063] Experimental materials:

[0064] Pullulan was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Glycerin (99% purity), agar powder, and sodium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd. Tryptone (LP0042) and yeast powder (LP0021) were purchased from Beijing Lamborghini Biotechnology Co., Ltd. Soy protein isolate was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Turmeric essential oil and orange essential oil were purchased from Jiangxi Hualong Plant Fragrance Co., Ltd. Escherichia coli CICC 10389 and Staphylococcus aureus CICC 10201 were both purchased from the China Industrial Microbial Culture Collection Center.

[0065] The particle size determination method for the emulsion prepared in this embodiment of the invention is as follows: the particle size of the emulsion is measured using a laser particle size analyzer (Nano-ZS90, Malvern Instruments Ltd, UK). The emulsion is diluted 1000 times with deionized water to prevent multiple scattering effects, and three tests are performed at 25°C.

[0066] The mechanical property testing method for the composite membrane material prepared in this embodiment of the invention is as follows: the membrane is cut into strips of 20mm×60mm, and its tensile strength (TS) and elongation at break (EAB) are measured using a physical property instrument. The testing speed is 1mm / s and the clamping distance is 30mm.

[0067] Tensile strength calculation formula: TS=F / (d×W)

[0068] Where: TS—tensile strength (N / mm) 2 F—the maximum tension (N) experienced when the membrane breaks; d—the membrane thickness (mm); W—the membrane width (mm).

[0069] Elongation at break formula: EAB=(L-L0)×100 / L0

[0070] Where: EAB—elongation at break (%); L—distance between the markings when the membrane breaks (mm); L0—original distance between the markings on the membrane (mm).

[0071] Example 1: Optimization of the ratio of deionized water, soy protein isolate, and turmeric essential oil in Sp / CEO crude emulsion

[0072] Deionized water, soy protein isolate, and turmeric oil were stirred at room temperature for 30 minutes until the soy protein isolate was completely dissolved. The solution was then homogenized at 12,000 rpm for 4 minutes to prepare a crude Sp / CEO emulsion. The ratios of deionized water, soy protein isolate, and turmeric oil were 89:1:10, 88:2:10, 87:3:10, 86:4:10, and 85:5:10.

[0073] The ratio of deionized water, soy protein isolate, and turmeric essential oil in Sp / CEO crude emulsion was optimized using particle size as an indicator. Figure 1 As shown, the particle size of the emulsion varies significantly with different ratios. The crude Sp / CEO emulsion prepared at a ratio of 85:5:10 has the smallest particle size, at 214.6 nm. Therefore, a crude Sp / CEO emulsion prepared at a ratio of 85:5:10 will be used for further research.

[0074] Example 2: Optimization of the ratio of deionized water, soy protein isolate, and orange essential oil in Sp / OEO crude emulsion

[0075] The method for optimizing the ratio of deionized water, soy protein isolate, and orange essential oil in Sp / OEO crude emulsion is the same as in Example 1, except that turmeric essential oil is replaced with orange essential oil, while other methods remain unchanged.

[0076] The ratio of deionized water, soy protein isolate, and orange essential oil in Sp / OEO crude emulsion was optimized using particle size as an indicator. Figure 1 As shown, the particle size of the emulsions varied significantly depending on the ratio. The crude Sp / OEO emulsion prepared at a ratio of 85:5:10 had the smallest particle size, at 222.5 nm. Therefore, a crude Sp / OEO emulsion at a ratio of 85:5:10 was prepared for further research.

[0077] Example 3: Optimization of Sp / CEO / US nanoemulsion addition amount in P / CEO / US composite membrane

[0078] (1) 85 mL of aqueous phase, 5 g of soy protein isolate, and 10 mL of turmeric essential oil were stirred at room temperature for 30 min until the soy protein isolate was completely dissolved. The solution was then homogenized at 12000 rpm for 4 min to prepare a crude Sp / CEO emulsion. The crude Sp / CEO emulsion was then ultrasonically treated for 10 min to prepare a Sp / CEO / US nanoemulsion. The ultrasonic power was 1200 W / L, and the interval was 3 s / 3 s.

[0079] (2) Stir 4g of pullulan polysaccharide at room temperature for 1 hour until it is completely dissolved, and prepare a polysaccharide solution with a concentration of 4g / 100mL. Add glycerol at a mass ratio of 15% of the total mass of pullulan polysaccharide and mix with the above polysaccharide solution at 50℃ for 30 minutes to obtain a uniform pullulan polysaccharide film-forming solution.

[0080] (3) Take 4, 6, and 8 mL of the prepared Sp / CEO / US nanoemulsion and mix them in 100 mL of pullulan polysaccharide film-forming solution. Stir and mix at room temperature for 30 min to obtain a uniform P / CEO / US film-forming solution. Then take 15 mL of the film-forming solution in a petri dish with a diameter of 9 cm and dry it at 50 °C for 6 h to obtain different P / CEO / US composite films.

[0081] The amount of Sp / CEO / US nanoemulsion added was optimized using mechanical parameters such as tensile strength (TS) and elongation at break (EAB). Figure 2 As shown, the amount of Sp / CEO / US nanoemulsion added has a significant impact on the mechanical properties of the composite membrane. The composite membranes prepared with 4, 6, and 8 mL of nanoemulsion added have TS and EAB values ​​of 6.41 N / mm². 2 9.30 N / mm 2 7.70 N / mm 2 The percentages were 300.24%, 332.88%, and 270.86%. The composite membrane exhibited optimal mechanical properties when the addition amount was 6 mL. Therefore, a Sp / CEO / US nanoemulsion addition amount of 6 mL was selected for further research.

[0082] Example 4: Optimization of Sp / OEO / US nanoemulsion addition amount in P / OEO / US composite membrane

[0083] The preparation method of the P / OEO / US composite membrane is the same as in Example 3, except that turmeric essential oil is replaced with orange essential oil, while the other methods remain unchanged.

[0084] The amount of Sp / CEO / US nanoemulsion added was optimized using mechanical parameters such as tensile strength (TS) and elongation at break (EAB). Figure 2 As shown, the amount of Sp / OEO / US nanoemulsion added has a significant impact on the mechanical properties of the composite membrane. The composite membranes prepared with 4, 6, and 8 mL of nanoemulsion added have TS and EAB values ​​of 5.75 N / mm². 2 9.69 N / mm 2 8.55 N / mm 2 The concentrations were 330.12%, 359.26%, and 300.15%. The composite membrane exhibited optimal mechanical properties when the addition amount was 6 mL. Therefore, a Sp / OEO / US nanoemulsion addition amount of 6 mL was selected for subsequent studies.

[0085] Comparative Example 1: Preparation of Pul composite membrane

[0086] 4g of pullulan was stirred at room temperature for 1 hour until completely dissolved, preparing a polysaccharide solution with a concentration of 4g / 100mL. Glycerol at 15% of the total mass of pullulan was added to the polysaccharide solution and mixed at 50℃ for 30 minutes to obtain a homogeneous pullulan film-forming solution. 15mL of the film-forming solution was placed in a 9cm diameter petri dish and dried at 50℃ for 6 hours to obtain the Pull composite membrane.

[0087] Comparative Example 2: Preparation of P / CEO Composite Membrane

[0088] (1) Stir 85 mL of aqueous phase, 5 g of soy protein isolate and 10 mL of turmeric essential oil at room temperature for 30 min until the soy protein isolate is completely dissolved. Then homogenize the solution at 12000 rpm for 4 min to prepare Sp / CEO crude emulsion.

[0089] (2) Stir 4g of pullulan polysaccharide at room temperature for 1 hour until it is completely dissolved, and prepare a polysaccharide solution with a concentration of 4g / 100mL. Add glycerol at a mass ratio of 15% of the total mass of pullulan polysaccharide and mix with the above polysaccharide solution at 50℃ for 30 minutes to obtain a uniform pullulan polysaccharide film-forming solution.

[0090] (3) Take 6 mL of the prepared Sp / CEO crude emulsion and mix it with 100 mL of pullulan polysaccharide film-forming solution. Stir and mix at room temperature for 30 min to obtain a uniform P / CEO film-forming solution. Then take 15 mL of the film-forming solution into a petri dish with a diameter of 9 cm and dry it at 50 °C for 6 h to obtain a P / CEO composite membrane.

[0091] Comparative Example 3: Preparation of P / OEO Composite Membrane

[0092] The P / OEO composite membrane was prepared according to Comparative Example 2, except that turmeric essential oil was replaced with orange essential oil, while other methods remained unchanged.

[0093] Experimental Example 1: Particle size, ζ-potential, and polymer dispersibility index of Sp / CEO and Sp / OEO emulsions prepared with different ultrasonic times

[0094] Experimental methods: The particle size, zeta potential, and polymer dispersibility index (PDI) of the emulsion were measured using a laser particle size analyzer (Nano-ZS90, Malvern Instruments Ltd, UK). The emulsion was diluted 1000 times with deionized water to prevent multiple scattering effects, and three tests were conducted at 25°C.

[0095] Figure 3 , Figure 4The particle size, zeta potential, and polymer dispersibility index of Sp / CEO and Sp / OEO emulsions prepared for different ultrasonic treatment times are shown. The initial particle sizes of the emulsions were 214.60±6.06 nm and 222.54±1.34 nm, respectively, which gradually decreased to 116.29±2.53 nm and 120.64±4.87 nm after ultrasonic treatment for 10 min. With the extension of ultrasonic treatment time, the range of destructive shear energy input is wider, resulting in a significant reduction in emulsion particle size. In addition, the zeta potential value significantly affects the stability of the emulsion system. Generally, a larger absolute value of zeta potential often indicates the existence of solid electrostatic attraction between particles, thereby preventing flocculation and coagulation and maintaining the stability of the emulsion system. The results showed that all samples of Sp / CEO and Sp / OEO emulsions exhibited low zeta potential values, ranging from -43.22±0.73 mV to -46.69±0.71 mV and -42.76±0.60 mV to -47.28±0.50 mV, respectively. This ensured the stability of the emulsion system, and continuous sonication did not significantly affect the zeta potential values. Furthermore, Sp / CEO and Sp / OEO emulsions were observed to have sufficiently low PDI values, ranging from 0.12±0.058 to 0.22±0.016.

[0096] Experimental Example 2: Microstructure of Sp / CEO and Sp / OEO emulsions prepared with different ultrasonic times

[0097] Experimental Methods: The microstructure of the emulsion was observed using a laser confocal scanning microscope (CLSM) (Leica TCS SP5, Germany). 10 μL of the emulsion was placed on a microscope slide, covered with a coverslip, and observed at 25°C using a 100× oil immersion lens. All images were taken at a scanning frequency of 200 Hz and a density of 1024 × 1024.

[0098] Microstructure is also crucial for verifying the stability of emulsion systems. To further elucidate the stability of emulsions, the microstructure of the emulsions was observed using laser confocal scanning microscopy (CLSM). Figure 5 In the CLSM images, oil droplets are depicted as green and proteins as red. The results indicate that oil droplets (green) are encapsulated by proteins (red), forming a network within the continuous phase. Furthermore, increasing the sonication time significantly reduces the emulsion particle size, resulting in a homogeneous shear-thinned system. The CLSM results are in good agreement with the particle size and zeta potential results.

[0099] Experimental Example 3: Storage stability of Sp / CEO and Sp / OEO emulsions prepared with different ultrasonic times

[0100] Experimental method: The storage stability of the emulsion was determined by monitoring the change in emulsion particle size over 60 days. The particle size was measured according to Experiment Example 1.

[0101] The stability of the emulsion is crucial for its use in subsequent film-forming processes. Figure 6 , Figure 7 The figures show the particle size differences of Sp / CEO and Sp / OEO emulsions on days 15, 30, and 60 during storage. The results indicate that the droplet size is significantly increased in the emulsion prepared by short-term ultrasonic treatment, while the nanoemulsion obtained from long-term ultrasonic treatment maintains the smallest droplet size during 60 days of storage. The change in emulsion particle size is mainly related to the decrease in surface area and free energy in a thermodynamically unstable system, which leads to flocculation and aggregation. Furthermore, storage stability is related to factors such as gravitational separation, aggregation, and flocculation that affect emulsion particle size; therefore, the emulsion prepared by 10 min of ultrasonic treatment exhibits better dynamic storage stability.

[0102] Experimental Example 4: Thickness of Different Composite Films

[0103] Experimental method: The thickness of the composite membrane sample was measured using a micrometer screw gauge. Six locations were randomly selected for measurement on each composite membrane sample, and the average value was taken.

[0104] The thickness of the composite film is the most critical parameter, as it affects the physical, optical, and mechanical properties of the composite film. For example... Figure 8 As shown, the composite membrane thickness ranges from 0.128±0.008 to 0.171±0.008 mm. The thicknesses of the P / CEO / US and P / OEO / US composite membranes are 0.169±0.004 mm and 0.171±0.008 mm, respectively, while the thicknesses of the Pul, P / CEO, and P / OEO composite membranes are 0.153±0.009 mm, 0.127±0.008 mm, and 0.144±0.003 mm, respectively. This phenomenon may be due to the smaller droplet size of the nanoemulsion after ultrasonic treatment, which reduces the instability of the film-forming solution and the loss of essential oils, thereby increasing the membrane thickness.

[0105] Experimental Example 5: Moisture Content (MC) and Water Vapor Permeability (WVP) of Different Composite Membranes

[0106] Experimental method: The MC was determined by drying the composite membrane at 105℃ to constant weight. The calculation formula is as follows:

[0107]

[0108] In the formula, M1 is the wet basis weight; M2 is the dry basis weight.

[0109] The w / v of the composite membrane was evaluated using gravimetric analysis. The composite membrane sample was placed tightly on the opening of a weighing bottle containing 10 g of CaCl2 particles (0% RH). The weighing bottle was then placed in a desiccator containing saturated KNO3 (93% RH). Weighing was performed at 25°C for 12 hours, with samples taken every 2 hours. W / v (gm / m³) was then calculated. 2 The formula for calculating (·s·Pa) is:

[0110]

[0111] In the formula, Δm is the mass increment, g; A is the water vapor permeation area, m. 2 Δt is the time interval, s; ΔP = 2.945 kPa.

[0112] like Figure 9 As shown, the moisture content of the P / CEO and P / OEO composite membranes (17.32±0.75% and 17.14±0.95%, respectively) was significantly higher than that of the Pul composite membrane (15.74±0.130%). This may be related to the low molecular weight arrangement of essential oils in the composite membrane matrix and the distortion of intermolecular compatibility. The moisture content of the P / CEO / US and P / OEO / US composite membranes was significantly lower, at 12.83±0.558% and 13.64±1.07%, respectively. This phenomenon is due to the smaller particle size of the emulsion prepared by ultrasound, which increases its relative solubility and leads to a more compact composite membrane structure.

[0113] Water vapor pressure (WVP) measures moisture transfer between a product and its environment through the internal structure of a bioactive membrane; therefore, a minimum WVP plays a crucial role in preventing food dehydration. Figure 9 As shown, compared with the Pul composite membrane, the WVP of the P / CEO and P / OEO composite membranes were significantly improved, reaching 17.285 × 10⁻⁶. -11 g cm -1 s -1 Pa -1 and 17.462×10 -11 g cm -1 s -1 Pa -1 These results indicate that oil distribution and emulsion particle size have a crucial impact on the wVP of the composite membrane. However, the wVP of the P / CEO / US and P / OEO / US composite membranes was significantly reduced, to 5.532 × 10⁻⁶. -11 g cm -1 s -1 Pa -1 and ×10 -11 g cm - 1 s -1 Pa-1 Consistent with previous studies, the nanoemulsions prepared by ultrasound exhibited the smallest particle size and enhanced solubility, resulting in a more compact composite membrane structure that limited the diffusion and transfer of water vapor.

[0114] Experimental Example 6: Mechanical Properties of Different Composite Membranes

[0115] Experimental method: The membrane was cut into strips of 20mm×60mm and its tensile strength (TS) and elongation at break (EAB) were measured using a physical property tester. The test speed was 1mm / s and the clamping distance was 30mm.

[0116] Tensile strength calculation formula: TS=F / (d×W)

[0117] Where: TS—tensile strength (N / mm) 2 F—the maximum tension (N) experienced when the membrane breaks; d—the membrane thickness (mm); W—the membrane width (mm).

[0118] Elongation at break formula: EAB=(L-L0)×100 / L0

[0119] Where: EAB—elongation at break (%); L—distance between the markings when the membrane breaks (mm); L0—original distance between the markings on the membrane (mm).

[0120] The strength and flexibility of composite membranes against external forces are evaluated using mechanical parameters such as tensile strength (TS) and elongation at break (EAB). Figure 10 As shown, the TS and EAB of the P / CEO / US and P / OEO / US composite membranes are 9.30 N / mm². 2 9.69 N / mm 2 The TS and EAB values ​​of the essential oils were 332.88% and 359.26%, respectively, significantly higher than those of the P / CEO and P / OEO composite membranes. The addition of essential oils can improve the plasticizing effect by increasing the fluidity and flexibility of the polymer chains and enhancing the EAB of the composite membrane. Ultrasonic treatment reduced the particle size of the emulsion, increasing the likelihood of interaction between the emulsion and the membrane matrix, thereby enhancing EAB and TS. However, to some extent, the addition of essential oils also weakened the internal structure and intermolecular interactions of the pullulan chains, resulting in a lower TS value relative to the Pul composite membrane.

[0121] Experimental Example 7: Fourier Transform Attenuated Total Reflectance Infrared Spectroscopy (ATR-FTIR) Analysis of Different Composite Films

[0122] Experimental method: An ATR-FTIR spectrometer was used at 4000-600 cm⁻¹ -1 The FTIR spectra of the thin film were recorded within a certain range, with 16 scans and a resolution of 4 cm⁻¹. -1 .

[0123] Figure 11 FTIR spectra (4000-650 cm⁻¹) of Pul, P / CEO, P / CEO / US, P / OEO, and P / OEO / US composite membranes. -1 The addition of essential oils to the Pul membrane resulted in some differences in the position and intensity of the characteristic peaks. The composite membrane containing essential oils showed some variation at 3288 cm⁻¹. -1 2928cm -1 and 1644cm -1 The peaks at these points correspond to the stretching vibrations of OH, bending vibrations of CH, and stretching vibrations of C=O, respectively. After the addition of essential oils, the OH characteristic peak increased from 3292 cm⁻¹. -1 Move to 3288cm -1 and 3287cm -1 This indicates the formation of hydrogen bond interactions between pullulan polysaccharide and essential oil. Furthermore, the amide I band at 1550 cm⁻¹... -1 The emergence of new peaks indicates that the interaction between the amino groups of the protein and the hydroxyl groups of pullulan polysaccharides alters the secondary structure of the pullulan membrane. The OH characteristic peaks of the P / CEO / US and P / OEO / US composite membranes also showed a significant shift, which is attributed to the smaller emulsion particle size and the formation of more hydrogen bonds with the membrane matrix. This further explains the improved mechanical properties of the P / CEO / US and P / OEO / US composite membranes.

[0124] Experimental Example 8: X-ray Diffraction Analysis of Different Composite Films

[0125] Experimental method: The XRD pattern of the composite film was obtained using an X-ray diffractometer in the 2θ range of 5°-80° at a scanning speed of 5° / min.

[0126] Figure 12 The X-ray diffraction patterns of the Pul, P / CEO, P / CEO / US, P / OEO, and P / OEO / US composite membranes are shown. The broad peak around 18° in the figure is related to the amorphous structure of pullulan. The five composite membranes exhibit similar curves, indicating that the addition of different types of emulsions did not change the crystallization mode of the polymer membrane. However, it is noteworthy that after adding the emulsion prepared by ultrasound, the diffraction peaks of the composite membrane became broader and flatter, indicating that the smaller particle size of the emulsion formed better intermolecular forces with the membrane matrix, enhancing the compatibility of macromolecules.

[0127] Experimental Example 9: Thermal Properties Analysis of Different Composite Films

[0128] Experimental methods: The thermal stability of the thin film was determined using a STA 449F3 Jupiter thermal analyzer, heated from 30°C to 550°C at a rate of 10°C / min under N2 atmosphere (N2 flow rate 20 mL / min).

[0129] Thermogravimetric curves (30-550℃) of Pul, P / CEO, P / CEO / US, P / OEO, and P / OEO / US composite membranes are as follows: Figure 13 and Figure 14 As shown, the first stage of thermal degradation occurred at 30-130℃, mainly due to the volatilization of water and a small amount of essential oil in the composite membrane. The second stage of thermal degradation was observed at 130-270℃, primarily due to the loss of a large amount of low-molecular-weight components and glycerol from the composite membrane matrix. The third stage of thermal degradation occurred at 270-365℃, mainly due to the disruption of the polymer network and the degradation of pullulan. In the second stage, the thermal stability of P / CEO and P / OEO composite membranes was slightly lower than that of the Pul membrane; this decrease in thermal stability may be related to the degradation and volatility of the emulsion. Furthermore, the thermal stability of P / CEO / US and P / OEO / US composite membranes was improved compared to P / CEO and P / OEO composite membranes. This is attributed to the reduced particle size of the emulsion after ultrasonic treatment, which increased the hydrogen bonding between the nanoemulsion and pullulan, enhancing intermolecular forces and thus limiting the degradation of essential oil. This also verifies the hypothesis that ultrasonic treatment in ATR-FTIR enhances the hydrogen bonding interactions between components in the composite membrane, which is consistent with the results of the composite membrane's mechanical properties.

[0130] Experimental Example 10: Retention Time of Essential Oils in Different Composite Membranes

[0131] Experimental method: A 4 cm diameter composite membrane sample was immersed in a test tube containing 10 mL of n-hexane and 5 mL of deionized water, and stirred vigorously at 25 °C for 12 h. The mixture was then centrifuged at 12000 rpm / min for 10 minutes. The absorbance was measured using a UV spectrophotometer. The essential oil loss rate in the membrane matrix was determined based on the ratio of the essential oil loss to the total amount of essential oil in the membrane dispersion.

[0132] Volatile oils are highly volatile and easily evaporate during drying and storage. Figure 15 The retention time of essential oils in the membrane matrix is ​​denoted as . The results showed that after 6 days of storage at room temperature, all samples experienced essential oil loss, while the P / CEO / US and P / OEO / US composite membranes exhibited lower essential oil loss rates. The retention capacity of essential oils is directly related to the membrane structure and intermolecular interactions. In our study, the difference in retention time may be due to stronger hydrogen bonding interactions between the ultrasound-prepared emulsion and the membrane matrix, resulting in a more compact composite membrane structure.

[0133] Experimental Example 11: Antibacterial Properties of Different Composite Films

[0134] Experimental methods: Escherichia coli and Staphylococcus aureus were cultured at 37°C and adjusted to a concentration of 1×10⁻⁶ with sterile physiological saline. 8 CFU / mL. 2g of membrane sample was placed in 10mL of LB medium, and 100μL of bacterial suspension was added. The mixture containing the bacterial sample was then incubated at 37°C for 36h. Every 3h, the absorbance of each sample group was measured at 650nm to reflect bacterial proliferation.

[0135] Figure 16 and Figure 17 The antibacterial properties of Pul, P / CEO, P / CEO / US, P / OEO, and P / OEO / US composite membranes against *Escherichia coli* and *Staphylococcus aureus* were evaluated. Turmeric essential oil is mainly rich in curcuminoids and curcuminones, while orange essential oil mainly contains limonene; both exhibit substantial inhibitory effects on Gram-positive and Gram-negative bacteria. The results show that the P / CEO, P / CEO / US, P / OEO, and P / OEO / US composite membranes all exhibited antibacterial effects, with the P / CEO / US and P / OEO / US composite membranes showing stronger antibacterial activity. This indicates that the smaller particle size of the emulsion results in a more uniform distribution within the membrane matrix, increasing the surface area of ​​the antibacterial agent and improving bacterial accessibility to the agent.

[0136] Application Example 1: Test of the preservation effect of composite film on fresh strawberries

[0137] The preparation of the Pul, P / CEO, P / CEO / US, P / OEO, and P / OEO / US composite membranes in the application examples follows the experimental examples. Undamaged or moldy strawberries were washed with deionized water, air-dried, and stored in a plastic container. The prepared composite membrane was then placed over the plastic container. The container was stored at 25°C for 7 days. The sample was weighed and photographed every 24 hours. The weight loss rate was the ratio of the weight lost by the strawberries to the total weight of the strawberries.

[0138] like Figure 18 As shown, the strawberry fruits in the P / CEO, P / CEO / US, P / OEO, and P / OEO / US composite film covered groups did not spoil and remained fresh. However, severe rotting occurred in the uncovered group and the Pul composite film covered group. The controlled damage in the fruit reveals the importance of the antibacterial activity of the essential oils in the composite film matrix. Figure 19 The results showed that the P / CEO / US and P / OEO / US composite films exhibited greater weight retention, suggesting that their structures are more compact and can effectively alleviate fruit moisture loss. This also supports the effectiveness of ultrasound in the food industry. Figure 20 The images show the physical specimens of the five composite membranes prepared in this invention.

Claims

1. An ultrasonic preparation method for a polysaccharide and vegetable preservation film loaded with nano-essential oil emulsion, characterized in that... Follow these steps: (1) Deionized water, soy protein isolate, and turmeric oil were stirred at room temperature for 30 min until the soy protein isolate was completely dissolved. The solution was then homogenized at 12,000 rpm for 4 min to prepare a crude emulsion of soy protein isolate / turmeric oil. (2) The crude emulsion of soy protein isolate / turmeric essential oil in step (1) is subjected to ultrasonic treatment to obtain soy protein isolate / turmeric essential oil nanoemulsion prepared by ultrasonication. (3) Stir pullulan at room temperature for 1 h until completely dissolved to prepare a polysaccharide solution; (4) Add a certain mass concentration of glycerol to the polysaccharide solution in step (3), place the blend in a water bath constant temperature magnetic stirrer, stir for 30 min at 50℃ to obtain pullulan polysaccharide film-forming solution. (5) Add a certain proportion of the soybean protein isolate / turmeric essential oil nanoemulsion prepared by ultrasound in step (2) to the film-forming solution in step (4), and stir the mixture at room temperature for 30 min to obtain the film-forming solution. (6) Take the film-forming solution in step (5) and form a film by casting. Dry it at 60°C for 8 hours. After the film is formed, take it out and cool it to room temperature to obtain a polysaccharide vegetable and fruit preservation film loaded with turmeric essential oil nanoemulsion. In step (1), the ratio of deionized water, soy protein isolate, and turmeric essential oil is (89-85):(1-5):10; In step (2), the ultrasonic power is 1200 W / L, the intermittent ratio is 3 s / 3 s, and the ultrasonic treatment time is 2.5-10 min. The mass concentration of pullulan polysaccharide solution in step (3) is 4 g / 100 mL; In step (4), the amount of glycerol added is 15% of the total mass of pullulan polysaccharide; The amount of soybean protein isolate / turmeric essential oil nanoemulsion added in step (5) by ultrasound preparation is 4%-8% of the volume ratio of pullulan polysaccharide film-forming solution.

2. The ultrasonic preparation method of the polysaccharide and vegetable preservation film loaded with nano-essential oil emulsion according to claim 1, characterized in that... In step (1), the ratio of deionized water, soy protein isolate, and turmeric essential oil is 85: 5:

10. The ultrasonic treatment time in step (2) is 10 min; The amount of soybean protein isolate / turmeric essential oil nanoemulsion added in step (5) by ultrasound preparation is 6% of the volume ratio of pullulan polysaccharide film-forming solution.

3. An ultrasonic preparation method for a polysaccharide and vegetable preservation film loaded with nano-essential oil emulsion, characterized in that... Follow these steps: (1) Deionized water, soy protein isolate, and orange essential oil were stirred at room temperature for 30 min until the soy protein isolate was completely dissolved. The solution was then homogenized at 12000 rpm for 4 min to prepare a crude emulsion of soy protein isolate / orange essential oil. (2) The crude emulsion of soy protein isolate / orange essential oil in step (1) is subjected to ultrasonic treatment to obtain ultrasonically prepared soy protein isolate / orange essential oil nanoemulsion. (3) Stir pullulan at room temperature for 1 h until completely dissolved to prepare a polysaccharide solution; (4) Add a certain mass concentration of glycerol to the polysaccharide solution in step (3), place the blend in a water bath constant temperature magnetic stirrer, stir for 30 min at 50℃ to obtain pullulan polysaccharide film-forming solution. (5) Add a certain proportion of the soybean protein isolate / orange essential oil nanoemulsion prepared by ultrasound in step (2) to the film-forming solution in step (4), and stir the mixture at room temperature for 30 min to obtain the film-forming solution. (6) Take the film-forming solution in step (5) and form a film by casting. Dry it at 60°C for 8 hours. After the film is formed, take it out and cool it to room temperature to obtain a polysaccharide vegetable and fruit preservation film loaded with orange essential oil nanoemulsion. In step (1), the ratio of deionized water, soy protein isolate, and orange essential oil is (89-85):(1-5):10; In step (2), the ultrasonic power is 1200 W / L, the intermittent ratio is 3 s / 3 s, and the ultrasonic treatment time is 2.5-10 min. The mass concentration of pullulan polysaccharide solution in step (3) is 4 g / 100 mL; the amount of glycerol added in step (4) is 15% of the total mass of pullulan polysaccharide. In step (5), the amount of soy protein isolate / orange essential oil nanoemulsion added during ultrasonic preparation is 4%-8% of the volume ratio of pullulan polysaccharide film-forming solution.

4. The ultrasonic preparation method of the polysaccharide and vegetable preservation film loaded with nano-essential oil emulsion according to claim 3, characterized in that... In step (1), the ratio of deionized water, soy protein isolate, and orange essential oil is 85: 5:

10. The ultrasonic treatment time in step (2) is 10 min; In step (5), the amount of soy protein isolate / orange essential oil nanoemulsion added during ultrasonic preparation is 6% of the volume ratio of pullulan polysaccharide film-forming solution.

5. The polysaccharide vegetable and fruit preservation film loaded with nano-essential oil emulsion prepared by the preparation method according to any one of claims 1-4 is used for the preservation packaging of strawberries.

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