Tea tree oil-thyme oil-chitosan composite membrane, its preparation method and application

Abstract

This invention discloses a tea tree oil-thyme oil-chitosan composite film, its preparation method, and its application, belonging to the field of food preservation film technology. The preparation method of the tea tree oil-thyme oil-chitosan composite film includes the following steps: dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution; adding glacial acetic acid, chitosan, plasticizer, and emulsifier to the polyvinyl alcohol and mixing evenly; then adding tea tree oil and thyme oil, homogenizing and mixing, and degassing to obtain a film-forming liquid; and drying the film-forming liquid to form a film to obtain the tea tree oil-thyme oil-chitosan composite film. The tea tree oil-thyme oil-chitosan composite film prepared by this invention has excellent antibacterial effects and improves the preservation time of sweet cherries.

Description

Technical Field

[0001] This invention relates to the field of food preservation film technology, and more specifically to a tea tree oil-thyme oil-chitosan composite film, its preparation method, and its application. Background Technology

[0002] Sweet cherries, also known as sweet cherries or large cherries, belong to the genus *Prunus* of the family Rosaceae in the order Rosales. Sweet cherries are characterized by their large size, abundant flesh, delicious taste, and vibrant color. They are also rich in iron, vitamins, organic acids, and other nutrients. However, sweet cherries have extremely thin skin, soft and juicy flesh, and are harvested during the hot and rainy season. Therefore, after harvesting, sweet cherries are susceptible to infection by microorganisms such as Penicillium, E. coli, and yeast, leading to dehydration, browning, and rotting. This reduces the nutritional value and quality of the cherries, affects their shelf life, and causes significant economic losses, hindering the development of the sweet cherry industry.

[0003] Currently, polyethylene cling film is a widely used preservation material. Its main component is polyethylene, which has advantages such as being non-toxic, odorless, resistant to low temperatures, and having good chemical stability. However, low-density polyethylene has poor permeability to water vapor and air. After harvesting, plants still undergo respiration, transpiration, and enzyme activity. Therefore, during storage, the respiration and transpiration of sweet cherries will cause water mist to appear on the inner surface of the polyethylene cling film, resulting in condensation. This causes changes in humidity inside the bag, which in turn nourishes bacteria, leading to rapid microbial reproduction, high fruit rot rate, loss of preservation effect, and a short shelf life. Summary of the Invention

[0004] To address the above problems, this invention provides a tea tree oil-thyme oil-chitosan composite membrane, its preparation method, and its application. The tea tree oil-thyme oil-chitosan composite membrane prepared by this invention has excellent antibacterial effects and improves the preservation time of sweet cherries.

[0005] The first objective of this invention is to provide a method for preparing a tea tree oil-thyme oil-chitosan composite membrane, comprising the following steps:

[0006] Polyvinyl alcohol is dissolved in water to obtain a polyvinyl alcohol solution.

[0007] After adding glacial acetic acid, chitosan, plasticizer and emulsifier to polyvinyl alcohol and mixing evenly, tea tree oil and thyme oil are added, and after homogenization and mixing, degassing is performed to obtain a film-forming solution.

[0008] The film-forming solution was dried to form a film, resulting in a tea tree oil-thyme oil-chitosan composite membrane.

[0009] It should be noted that when drying to form a film, the drying temperature mainly considers the drying time and the volatility of the essential oil. If the drying temperature is too low, the drying effect will be poor, resulting in a poor film formation effect; if the drying temperature is too high, the essential oil will evaporate excessively, affecting the antibacterial effect. Preferably, the drying and film formation of this invention is carried out at 35°C for 4 hours.

[0010] In a preferred embodiment of the present invention, the ratio of chitosan to tea tree oil is 1g:1mL~3mL; for example, the ratio of chitosan to tea tree oil is 1g:1mL, 1g:2mL, 1g:3mL, etc.

[0011] In a preferred embodiment of the present invention, the volume ratio of tea tree oil to thyme oil is 1:1.

[0012] In a preferred embodiment of the present invention, the ratio of polyvinyl alcohol to glacial acetic acid is 3g:1mL, and the mass ratio of polyvinyl alcohol to chitosan is 6:1.

[0013] In a preferred embodiment of the present invention, the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 3%.

[0014] In a preferred embodiment of the present invention, the ratio of chitosan to plasticizer is 1g:4mL.

[0015] In a preferred embodiment of the present invention, the plasticizer is propylene glycol. Propylene glycol used in this invention has low toxicity and is inexpensive.

[0016] In a preferred embodiment of the present invention, the emulsifier is Tween 80. Tween 80 used in this invention has low toxicity and is inexpensive.

[0017] The second objective of this invention is to provide a tea tree oil-thyme oil-chitosan composite membrane prepared by the above-described preparation method.

[0018] The third objective of this invention is to provide the application of the above-mentioned tea tree oil-thyme oil-chitosan composite film in the preservation of sweet cherries.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) In this invention, glacial acetic acid is used as a solvent to dissolve chitosan. Polyvinyl alcohol and chitosan are used as raw materials for preparing composite membranes. Tea tree oil and thyme oil work together to inhibit pathogens. Plasticizers and emulsifiers are added to help tea tree oil and thyme oil mix evenly with water. Homogenization allows for better emulsification. Finally, the membrane is dried to obtain a tea tree oil-thyme oil-chitosan composite membrane. The prepared tea tree oil-thyme oil-chitosan composite membrane reduces oxygen concentration and increases carbon dioxide concentration, inhibits plant respiration and enzyme activity, thereby inhibiting water loss from sweet cherries, delaying spoilage caused by bacteria, yeast and mold, inhibiting the intensity of respiration, slowing down the decrease in soluble solids, measurable acids and vitamin C content in sweet cherries, maintaining their nutritional value, improving preservation effect and extending the storage time of sweet cherries.

[0021] (2) In the preparation process of this invention, chitosan has good film-forming properties and high safety, but it has poor fluidity and high price when used alone. The addition of polyvinyl alcohol helps to reduce costs, and the addition of tea tree oil and thyme oil can increase fluidity and enhance mechanical properties.

[0022] (3) When the volume ratio of tea tree oil to thyme oil is 1:1, the tea tree oil-thyme oil-chitosan composite film has suitable tensile strength and flexibility, and has a good preservation effect on sweet cherries. Attached Figure Description

[0023] Figure 1 The effect of chitosan composite membranes with different amounts of essential oils on the total bacterial count in sweet cherry.

[0024] Figure 2 The effect of adding different amounts of essential oils to chitosan composite membranes on the number of sweet cherry molds and yeasts.

[0025] Figure 3 The effect of chitosan composite membranes with different amounts of essential oils on the soluble solids of sweet cherries.

[0026] Figure 4 The effect of different amounts of essential oils added to chitosan composite membranes on the measurable acidity content of sweet cherries.

[0027] Figure 5 The effect of adding different amounts of essential oils to chitosan composite membranes on the vitamin C content of sweet cherries.

[0028] Figure 6 The effect of chitosan composite membranes with different amounts of essential oils on the weight loss rate of sweet cherries.

[0029] Figure 7 The effect of chitosan composite membranes with different amounts of essential oils on the rot rate of sweet cherries.

[0030] Figure 8Sensory changes in sweet cherries in chitosan composite membranes with different amounts of essential oils added. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The fresh sweet cherries used in this invention were obtained from Yuanhua Planting Professional Cooperative in Hanyuan County, Ya'an City, Sichuan Province, and the variety was Black Pearl. Tea tree oil was purchased from Yunnan Jingyu Biotechnology Co., Ltd.; thyme oil and chitosan were purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with a molecular weight of 150,000 for the chitosan. Plate counting agar and Bengal red agar were purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; Tween 80, sodium chloride, polyvinyl alcohol 1799, glacial acetic acid, and propylene glycol were all analytical grade and purchased from Chengdu Kelong Chemical Co., Ltd.; 2,6-dichlorophenolindophenol was analytical grade and purchased from Tanmo Quality Inspection Technology Co., Ltd. Staphylococcus aureus ATCC6538, batch number C0683KJ, was purchased from Guangdong Huankai Biotechnology Co., Ltd.; Pseudomonas aeruginosa ATCC9027, batch number 230918S04, was purchased from Guangdong Huankai Biotechnology Co., Ltd.; Candida albicans ATCC10231, batch number J0070DX, was purchased from Guangdong Huankai Biotechnology Co., Ltd.

[0033] To investigate the bactericidal and bacteriostatic effects of tea tree oil and thyme oil, a total of 0.21g of tea tree oil and thyme oil were weighed and subjected to bacteriostatic and bactericidal effects against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans at mass ratios of 1:1, 1:2, and 2:1, respectively. The specific operating steps are as follows.

[0034] After resuscitation, the bacterial strains were transferred to agar plates. Then, single colonies of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were picked from the plates to prepare bacterial suspensions. These suspensions were diluted with sterile physiological saline to a concentration of 0.5 McFarland units, with a bacterial count equivalent to 1.0–1.5 × 10⁸ CFU / mL, and kept for later use.

[0035] To investigate the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of thyme essential oil and tea tree essential oil against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, the study examined whether a mixture of the two essential oils in a certain proportion had a synergistic effect on the inhibition and bactericidal activity of these three bacteria.

[0036] The bacteria under investigation were divided into three major experimental groups, with fifty sterile test tubes in each group. The test tubes for Staphylococcus aureus and Pseudomonas aeruginosa were each inoculated with 1 mL of brain-heart extract liquid medium. The test tubes for Candida albicans were inoculated with 1 mL of Sabouraud dextrose liquid medium. Each group of fifty test tubes was divided into subgroups of ten. In the first tube, 1 mL of the prepared essential oil emulsion in different proportions was added and mixed. Then, 1 mL was transferred from the first tube to the second tube and mixed. This process was repeated until the tenth tube was reached. 1 mL of the tenth tube was then discarded, creating a two-fold dilution series with concentrations of 11.6667 mg / mL, 5.8333 mg / mL, 2.9167 mg / mL, 1.4584 mg / mL, 0.7292 mg / mL, 0.3646 mg / mL, 0.1823 mg / mL, 0.09114 mg / mL, 0.04557 mg / mL, and 0.02279 mg / mL, and these concentrations were numbered sequentially. Finally, 0.1 mL of the corresponding prepared bacterial suspension was added to each test tube; for example, 0.1 mL of Staphylococcus aureus was added to the Staphylococcus aureus group. The same procedure was performed for the Pseudomonas aeruginosa group and the Candida albicans group. Staphylococcus aureus and Pseudomonas aeruginosa groups were incubated at 36℃ for 24–48 h, while Candida albicans group was incubated at 26℃ for 48–72 h. Clear test tubes were then transferred to the corresponding plates, and the presence or absence of colony growth was observed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).

[0037] Regarding the antibacterial results against Staphylococcus aureus: In the experiments with tea tree oil alone and tea tree oil and thyme oil in a mass ratio of 2:1, the first to fifth vials were completely clear, while the sixth vial began to become cloudy. In the experiments with thyme oil alone, tea tree oil and thyme oil in a mass ratio of 1:1, and tea tree oil and thyme oil in a mass ratio of 1:2, the first to seventh vials were completely clear, while the eighth vial began to become cloudy.

[0038] The results showed that the minimum inhibitory concentration (MIC) of tea tree oil alone and the minimum bactericidal concentration (MBC) of tea tree oil and thyme oil in a mass ratio of 2:1 against Staphylococcus aureus were 0.7292 mg / mL and 1.4584 mg / mL, respectively.

[0039] The minimum inhibitory concentration (MIC) of thyme essential oil, tea tree essential oil, and thyme essential oil in a 1:1 mass ratio, and the minimum bactericidal concentration (MBC) of tea tree essential oil and thyme essential oil in a 1:2 mass ratio against Staphylococcus aureus was 0.1823 mg / mL and 0.3646 mg / mL, respectively.

[0040] Regarding the antibacterial results for Candida albicans, the overall sensitivity of Candida albicans to both essential oils was relatively weaker than that to Staphylococcus aureus, while it was relatively more sensitive to thyme essential oil. Specifically, in experiments with tea tree oil added alone and with a tea tree oil to thyme essential oil ratio of 2:1, the first three bottles were completely clear, while the fourth bottle began to become cloudy.

[0041] In experiments where thyme essential oil, tea tree essential oil, and thyme essential oil were added separately in a 1:1 mass ratio, and tea tree essential oil and thyme essential oil in a 1:2 mass ratio, the first to fifth bottles were completely clear, while the sixth bottle began to become cloudy.

[0042] The results showed that the minimum inhibitory concentration (MIC) of tea tree oil alone, and the MIC of tea tree oil and thyme oil in a 2:1 mass ratio against Candida albicans, was 2.9167 mg / mL, and the minimum bactericidal concentration (MCC) was 5.8333 mg / mL. The MICs of thyme oil alone, tea tree oil, and thyme oil in a 1:1 mass ratio, and the MIC of tea tree oil and thyme oil in a 1:2 mass ratio against Candida albicans, were 0.7292 mg / mL and the MCC was 1.4584 mg / mL.

[0043] Regarding the antibacterial results against Pseudomonas aeruginosa, the two essential oils showed relatively weak antibacterial activity. In the experiments with tea tree oil alone and tea tree oil and thyme oil in a 2:1 mass ratio, the first three bottles were completely clear, while the fourth bottle began to become cloudy.

[0044] In experiments where thyme essential oil, tea tree essential oil, and thyme essential oil were added separately in a 1:1 mass ratio, and tea tree essential oil and thyme essential oil in a 1:2 mass ratio, the first and second bottles were completely clear, while the third bottle began to become cloudy.

[0045] The results showed that the minimum inhibitory concentration (MIC) of tea tree oil alone, and the MIC of tea tree oil and thyme oil in a 2:1 mass ratio, against *Pseudomonas aeruginosa* was 2.9167 mg / mL, and the minimum bactericidal concentration (MCC) was 5.8333 mg / mL. The MIC of thyme oil alone, the MIC of tea tree oil and thyme oil in a 1:1 mass ratio, and the MIC of tea tree oil and thyme oil in a 1:2 mass ratio against *Pseudomonas aeruginosa* was 5.8333 mg / mL, and the MCC was 11.6667 mg / mL. *Staphylococcus aureus* was sensitive to both essential oils, while *Pseudomonas aeruginosa* was more sensitive to tea tree oil, and *Candida albicans* was relatively more sensitive to thyme oil. In conclusion, the tea tree oil and thyme oil selected in this invention have excellent bactericidal and bacteriostatic effects. Adding tea tree oil and thyme oil in a 1:1 ratio to the composite membrane can effectively and comprehensively ensure the membrane's antibacterial and bactericidal effects.

[0046] It should be noted that during the bactericidal and bacteriostatic experiments, it was found in five groups of experiments that tea tree oil and thyme oil at a mass ratio of 1:1 provided a more comprehensive and synergistic bactericidal or bacteriostatic effect. Considering that in the preparation of the composite membrane, regardless of whether the addition ratio of the two essential oils is by mass or volume, as long as they are added to the composite membrane at a 1:1 ratio and reach a certain concentration, an antibacterial effect can be achieved, this invention uses a 1:1 volume ratio when preparing the tea tree oil-thyme oil-chitosan composite membrane.

[0047] Example 1

[0048] Weigh 3g of polyvinyl alcohol 1799 and add 100mL of boiling water. Stir in an 80℃ water bath until the polyvinyl alcohol 1799 is completely dissolved. Add 1mL of glacial acetic acid and 0.5g of chitosan, stir to dissolve, add 2mL of propylene glycol and 1mL of Tween 80, and add 0.5mL of tea tree oil and 0.5mL of thyme oil. Mix in a homogenizer at 900r / min for 5min and degas for 1h to obtain the film-forming solution. Pour 50mL of the film-forming solution into a 900mm film-forming plate and dry in a 35℃ drying oven for 4h. After the film cools to room temperature, peel it off to obtain a tea tree oil-thyme oil-chitosan composite membrane, and store it in a desiccator at room temperature for later use. The tea tree oil-thyme oil-chitosan composite membrane prepared in this example is denoted as 1%TTO-TEO.

[0049] Example 2

[0050] Weigh 3g of polyvinyl alcohol 1799 and add 100mL of boiling water. Stir in an 80℃ water bath until the polyvinyl alcohol 1799 is completely dissolved. Add 1mL of glacial acetic acid and 0.5g of chitosan, stir to dissolve, add 2mL of propylene glycol and 2mL of Tween 80, and add 1.0mL of tea tree oil and 1.0mL of thyme oil. Mix in a homogenizer at 900r / min for 5min and degas for 1h to obtain the film-forming solution. Pour 50mL of the film-forming solution into a 900mm film-forming plate and dry in a 35℃ drying oven for 4h. After the film cools to room temperature, peel it off to obtain a tea tree oil-thyme oil-chitosan composite membrane, and store it in a desiccator at room temperature for later use. The tea tree oil-thyme oil-chitosan composite membrane prepared in this example is denoted as 2%TTO-TEO.

[0051] Example 3

[0052] Weigh 3g of polyvinyl alcohol 1799 and add 100mL of boiling water. Stir in an 80℃ water bath until the polyvinyl alcohol 1799 is completely dissolved. Add 1mL of glacial acetic acid and 0.5g of chitosan, stir to dissolve, add 2mL of propylene glycol and 3mL of Tween 80, and add 1.5mL each of tea tree oil and thyme oil. Mix in a homogenizer at 900r / min for 5min, degas for 1h, and obtain the film-forming solution. Pour 50mL of the film-forming solution into a 900mm film-forming plate and dry in a 35℃ drying oven for 4h. After the film cools to room temperature, peel it off to obtain a tea tree oil-thyme oil-chitosan composite membrane, and store it in a desiccator at room temperature for later use. The tea tree oil-thyme oil-chitosan composite membrane prepared in this example is denoted as 3%TTO-TEO.

[0053] The following characterization tests were performed on the tea tree oil-thyme oil-chitosan composite membrane prepared in the examples.

[0054] Mechanical properties: The determination of tensile strength and nominal strain at break was performed according to GB / T1040.3—2006 "Determination of tensile properties of plastics—Part 3: Test conditions for thin films and sheets". The composite film was fixed on a universal testing machine with an initial gauge length of 50 mm and a test rate of 50 mm / min. The tensile load and nominal strain at break of the film were measured. Five sets of tests were performed, and the average value was taken.

[0055] Preservation performance: Freshly harvested sweet cherries were selected, choosing those that were plump, uniform in size, and in good condition. Each cherry was placed in a food-grade open-top polyester preservation box (approximately 250g) and randomly divided into five groups of 57 boxes each. Group 1 used the 1% TTO-TEO sealed preservation box prepared in Example 1; Group 2 used the 2% TTO-TEO sealed preservation box prepared in Example 2; Group 3 used the 3% TTO-TEO sealed preservation box prepared in Example 3; Group 4 used a food-grade polyethylene preservation film sealed preservation box (referred to as polyethylene preservation); and Group 5 did not use a composite film sealed preservation box (referred to as the blank control group). The sweet cherries were stored in a refrigerator at 4±1℃. Microbial count, physicochemical properties, and sensory quality were measured on days 0, 2, 4, 6, 8, 10, 12, 14, and 16 of storage.

[0056] Determination of total bacterial count: In accordance with GB4789.2-2022 "National Food Safety Standard for Microbiological Examination of Food - Determination of Total Bacterial Count", 25.0g of each sample was randomly weighed into 225mL of physiological saline, mixed well, and 1.00mL was taken into two plates. Approximately 20mL of plate counting agar at about 45℃ was added. After cooling and solidification, the plates were inverted and incubated in an incubator for 48h before counting.

[0057] Determination of molds and yeasts: In accordance with GB4789.15-2016 "National Food Safety Standard for Microbiological Examination of Food - Counting of Molds and Yeasts", 25.0 g of each sample was randomly weighed into 225 mL of physiological saline, mixed well, and 1.00 mL was taken into two plates. About 20 mL of Bengal Red Agar at about 45℃ was added. After cooling and solidification, the plates were inverted and incubated at 28℃ for 5 days before counting.

[0058] Determination of soluble solids content: Referring to the article "The Influence of Cutting Method on the Storage Quality and Microorganisms of Fresh-Cut Cucumbers" published by Wang Haidan et al. in the 10th issue of *Northern Horticulture* in 2022, the soluble solids content was determined. Specifically, sweet cherries were pitted and pulped, filtered through four layers of gauze, and the filtrate was used to determine the soluble solids content using a digital Abbe refractometer. Three parallel experiments were performed for each group, and the average value was taken.

[0059] Determination of measurable acid content: Remove the pits from sweet cherries, crush them using a tissue homogenizer, mix well, weigh 5g of sweet cherry pulp using an electronic balance and place it in a 150mL Erlenmeyer flask, add 10mL of carbon dioxide-free water at 80℃, mix well, and boil in a boiling water bath for 30 minutes, shaking 3 times during the process to ensure all organic acids are dissolved in the solution. Remove from the water bath, cool to room temperature, and dilute to 50mL with carbon dioxide-free water. Filter through rapid filter paper, collect the filtrate, accurately pipette 10.0mL of the filtrate, add 30mL of carbon dioxide-free water, start the fully automatic potentiometric titrator, turn on the stirrer, and quickly titrate with 0.09938mol / L NaOH standard titration solution until the solution pH reaches 8.2. Record the volume of NaOH standard titration solution consumed. Take three parallel samples for each control group, and perform a blank test simultaneously. Measurable acids are expressed as a percentage of malic acid content, specifically referring to GB12456-2021 National Food Safety Standard for the Determination of Total Acids in Food.

[0060] Vitamin C content determination: Following the method described by Cao Jiankang et al. in their 2007 publication, *Guide to Postharvest Physiological and Biochemical Experiments in Fruits and Vegetables*, the 2,6-dichlorophenolindophenol titration method was used. In this experiment, only the pulp of sweet cherries was used to determine the vitamin C content. Each treatment group was measured three times, and the results were averaged and expressed in mg / 100g.

[0061] Determination of weight loss rate: The weight loss rate of sweet cherries during storage was determined by weighing and calculated according to formula (1).

[0062] (1).

[0063] Determination of rot rate: During the storage of sweet cherries, the number of rotten fruits is observed and counted regularly, and the result is calculated according to formula (2).

[0064] (2).

[0065] Sensory evaluation: Sensory evaluators were recruited and trained according to the methods in GB / T 16291.1-2012 "General Guidelines for the Selection, Training and Management of Sensory Evaluators Part 1: Selection of Evaluators". A sensory evaluation team of 10 people of different genders and ages was selected through final assessment. A 100-point scoring system was used, and the sensory evaluation standards in Table 1 were applied to the sensory evaluation of each group of sweet cherries during the storage period. Higher scores indicate better quality sweet cherries. Sweet cherries with scores below 60 points lose their commercial value; those with scores below 40 points lose their edible value.

[0066] Table 1 Sensory Evaluation Criteria for Sweet Cherries

[0067]

[0068] Mechanical properties are one of the essential properties that all packaging films should possess. Tensile strength and nominal strain at break are important reference standards for evaluating the mechanical properties of packaging films, reflecting the tensile strength and flexibility of composite films. Using the tensile strength and nominal strain at break of composite films as indicators, the preparation process of composite films was examined to obtain the composite film with the best overall performance. The mechanical property analysis results of the three tea tree oil-thyme oil-chitosan composite films prepared in Examples 1-3 with different essential oil addition amounts are shown in Table 2.

[0069] Table 2 shows that the tensile strength of the composite membrane increases and then decreases with increasing essential oil content, reaching its maximum at an essential oil content of 2%. This may be because an appropriate amount of essential oil can increase fluidity and improve membrane extensibility, but excessive essential oil may lead to increased intermolecular repulsion, larger intermolecular gaps, and decreased tensile strength. The nominal fracture strength of the composite membrane should gradually decrease with increasing essential oil content, reaching its maximum at 1%. This may be due to the hydrophobicity of the essential oil altering intermolecular forces and molecular arrangement. Mechanical property analysis shows that the composite membrane possesses certain extensibility and flexibility.

[0070] Table 2 Mechanical properties of chitosan composite membranes with different essential oil addition amounts

[0071]

[0072] Total bacterial count is an indicator of the degree of food contamination. Observing the dynamics of bacterial reproduction during food storage can provide a basis for the hygienic evaluation of food. Figure 1As shown, the initial total bacterial count of sweet cherries during storage was 3.92 log CFU / g. The total bacterial count increased with prolonged storage, but decreased in the later stages of storage. This is because spoilage leads to nutrient loss and moisture reduction, which is unfavorable for bacterial growth and reproduction. The blank control group showed the fastest bacterial growth and reproduction rate, reaching a total bacterial count of 7.20 log CFU / g on day 12. All membrane-sealed groups exhibited some antibacterial effect, with the composite membrane showing superior performance compared to ordinary polyethylene preservation film. The 2% TTO-TEO and 3% TTO-TEO groups showed the best antibacterial and preservation effects, effectively extending the storage time of sweet cherries. This may be because the plant essential oils and chitosan in the composite membrane have antibacterial activity.

[0073] Yeast and mold are widespread in the natural environment and are among the causes of food spoilage. When yeast and mold contaminate food, they not only alter its color, aroma, taste, and shape, but also cause the loss of nutrients, reducing its edible value, and may even produce toxins that harm human health. Therefore, yeast and mold are indicator bacteria for evaluating food hygiene quality. Figure 2 As shown, the initial total number of mold and yeast cells in sweet cherries was 0.69 log (CFU / g). With prolonged storage, the total number of mold and yeast cells initially increased and then decreased. This may be because the loss of nutrients and moisture, along with changes in the environmental pH caused by early growth metabolites, were unfavorable for the continued growth of yeast and mold. On day 12, the total number of mold and yeast cells reached its highest point: 4.3 log CFU / g in the blank control group and the ordinary polyethylene film group, 3.3 log CFU / g in the 1% TTO-TEO group, and 2.5 log CFU / g and 2.6 log CFU / g in the 2% TTO-TEO group and 3% TTO-TEO group, respectively. The addition of tea tree oil and thyme oil effectively inhibited the growth of mold and yeast, with the 2% TTO-TEO group showing the strongest inhibitory effect and the best preservation effect.

[0074] Figure 3 The effect of different amounts of essential oils added to chitosan composite membranes on the soluble solids content of sweet cherries. Figure 3It was found that the soluble solids content of sweet cherries decreased during storage. This is because some soluble solids participated in the plant's respiration. Cherries are non-climacteric fruits, producing very little ethylene, but the pedicel is a climacteric. As storage time increased, the pedicel primarily produced ethylene, promoting respiration and leading to an overall decrease in the soluble solids content of sweet cherries. The loss of soluble solids content in the composite film group was slower than that in the blank control group and the ordinary polyethylene preservation film group. The 2% TTO-TEO group showed the slowest loss of soluble solids content, with a soluble solids content of 17.60% on day 16, which was 4.60 percentage points higher than the blank control group and 3.60 percentage points higher than the ordinary polyethylene preservation film group. This may be because the composite film can effectively reduce oxygen concentration, increase carbon dioxide concentration, inhibit plant respiration, reduce soluble solids loss, and extend shelf life.

[0075] Figure 4 The effect of different essential oil addition amounts on the measurable acid content of chitosan composite membranes in sweet cherries. Figure 4 It was found that the measurable acid content decreased with prolonged storage time, while the measurable acid content in the composite membrane sealing treatment group remained at a relatively high level. At day 8, the differences in measurable acid content began to widen. The blank control group showed the most significant decrease in measurable acidity, from 0.87% to 0.51%, while the 2% TTO-TEO group showed the most gradual decrease in measurable acidity throughout the storage period, from 0.87% to 0.74%. This is because the composite membrane sealing treatment inhibits cherry respiration and metabolism, reduces the consumption of organic acids, and maintains the sweet cherry flavor during storage.

[0076] Vitamin C is one of the most important nutrients and antioxidants in fruits and vegetables, playing various biological roles in the human body and scavenging free radicals generated during production. The effect of different amounts of essential oils added to chitosan composite membranes on the vitamin C content of sweet cherries is as follows: Figure 5 As shown, the vitamin C content decreased during the post-harvest storage of sweet cherries. This is because organic acid vitamin C is typically used as a major substrate for respiration and other metabolic processes. During storage, the vitamin C content in all experimental groups was significantly higher than that in the control group. This indicates that the composite film sealing treatment can inhibit the decomposition of vitamin C in sweet cherries. This is because the oxygen barrier properties of the composite film reduce the oxygen concentration inside the fruit, decrease its respiration intensity and related enzyme activity, and slow down the fruit's own water transpiration and metabolism, thereby reducing vitamin C decomposition. The tea tree oil-thyme oil-chitosan composite film has better gas and moisture barrier properties than ordinary polyethylene preservation film, further improving the storage quality of sweet cherries. Among them, the 2% TTO-TEO group showed the lowest vitamin C content loss and the best preservation effect.

[0077] Figure 6 The effect of different essential oil addition amounts on the weight loss rate of sweet cherries. Figure 6 It was found that the weight loss rate of all groups increased during the storage of sweet cherries. The control group had the highest weight loss rate, reaching 28.29% on day 16. Compared with the control group, the composite film covering treatment effectively reduced weight loss, and the composite film had a better water retention effect than ordinary polyethylene film. The 2% TTO-TEO group had a weight loss rate of 10.21% on day 16, which was 18.08 percentage points lower than the control group, showing the best inhibitory effect on the loss of water and other substances in sweet cherries. The chitosan in the composite film has multiple hydroxyl and amino groups, which have certain water retention properties; the plant essential oils enhanced the hydrophobic properties of the composite film, further isolating it from external air and reducing moisture loss.

[0078] The changes in the rot rate of sweet cherries during storage in each experimental group are shown in the figure. Figure 7 As can be seen, the blank control group experienced the fastest decay rate, with a rapid increase in decay rate during the later stages of storage, reaching 49.10% on day 16. This is likely due to the direct contact between the sweet cherries and environmental microorganisms in this group. The ordinary polyethylene film group also showed a relatively fast decay rate, reaching 41.61% on day 16. Compared to the blank control group and the ordinary polyethylene film group, the composite film group was more effective in inhibiting the decay rate of sweet cherries. Among them, the 2% TTO-TEO group showed the best preservation effect, with a decay rate of 3.50% on day 16, while the 1% TTO-TEO group showed a poorer preservation effect, with a decay rate of 13.37% on day 16. This is because thyme essential oil and tea tree essential oil are rich in antibacterial active substances such as alkenes and phenols, which can effectively disrupt the cell membrane structure of microorganisms, thereby reducing the decay rate of sweet cherries.

[0079] Figure 8Sensory changes in sweet cherries were investigated in groups with different essential oil additions (chitosan composite film, ordinary polyethylene preservation film, and blank control). Throughout the storage period, the sweet cherries in the blank control group and the ordinary polyethylene preservation film group showed the greatest sensory changes. By day 6, their sensory evaluation scores were all below 60 points, rendering them commercially worthless. By days 8 and 10, respectively, the fruit exhibited slight browning and softening, with a mild off-odor, rendering them inedible. The 2% TTO-TEO group and the 3% TTO-TEO group achieved sensory scores of 71 and 67 points, respectively, by day 16. The fruit color had slightly darkened, the pulp tissue had slightly softened, and there was no off-odor, retaining some commercial value. The 1% TTO-TEO group showed a significant darkening of the fruit color and a slightly increased degree of pulp softening by day 12, rendering it commercially worthless. The composite film prepared in this invention has excellent barrier properties, which can regulate the concentration of carbon dioxide and oxygen within the packaging, inhibiting the respiration of sweet cherries, reducing nutrient consumption, and maintaining their nutritional value. The oils in the composite film can form a hydrophobic layer on its surface, reducing the contact between water vapor and the film. This forces the water vapor to remain around the sweet cherries, minimizing moisture loss and keeping the fruit plump and glossy, thus maintaining its sensory quality. Thyme oil, tea tree oil, and chitosan have certain antibacterial effects, not only inhibiting the growth and reproduction of microorganisms within the composite film itself, but also mitigating the rotting of sweet cherries due to the volatility and diffusion of their active substances. This indicates that the composite film prepared in this invention has a significant preservation effect on sweet cherries, with the 2% TTO-TEO group showing the best results.

[0080] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0081] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a tea tree oil-thyme oil-chitosan composite membrane, characterized in that, Includes the following steps: Polyvinyl alcohol is dissolved in water to obtain a polyvinyl alcohol solution; After adding glacial acetic acid, chitosan, plasticizer and emulsifier to polyvinyl alcohol and mixing evenly, tea tree oil and thyme oil are added, and after homogenization and degassing, a film-forming solution is obtained; the ratio of chitosan to tea tree oil is 1g:1mL~3mL; the volume ratio of tea tree oil to thyme oil is 1:

1. The film-forming solution was dried to form a film, resulting in a tea tree oil-thyme oil-chitosan composite membrane. The ratio of polyvinyl alcohol to glacial acetic acid is 3g:1mL, and the mass ratio of polyvinyl alcohol to chitosan is 6:

1. Tea tree oil and thyme oil are used to increase fluidity and enhance the mechanical properties of the tea tree oil-thyme oil-chitosan composite film.

2. The method for preparing a tea tree oil-thyme oil-chitosan composite membrane according to claim 1, characterized in that, The concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 3%.

3. The method for preparing a tea tree oil-thyme oil-chitosan composite membrane according to claim 1, characterized in that, The ratio of chitosan to plasticizer is 1g:4mL.

4. The method for preparing a tea tree oil-thyme oil-chitosan composite membrane according to claim 1, characterized in that, The plasticizer is propylene glycol.

5. The method for preparing a tea tree oil-thyme oil-chitosan composite membrane according to claim 1, characterized in that, The emulsifier is Tween 80.

6. A tea tree oil-thyme oil-chitosan composite membrane prepared by the preparation method according to any one of claims 1-5.

7. The application of the tea tree oil-thyme oil-chitosan composite membrane as described in claim 6 in the preservation of sweet cherries.

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