Application of tea tree oil active ingredients in preparation of fruit and vegetable fresh-keeping preparation

Abstract

The application belongs to the field of preservation technology, and particularly relates to application of tea tree oil active ingredients in preparation of fruit and vegetable preservation preparation. The tea tree oil active ingredients are 4-terpineol, p-cymene or alpha-muurolene. The preparation is used for inhibiting common rot fungi of fruits and vegetables after harvesting, and the rot fungi include Alternaria spp., Botrytis spp., Penicillium spp., Fusarium spp. and the like. The preparation can also reduce softening of fruits and vegetables, and reduce the degree of membrane lipid peroxidation of fruit and vegetable tissues. The preparation is specifically a fumigation preparation or an atomization preparation. The tea tree oil active ingredients are used for fumigation treatment or atomization treatment of fruits and vegetables in a relatively closed space, and the treated fruits and vegetables are stored. The incidence of the treated fruits and vegetables is significantly reduced, aging is delayed, and the quality of the fruits is maintained.

Description

Technical Field

[0001] This invention belongs to the field of preservation technology, specifically relating to the application of active ingredients in tea tree oil in the preparation of fruit and vegetable preservation agents. Background Technology

[0002] Fruits and vegetables are stored in concentrated areas after harvest and face multiple threats, including rapid aging, logistical damage, and microbial contamination. The loss rate is generally around 20%, and improper handling can lead to even greater losses. Berries, in particular, are among the most difficult fruits and vegetables to store and transport due to their thin skins, which are easily damaged by mechanical forces and susceptible to contamination by putrefactive microorganisms.

[0003] To reduce the harm of microorganisms during the storage and transportation of harvested fruits and vegetables, current technologies mostly employ fumigation with sulfur dioxide, chlorine dioxide, or ozone. These treatments produce a pungent odor during processing and often leave unpleasant smells afterward. Some treated fruits also suffer from poor flavor, bitterness, or strange tastes. Therefore, actively seeking natural preservation methods is currently a hot topic in post-harvest research on fruits and vegetables.

[0004] Plant essential oils are a class of highly volatile, deeply penetrating plant secondary metabolites that are less prone to developing resistance. They possess insecticidal, antibacterial, antifungal, antioxidant, and anticancer properties. In recent years, there has been considerable research on the use of plant essential oils as preservatives in postharvest preservation of fruits and vegetables. While their antibacterial effects are excellent, the unpleasant odor of essential oils hinders widespread adoption. The applicant team initially selected tea tree oil by evaluating its antibacterial effect against common postharvest spoilage pathogens in fruits and vegetables, as well as its pleasant odor.

[0005] Tea tree oil has a mild aroma and is widely used in the cosmetics and food flavoring industries. Reports indicate that tea tree oil, used alone or in combination with other ingredients, can improve the quality of postharvest bananas, lilies, strawberries, and areca nuts. There are also reports that treatment with tea tree oil can reduce the damage caused by postharvest peach brown rot (Monilinia fructicola), tomato early blight (Alternaria solani), and strawberry gray mold (Botrytis cinerea). However, there are no reports of fumigation treatments of fruits and vegetables for disease resistance studies, and the specific active ingredients in the oil that inhibit fungi are unknown. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide the application of active ingredients of tea tree oil in the preparation of fruit and vegetable preservation agents, as well as a method for post-harvest preservation of berries.

[0007] The present invention adopts the following technical solution:

[0008] On the one hand, the present invention provides the application of active ingredients of tea tree oil in the preparation of postharvest preservatives for fruits and vegetables, wherein the active ingredients of tea tree oil are 4-terpineol, p-cymene, or α-terpinene.

[0009] As a preferred application of the present invention, the preparation is a fumigation preparation or atomized preparation. Currently, there is a great deal of research on natural plant essential oils, and certain results have been achieved. However, the commercial application of natural plant essential oils is still a long way off. Existing application technologies mostly involve soaking or spraying fruits with essential oils, which can easily damage the sensory indicators of fruits and vegetables. Furthermore, post-harvest processing procedures are cumbersome and increase labor costs. Since plant essential oils are volatile, fumigation or atomization methods can be considered to avoid direct contact with fruits and vegetables.

[0010] As a preferred application of the present invention, the formulation is used to inhibit putrefactive fungi, including fungi of the genera *Alternaria*, *Botrytis*, *Penicillium*, and *Fusarium*.

[0011] As a preferred application of the present invention, the formulation is used to reduce fruit and vegetable rot caused by the putrefactive fungi.

[0012] As a preferred application of the present invention, the formulation is used to alleviate the softening of fruits and vegetables caused by the putrefactive fungi.

[0013] As a preferred application of the present invention, the formulation is used to reduce the degree of lipid peroxidation in fruit and vegetable tissue membranes.

[0014] As a preferred application of the present invention, the fruits and vegetables include blueberries, raspberries, cherries, mulberries, honeysuckle berries, currants, plums, hardy kiwifruit, kiwifruit, grapes, tomatoes, sea buckthorn, cranberries, dates, and small apples.

[0015] In another aspect, the present invention provides a method for post-harvest preservation of berries, including the steps of using the active ingredients of the tea tree oil to fumigate or atomize the fruits and vegetables in a closed environment and then storing them.

[0016] As a preferred embodiment of the method described in this invention, the concentration of volatile active ingredients in the tea tree oil in the enclosed environment is 1–2000 μL / L.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention utilizes 4-terpineol and 4-terpineol-type tea tree oil to fumigate postharvest berries, aiming to reduce the occurrence of postharvest diseases in fruits and vegetables and improve their disease prevention and preservation effects. Fruits treated with this method, when stored under suitable conditions, show a significant reduction in disease incidence, as well as a significant increase in firmness and soluble solids content. This invention's postharvest disease prevention and preservation method for fruits and vegetables effectively reduces diseases during postharvest storage, significantly lowers the incidence rate, and improves disease prevention and preservation effects. The method is safe, environmentally friendly, easy to operate, and readily applicable. Attached Figure Description

[0019] Figure 1 The treatment method is flat-plate fumigation.

[0020] Figure 2 To assess the antibacterial effects of different essential oils on Alternaria target bacteria, AJ represents: perilla essential oil, tea tree essential oil, grapefruit peel essential oil, arborvitae essential oil, thyme essential oil, Sichuan pepper essential oil, mugwort essential oil, rosemary essential oil, and sweet orange essential oil.

[0021] Figure 3 The antibacterial effects of different components of tea tree oil on Alternaria alternifolia.

[0022] Figure 4 The antibacterial effects of different components of tea tree oil on Botrytis cinerea fungus.

[0023] Figure 5 The quality of blueberries treated with 400 μL / L 4-terpineol and then inoculated with Alternaria alternata was measured after storage at 25°C for 5 days (left image is the control group, right image is the 400 μL / L 4-terpineol treatment group).

[0024] Figure 6 The quality of blueberries after fumigation with 400 μL / L 4-terpineol followed by inoculation with Botrytis cinerea and storage at 25°C for 5 days (left image is control, right image is 400 μL / L 4-terpineol treatment group). Detailed Implementation

[0025] The present invention will be described in detail below with reference to specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention's description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0027] Unless otherwise stated or in case of contradiction, the terms or phrases used in this invention shall have the following meanings:

[0028] The optional range of the term "and / or" as used herein includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0029] In this invention, "preferred" is merely a description of a more effective implementation method or embodiment, and should be understood as not constituting a limitation on the scope of protection of this invention.

[0030] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0031] In this invention, numerical ranges are involved, and unless otherwise specified, they include the two endpoints of the numerical range.

[0032] Unless otherwise specified, the percentage content involved in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures and gas-phase-gas mixtures.

[0033] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0034] The perilla essential oil, tea tree essential oil, grapefruit peel essential oil, arborvitae essential oil, thyme essential oil, Sichuan pepper essential oil, artemisia essential oil, rosemary essential oil, and sweet orange essential oil involved in the following embodiments of the present invention were purchased from Guangzhou Yimei Raw Materials Co., Ltd.; 4-terpineol, p-cymene, and α-terpinene were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0035] The putrefactive fungi involved in the following embodiments of the present invention were isolated by our team. The disease samples were post-harvest rotten fruits and vegetables. After the DNA of the purified putrefactive fungi was extracted, the ITS sequence was amplified and sent to bioengineering sequencing. The sequence was then compared with the NCBI database (blast.ncbi.nlm.nih.gov). The genus-level identification results of the fungus were determined by combining the morphological observation of the pathogen.

[0036] In the following embodiments of the present invention, the fumigation treatment of putrefactive fungal colonies all employs the plate fumigation method. After culturing the target bacteria for 5 days, a 5 mm diameter fungal cake is taken under aseptic conditions and placed in the center of the PDA culture medium at the bottom of a dish. A filter paper is placed on the dish lid, and a certain concentration of essential oil or its main component is dripped onto the center of the filter paper. The dish is then inverted onto the lid. A control group is prepared by adding water (see...). Figure 1 The plates were sealed with sealing film, inverted, and each treatment was repeated three times. The volatile concentration was determined by the ratio of the volume of the added essential oil or active ingredient to the volume of the sealed container.

[0037] The inhibition rate (%) of plate fumigation method = [(diameter of fungal colonies in control group - diameter of fungal colonies in treatment group) / (diameter of fungal colonies in control group)] × 100%;

[0038] The following are specific examples. Unless otherwise specified, the raw materials used in the examples are all commercially available products.

[0039] This invention evaluates the inhibitory effects of different essential oils on postharvest rot-causing fungi. Using *Alternaria tenuissima*, a common rot-causing fungus in harvested fruits and vegetables, as the target fungus, the antibacterial effects of different essential oils were determined using the plate fumigation method (see [link to article]). Figure 2 The results showed that perilla and tea tree oils had good antibacterial effects. Based on their antibacterial effects, tea tree oil with a light and pleasant aroma was selected through aroma evaluation.

[0040] To prevent inconsistencies in the antibacterial effects of tea tree oil from different batches and manufacturers, this invention further determined the antibacterial effects of three main volatile components in tea tree oil: 4-terpineol, cymene, and α-terpinene (see...). Figure 3 , Figure 4 (Tables 1 and 2). This assay used the plate fumigation method, targeting *Alternaria* sp. and *Botrytis* sp. fungi, with the three main components used at a concentration of 100 μL / L. The results showed that 4-terpineol exhibited the best antibacterial effect.

[0041] Table 1. Antibacterial effects of different components of tea tree oil on Alternaria fungi.

[0042]

[0043] Table 2. Antibacterial effects of different components of tea tree oil on Botrytis cinerea fungi.

[0044]

[0045] Based on the above findings, this invention utilizes 4-terpineol, p-cymene, and α-terpinene for postharvest disease prevention and preservation treatment of fruits and vegetables.

[0046] The following are specific examples. Unless otherwise specified, the raw materials used in the examples are all commercially available products.

[0047] Example 1

[0048] Different concentrations of 4-terpineol were investigated for their effects on common postharvest rot-causing fungi of the genera *Alternaria*, *Botrytis*, *Penicillium*, and *Fusarium*. A plate fumigation method was used with 4-terpineol concentrations of 10, 50, 100, 200, and 400 μl / L, with plates without added 4-terpineol serving as a control.

[0049] The results showed that 4-terpineol had good inhibitory effects on all the tested target bacteria. Specifically, 100 μl / L of 4-terpineol achieved an inhibition rate of 97% against Alternaria alternata (see Table 3), 50 μl / L of 4-terpineol achieved an inhibition rate of 95% against Botrytis cinerea (see Table 4), 50 μl / L of 4-terpineol achieved an inhibition rate of 98% against Penicillium (see Table 5), and 50 μl / L of 4-terpineol achieved an inhibition rate of 88% against Fusarium (see Table 6).

[0050] Table 3. Inhibition rate of different concentrations of 4-terpineol against Alternaria alterniflora.

[0051]

[0052] Table 4. Inhibition rate of different concentrations of 4-terpineol against the tested Staphylococcus aureus.

[0053]

[0054] Table 5. Inhibition rate of different concentrations of 4-terpineol against the tested Penicillium species.

[0055]

[0056] Table 6. Inhibition rate of different concentrations of 4-terpineol against the tested Fusarium spores.

[0057]

[0058] Example 2

[0059] In this embodiment, blueberry fruits were treated with different concentrations of 4-terpineol (100, 200, 300, and 400 μL / L). The treated fruits were then inoculated with *Alternaria sp.* to evaluate the fruit rot index, firmness, and malondialdehyde (MDA) content under *Alternaria sp.* stress. MDA is a commonly used indicator for measuring the degree of oxidative stress and reflects the extent of damage to plant cell membranes.

[0060] The method described in this example specifically includes the following steps:

[0061] (1) Select blueberries that are uniform in maturity, size, and free from pests, diseases, and mechanical damage;

[0062] (2) Place the fruit from step (1) in an 8L preservation box, attach filter paper to the lid, and calculate the volume of 4-terpineol to be added based on the volume of the preservation box, so that the final concentration of 4-terpineol volatiles in the preservation box reaches 100, 200, 300, and 400 μL / L, respectively, with water added as a control group. Place the blueberries in the above-mentioned different preservation boxes and treat them under relatively sealed conditions for 3 hours. After that, remove the blueberries, treat them by dispersing for 30 minutes, and then inoculate the stem end with 10 μL of Alternaria spore suspension (concentration of 1×10⁻⁶). 5 After inoculation, the blueberries were stored at 25°C and relative humidity of 5-95% for 5 days before observation.

[0063] Fruit rot grading standards (0-4): The higher the value, the more severe the disease. Blueberry fruit without disease is grade 0; fruit lesion diameter ≤ 3 mm is grade 1; fruit lesion diameter ≤ 6 mm is grade 2; fruit lesion diameter ≤ 10 mm is grade 3; fruit lesion diameter ≤ 10 mm is grade 4.

[0064] Fruit rot index = ∑(number of rotten fruits at each level × value of that level) / (total number of fruits surveyed × highest level value) × 100

[0065] Hardness was measured using a texture analyzer.

[0066] Method for determining malondialdehyde (MDA) content: Accurately weigh 3.0 g of blueberry pulp tissue, add 5.0 mL of 100 g / L trichloroacetic acid (TCA) solution, grind into a homogenate, centrifuge at 4℃ and 10000×g for 10 min, collect the supernatant, and store at 4℃ for later use. Take 2.0 mL of sample supernatant (CK blank control group) and add 2.0 mL of 100 g / L TCA solution. Add 2.0 mL of 0.67% thiobarbituric acid (TBA) solution to each sample, shake to mix, boil in a water bath at 100℃ for 10 min, remove and cool on ice, then centrifuge again at 4℃ and 10000×g for 10 min. Measure the absorbance of the supernatant at wavelengths of 450 nm, 532 nm, and 600 nm. Repeat 3 times.

[0067] MDA content (mmol / Kg) = 6.45 × (OD) 532 -OD 600 -0.56×OD 450

[0068] The results showed that on day 0 of storage at 25 degrees Celsius, the fruit rot index of all groups was 0, the firmness was 1.52 N, and the MDA content was 0.59 mmol / Kg. The fruit quality of each group after 5 days of storage is shown in Table 7.

[0069] Table 7 Quality Indicators of Blueberries Inoculated with Alternaria sp.

[0070]

[0071] Table 7 shows that, compared with the water control, fumigation of blueberries with 4-terpineol significantly reduced Alternaria sp. rot within the same storage time, alleviated fruit softening, and decreased malondialdehyde (MDA) content. The effect was even better as the concentration of 4-terpineol volatiles increased within the range of 100–400 μL / L. The preservation effect of 400 μL / L 4-terpineol treatment on blueberries is shown in Table 7. Figure 5 .

[0072] Example 3

[0073] This embodiment is basically the same as embodiment 2 in operation, except that 10 μL of a suspension of *Botrytis* sp. fungi (concentration 1×10⁻⁶) is inoculated at the fruit stem. 5 (CFU / mL)

[0074] The results showed that on day 0 of storage at 25 degrees Celsius, the fruit rot index of all groups was 0, the firmness was 1.52 N, and the MDA content was 0.59 mmol / Kg. The fruit quality of each group after 5 days of storage is shown in Table 8.

[0075] Table 8. Quality indicators of blueberries inoculated with *Botrytis sp.*

[0076]

[0077] As shown in Table 8, compared with the water control, fumigation treatment of blueberries with 4-terpineol significantly reduced Botrytis sp. rot within the same storage time, alleviated fruit softening, and decreased malondialdehyde (MDA) content. The effect was even better as the concentration of 4-terpineol volatiles increased within the range of 100–400 μL / L. The preservation effect of 400 μL / L 4-terpineol treatment on blueberries was shown in Table 8. Figure 6 .

[0078] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0079] 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.

[0080] 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. The application of active ingredients from tea tree oil in the preparation of postharvest preservatives for blueberries, characterized in that, The active ingredient in the tea tree oil is 4-terpineol. The application includes the step of fumigating and storing blueberries with the active ingredient in the tea tree oil in a closed environment, wherein the concentration of the volatile components of the active ingredient in the tea tree oil in the closed environment is 100-400 μL / L. The formulation is used to inhibit putrefactive fungi, which are Alternaria species (Alternaria). Alternaria spp.) or Botrytis ( Botrytis (spp.); The formulation is used to reduce blueberry rot or softening caused by the putrefactive fungi and to reduce the malondialdehyde content of blueberries.

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