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. 2025 Mar 8;34(9):1921–1933. doi: 10.1007/s10068-024-01812-9

Application of tea tree oil nanoemulsion pads in Lateolabrax japonicas fillets

Qian Feng 1, Ying Song 1,2, Xinyu Liu 3, Yanru Huang 1, Junyi Yang 1, Yingchang Li 1,✉, Qiuying Li 1, Tong Sun 1,✉
PMCID: PMC11972239  PMID: 40196340

Abstract

Abstract

To improve the efficiency of biological preservatives, a novel slow-release system was constructed. The oil-in-water (O/W) nanoemulsions were prepared with tea tree essential oil (TTO) and its main components, 1,8-cineole (CN) and terpinen-4-oil (T4O) as core materials, and with tea saponin as surfactant. The preservation properties of the pad containing nanoemulsion slow-release system on Lateolabrax japonicus fillets were measured. The results showed that the nanoemulsion had good stability and can delay the release of essential oil, and the cumulative release percentage of TTO was as high as 81 % at 72 h. The establishment of nanoemulsions slow-release system effectively improved the preservation properties of the pad, and TTO nanoemulsion pad (TTO-NE-P) had the optimal preservation properties due to the synergistic effect of preservative ingredients and the sustained release system of the nanoemulsion. This study can provide technical support for the combined application of biological preservative agent and aquatic product pads.

Graphical abstract

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Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-024-01812-9.

Keywords: Tea tree essential oil, Nanoemulsion, Preservation pad, Lateolabrax japonicus

Introduction

The living standards of the public are gradually improving, and the requirements for the freshness of aquatic products are higher. Lateolabrax japonicas meat is rich in protein, polyunsaturated fatty acids such as docosahexaenoic acid (DHA), which is highly favored by the consumers (Chen et al., 2024). However, it is easy to corrupt under the action of microorganisms and endogenous enzymes during storage, processing, and sales (Lan et al., 2024). Therefore, adopting appropriate preservation techniques to enhance the commercial value of fish fillets has become one of the hotspots of attention both domestically and internationally.

Traditional food packaging can only isolate food from the external environment and cannot meet people’s needs for food preservation. Active packaging can not only meet people’s packaging needs for food, but also optimize the internal environment of the packaging (Sul et al., 2023). It can effectively protect the food quality and extend shelf life. The preservation pad is a type of active packaging with a porous structure and high specific surface area and is an ideal carrier for loading antibacterial agents. The pad is placed in the fresh food product tray package to absorb the exudate to alleviate the infection of microorganisms.

Antibacterial and antioxidant preservatives can also be added into the pad to improve the preservation effect (Jiao et al., 2023). Chemical preservation technology is the use of chemical drugs to extend the shelf life of food products. However, most chemical preservatives are easy to remain, their application scope is limited. Biological preservatives are a class of substances with antibacterial and antioxidant activity, naturally occurring in animals, plants and microorganisms or their products, non-toxic and harmless.

Tea tree essential oil (TTO) is a colorless to yellowish light oily liquid get past steam distilled from fresh foliage of Melaleuca alternifolia Cheel (Hammer, 2015). It is mild and safe and has broad-spectrum antibacterial properties, its chemical composition is very complex. Among them, terpinen-4-ol (T4O) and 1,8-cineole (CN) are the characteristic components of TTO (Brun et al., 2019). T4O has excellent biological activity and broad-spectrum antimicrobial activity. CN has a variety of biological and pharmacological activities and antibacterial, insecticidal, and anti-inflammatory activities (Hoch et al., 2023) However, low stability and poor water solubility limit their effectiveness in practical applications. To extend the preservation effect of preservatives added to the pad, it is urgent to build a slow-release system.

Nanoemulsion is an idea slow-release system due to its advantages of good stability and slow-release properties for embedded active substances (Wang et al., 2024). The water solubility, stability, and biocompatibility of essential oil can be achieved by nanoemulsion, thus promoting its application in the food field. Nanoemulsion treatment can delay the spoilage of fresh food, to ensure its quality and safety (da Silva et al., 2022). Tea saponin is a natural non-ionic surfactant (Zhao et al., 2024). It has excellent foam stability, dispersibility, emulsification, antibacterial and other functions. It has been widely used in food, chemical industry, and other industries (Xie et al., 2023).

In this work, to extend the effect of biological preservative and improve the preservation properties of the pad with biological preservative, a nanoemulsion slow-release system were constructed in the preservative pad, and the slow-release behavior of the preservative agent from the nanoemulsion were studied. TTO and its main components were used as preservatives to study whether there was a synergistic effect between the main components. Taking L. japonicus fillets as preservation objects, the preservation properties of the nanoemulsion preservation pad were verified, and the preservation mechanism was clarified (Fig. 1). The technology of adding the slow-release system to the preservation pad proposed in this study has a good application prospect.

Fig. 1.

Fig. 1

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The general schematic diagram of the preparation of nanoemulsion, the mechanism of unidirectional water-conducting preservation pad, there lease of antibacterial agent and the antibacterial mechanism

Materials and methods

Fresh L. japonicus of 1.80 ± 0.10 kg, was purchased from Jinzhou Aquatic Product Market (Liaoning, China). TTO was purchased from Yisenyuan Plant Fragrance Co., Ltd. (Jiangxi, China). CN (99%), T4O (98%) were purchased from Meiruier Chemical Technology Co., Ltd. (Shanghai, China). Tea saponin was purchased from Jinsui Biotechnology Co., Ltd. (Shanghai, China). Medium chain triglycerides (MCT) were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Hematoxylin–eosin kit was purchased from Solaibao Technology Co., Ltd. (Beijing, China). Shewanella putrefaciens and Pseudomonas fluorescens were isolated from salmon and preserved in the laboratory. All other chemical and biological reagents were analytical grade.

Preparation of the tea tree essential oil nanoemulsions

Oil-in-Water (O/W) nanoemulsion was prepared by ultrasonic emulsification method. Tea saponin of 2 g was dissolved in 90.00 g deionized water as the water phase. Ten g MCT, 2.00 g MCT and 8.00 g CN, 2.00 g MCT and 8.00 g T4O, 2.00 g MCT and 8.00 g TTO were mixed and used as four different oil phases to prepare 4 different nanoemulsions, respectively. The water phase is slowly added to the oil phase, and the coarse emulsion is formed by high-speed homogenization (10,000 r/min, 3 min). The coarse emulsion was put in the ultrasonic cell crusher (SCIENTZ-IID, China) for ultrasonic 10 min, and the nanoemulsion was cooled in ice water bath, then the coreless nanoemulsion (NE), CN nanoemulsion (CN-NE), T4O nanoemulsion (T4O-NE) and TTO nanoemulsion(TTO-NE) were prepared respectively. The samples were stored at 4 °C in sterile screw cap glass reagent bottles for stand.

Preparation of the nanoemulsion preservation pads

TTO-NE composite loaded core paper was prepared by loading 10 g TTO-NE on 13 cm × 18 cm absorbent paper and then was dried at room temperature. The bottom film supported by absorbent agent was prepared by uniformly loading 0.45 g bentonite and 0.85 g cotton on the surface of 15 cm × 20 cm absorbent paper. The food grade adhesive was adhere to the front and back sides of the TTO-NE loaded core paper. One side is an absorbent loaded bottom film with equal area, and the other side is a non-woven fabric with equal area and permeability, and then the TTO-NE preservation pad (TTO-NE-P) was prepared after press together. The above-mentioned TTO-NE is replaced with NE, CN-NE, T4O-NE, and TTO ethanol solution with the same concentration as TTO-NE respectively, the corresponding preservation pads were prepared. Respectively named as NE-P, CN-NE-P, T4O-NE-P, TTO-P. The blank absorbent preservation pad (PAD) was prepared with blank absorbent paper as the core materials.

Characterization and physicochemical properties of the nanoemulsions

The microstructure of nanoemulsion was observed by confocal laser scanning microscope (CLSM, Leica SP8, Germany). The diameter distribution of nanoemulsion was calculated by ImageJ software (1.8.0, National Institutes of Health, USA). The apparent shape and size of the nanoemulsion were visualized by field emission transmission electron microscopy (TEM, Jem-2100F, Japan Electronics Co., Ltd, Japan).

The zeta potential of nanoemulsion was determined by zeta potential and nano-particle size analyzer (ZS90, Malvern, England). The polydispersity index (PDI) was determined by laser particle size analyzer (S3 plus, Dandong Bettersize Instruments Ltd., China). The pH of emulsion is measured with a pH-meter (FE20, Zhengzhou Great Wall Branch Industry and Trade, China). The static contact angle of nanoemulsion was measured at room temperature by video optical contact angle meter (OCA 25, Data Physics Inc., Feldstadt, Germany). Referring to the method of Sahafi et al. (2018) to determine the turbidity. The L, a and b values of the nanoemulsion were measured with a colorimeter (CR400, KONICA MINOLTA, China). The whiteness index (WI) is calculated as below:

WI=100-100-L2+a2+b2 1

The thermal stability of the nanoemulsion was measured by differential scanning calorimeter (Q2000, TA Instruments, USA), protected by N2, with a heating rate of 10 °C/min at 80–180 °C. The rheological properties of the nanoemulsion were measured by Rheometer (Discovery DHR-1, ARES-RFS, England).

The nanoemulsion of 4.0 mL was placed in a dialysis bag, and the dialysis bag was placed in 100 mL PBS phosphate buffer (5 mmol/L, pH = 7.2) at 4 °C. At regular intervals, 2 mL of dialysate was removed and an equal volume of PBS solution was added. The essential oil content at different sampling times was determined by high performance liquid chromatography (LC-2030, Shimadzu‌, Japan), and the cumulative release of essential oil from the nanoemulsion was calculated according to Cheng et al. (2019).

Referring to Li et al. (2023) method, the average particle size, PDI, and zeta potential were measured after exposing the nanoemulsions to each treatment, and the effects of different temperature (4, 15, and 25 °C), centrifugation (centrifugal speed 10,000 r/min, centrifugal time 10 min), and pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) on the stability of the nanoemulsions were studied.

The antioxidant activity of the nanoemulsion was determined by 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) rapid assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay (Zhang et al., 2024a). Using Shewanella putrefaciens and Pseudomonas fluorescens as model bacteria, adding 200 μL nanoemulsion, the antibacterial activity was determined by Oxford cup method.

Determination of the freshness of L. japonicus fillets

Living L. japonicas was sudden death in ice, and the fish fillets were taken on both sides of the dorsal fish, each weighing 100 ± 5 g. The fish fillets were cleaned with sterile deionized water, and the surfaces were wiped with sterile filter paper. The fillets were placed in plastic crisper containers with different pads. The unpadded plastic crisper container was used as the control one and named CK. The fillets were refrigerated at 4 °C, and the relevant preservation indexes were measured every 3 d.

According to a previous study, L. japonicus fillets were evaluated by using the weighted method (Liu et al., 2021). The maximum score was 10, the acceptable minimum was 3. Referring to Mai’s method, the total viable count (TVC) of fish fillets shall be processed, cultured, and calculated (Mai et al., 2024). Referring to Hou’s method, the pH of the sample was measured (Hou et al., 2024). Thiobarbituric acid (TBA) value of the samples was determined using Huang et al. (2023) method. Referring to Cao’s method, the Total Volatile Base Nitrogen (TVB-N) value of the sample was determined with Kieldahlazotometer (K9840 + SH220F, Haineng Instrument Co., Ltd, China) (Cao et al., 2019). Cutting the L. japonicus fillets into small cubes of 1 cm × 1 cm × 1 cm and frozen at -80 °C. The slices were sliced using a frozen slicer (CM1850, Leica, Germany) and stained with a Hematoxylin–eosin kit. The muscle structure of the fillets was observed 40 times larger under an optical microscope (80-i, Nikon, Japan).

Statistical analysis

All data were measured at least three times, and the results were expressed as “means ± standard deviation”. The plots were performed with Origin 8.5 software, and the one-way analysis of variance were performed with SPSS (version19.0) software. The differences among groups were evaluated by performing Duncan’s test, and the significance level was P < 0.05.

Results and discussion

Characterization and properties of the nanoemulsions

The micromorphologies of the nanoemulsions

The microstructure and droplet distribution of the nanoemulsions can be observed by CLSM. Figure 2 shows the nanoemulsion as a relatively regular spherical drop with a size of 22–60 nm. The average particle size of NE is 42.09 nm, the droplet has a large amount of aggregation, and the emulsion system is not stable. The droplets of CN-NE are few and unclear, which may be related to the properties of the samples themselves. Due to its small particle size, the formed droplet is also small, resulting in faster fluorescence quenching, and the observed droplet is not obvious. Compared with NE, the average particle size of the nanoemulsion with T4O as the core material is reduced by 8.90 nm, and the droplet dispersion is relatively uniform. Compared with NE, the average particle size of TTO-NE is increased by 0.84 nm, and the droplets are spherical and monodisperse. After the addition of core material, the dispersion of nanoemulsions is uniform, indicating that the surfactant tea saponin can cover TTO and its main components and form nanoemulsion with particle size under ultrasonic action, so the flocculation and aggregation between droplets are effectively prevented. Hollow micelles are formed only by the non-polar groups of tea saponin during the formation of emulsion in the coreless material system, and the droplets formed are larger. When TTO or its main component is used as the core material, the non-polar groups of tea saponin in the emulsification process are centered on TTO or its main component, and the micelle droplets formed are smaller.

Fig. 2.

Fig. 2

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CLSM images (1), TEM images (2) and particle size distribution (3) of the nanoemulsions. a NE; b CN-NE; c T4O-NE; d TTO-NE

Physicochemical properties of the nanoemulsions

As shown in Fig. 3(1), the value of zeta potential of all nanoemulsions is negative, and there is electrostatic repulsion between emulsion droplets, and the higher the absolute potential (≥ 30 mV), the more stable the emulsion system (Akbas et al., 2018). The value of zeta potential of NE is − 36.6 ± 0.9 mV. Compared with NE, the zeta potential absolute value of CN-NE is increases by 11.6, which may be related to its molecular structure. CN has better lipophilicity, and the combination of tea saponin is more tightly, which enhances the stability of the nanoemulsion. Compared with NE, there is no significant difference in the potential and stability of T4O-NE, which may be due to the hydrophilicity of T4O because it contains a hydroxyl group. The zeta potential absolute value of TTO-NE is between CN-NE and T4O-NE, indicating that the molecular structure of the core material had an effect on the stability of the nanoemulsion. It is generally believed that the PDI index is less than 0.3, and the nanoemulsion has good uniformity and dispersion(Gul et al., 2018). Figure 3(2) shows that the highest PDI value of NE is 0.314 ± 0.011, indicating that the dispersion system is not uniform and the particle size is different. The dispersion system of the nanoemulsion with CN, T4O and TTO as the core materials is relatively uniform, and the particle size distribution is relatively uniform. When the core material has only one component, the PDI value of the nanoemulsion is smaller, that is, the dispersion system is more uniform, which is consistent with the particle size analysis results. The pH of the four nanoemulsions prepared in this study are all in the range of 5.27–5.40 and were acidic, and there is no significant difference (P > 0.05), indicating that the CN, T4O and TTO have no significant effect on the pH of the nanoemulsions (Fig. 3(3)).

Fig. 3.

Fig. 3

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Physicochemical properties of the nanoemulsions (1) zeta-potential;(2) PDI; (3) pH; (4) The static contact angle;(5) Turbidity; (6) Whiteness Index; (7) Chemical structure of CN; (8) Chemical structure of T4O; (9) Chemical structure of tea saponin. Different lowercase letters (a, b, c, etc.) in the figures indicate significant differences among these groups (p < 0.05)

It is generally believed that the smaller the contact angle and the lower the interfacial tension, the better the emulsifying ability of the emulsifier. As shown in Fig. 3(4), the maximum contact angle of CN-NE is 36.53 ± 2.85°, and the minimum contact angle of T4O-NE is 16.17 ± 2.38°. CN has no hydroxyl group and binds well to the hydrophobic part, resulting in a small number of hydroxyls on the nanoemulsion surface and a large contact angle (Cai et al., 2021). According to the principle of similar phase dissolution, both T4O and tea saponin have six-membered rings, which makes the emulsion system formed by them more stable and has a small contact angle. Since CN and T4O are the main components of TTO, the contact angle of TTO-NE is between them.

The larger the turbidity of emulsion causes the larger the number of droplets, and the more unstable the emulsion system (Acevedo-Fani et al., 2015). As seen in Fig. 3(5), the turbidity of the NE is the largest, which is 11,952 ± 13 cm−1, which may be due to droplet agglomeration. The turbidity of the nanoemulsion with TTO and its main components as a core material decreases significantly, the turbidity of CN-NE, T4O-NE, and TTO-NE are 317 ± 8, 382 ± 8 and 2174 ± 13 cm−1, respectively. The results are consistent with the particle size. WI is closely related to the size of the emulsion droplets (Manzoor et al., 2021). The WI of NE is the highest. The nanoemulsion with TTO and the main components as the core material has a smaller particle size, which effectively reduces the light scattering and reduces the WI of the nanoemulsion. The apparent particle size of CN-NE is the smallest, and its WI is the lowest.

Thermal stability and rheological properties of nanoemulsions

The thermal stability of nanoemulsion is one of the important factors to measure its stability. In the DSC diagram (Fig. S1(1)), the glass transition temperature (Tg) of the NE is 156.13 °C. The position of the heat absorption peak of the nanoemulsion moves to a lower temperature when the TTO and the main component are added as the core materials. Studies have shown that the melting temperature of particles decreases with the decrease of particle size. Compared with NE, CN-NE, T4O-NE and TTO-NE have smaller particle size and higher surface energy, which can reduce the heat required to melt lipids, and thus reduce the Tg of the nanoemulsions to a certain extent (Ali et al., 2010).

The rheological properties of nanoemulsion are closely related to its internal system structure, and the dynamic stability of nanoemulsion can be measured (Ling et al., 2024). The trend of the rheological curves of all nanoemulsions is consistent (Fig. S1(2)), and the apparent viscosity of the samples is negatively correlated with the shear rate. When the shear rate changes in the lower range, the apparent viscosity of the sample decreases rapidly with the shear rate increasing. When the shear rate of the emulsion is large enough, the apparent viscosity of the sample remains basically unchanged and approaches 0. It shows a typical shear thinning phenomenon, which is characteristic of non-Newtonian fluids. When the shear rate is 0–4 s−1, the apparent viscosity of NE, T4O-N and TTO-NE is not significantly different (P > 0.05), and the apparent viscosity of CN-NE is the lowest. This is consistent with the results of turbidity and whiteness index of the nanoemulsion.

The release kinetics of essential oils from the nanoemulsions

To determine the sustained release of the essential oil in the nanoemulsions, the release behavior was studied. Fig. S2 shows the cumulative release rates of CN, T4O, and TTO from nanoemulsions in PBS. Each release curve shows a slight rapid release within 2 h, and then the release rate slows to equilibrium. About 24–28 % of essential oils are released in the first 2 h, mainly because some essential oils are not embedded, poor embedding, or adsorbed on the surface of nanomicelles. After 24 h, the release of essential oil from nanoemulsion is close to equilibrium. At 72 h, the cumulative release percentages of CN, T4O and TTO reach 75, 77 and 81 % respectively.

To study the release mechanism of CN, T4O and TTO from nanoemulsion, the release curves are fitted by ExpDecl, Higuchi, Logistic and Riter-Peppas models. As shown in Table S1, the release behaviors of CN, T4O and TTO are consistent with ExpDecl (R2 > 0.980) and Logistic (R2 > 0.977) models. In other words, the release of the core materials from the nanoemulsion conforms to the first-order release kinetic model and Logistic model, indicating that the release of essential oil from the nanoemulsion is a simple diffusion behavior (Li et al., 2020), accompanied by complex disintegrating and dissolving behaviors (Yang et al., 2020).

Stability of the nanoemulsion under different conditions

The storage stability of the nanoemulsion during 18 days

The long-term stability of essential oil nanoemulsion should be considered in practical application (Ferraz et al., 2022). As shown in Fig. 4, during storage time, the NE becomes larger under different storage conditions, while the nanoemulsions with TTO and the main components as core materials do not change significantly. The reason is that the condensation probability of small particle size nanoemulsion under the influence of gravity is low (Rao and McClements, 2012), indicating that the nanoemulsion system with core material is more stable. During the whole storage period, the PDI of the NE changes greatly, and the PDI of the nanoemulsions with TTO and the main component as the core material changes little and is always less than 0.3. The variation trend of zeta potential of the samples is consistent with that of PDI (Fig. 4(2)). The results at different temperatures indicate that the essential oil nanoemulsion stored at 4 °C has a slight advantage in stability, and the fluctuation of particle size and PDI is small, which may be since the nanoemulsion is a thermodynamically unstable system. With the increase of storage temperature, the molecular motion rate increases, and the collision and aggregation of particles occur, resulting in the increase of the particle size of the nanoemulsion, and the change of PDI and zeta potential. In summary, the prepared nanoemulsionsare more suitable for storage and use at 4 °C. This provides the basis for its application in the preservation of L. japonicus fillets.

Fig. 4.

Fig. 4

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Changes in particle size (1), PDI (2) and zeta potential (3) of nanoemulsions under 4 °C (A), 15 °C (B), and 25 °C (C). The significant differences between groups are represented by capital letters (A, B, C, etc.), while the significant differences within groups are represented by different lowercase letters (a, b, c, etc.) (p < 0.05)

Centrifugal stability of the nanoemulsions

The delamination process of nanoemulsions was simulated by centrifugation. The stratification of the nanoemulsions before and after centrifugation was observed. The upper layer is the emulsion layer with no obvious fluidity, while the lower layer is the water layer, indicating that centrifugation results in the destruction of some large droplet aggregates and the lower layer is the relatively stable small droplet aggregates. As shown in Fig. S3(A), after centrifugation, both particle size and PDI of NE decrease, while the absolute value of zeta potential increases. The results show that centrifugation destroys most of the large droplet aggregates in the NE, and leaves the emulsion system, which reduces the factors affecting the stability of the emulsion and makes the remaining emulsion system stable. The particle size of the nanoemulsion with TTO and its main component as the core materials decreases to different degrees after centrifugation, and the PDI increases slightly, but in a stable range. At the same time, the absolute value of zeta potential also decreases to some extent compared with that before centrifugation, but it does not affect the stability of the emulsion. Before and after centrifugation, the PDI, zeta potential and particle size of CN-NE, T4O-NE and TTO-NE show that the nanoemulsion still has good stability after high-speed centrifugation.

pH stability of the nanoemulsions

The pH of different food systems is different, so it is of significance to study the effect of pH on the stability of nanoemulsion (Zhang et al., 2024,b). When pH increases from 3.0 to 8.0, the PDI, zeta potential and mean particle size of NE change less. When pH increases from 4.0 to 7.0, the PDI, zeta potential and mean particle size of TTO and its main component nanoemulsions change little, indicating that the nanoemulsions have good acid resistance. When pH increases from 8.0 to 9.0, the mean particle size, PDI and zeta potential of each group of nanoemulsions change greatly, indicating that the alkaline resistance of the emulsions is poor.

When pH = 7.0, the nanoemulsion system is relatively stable. The reason may be that in an acidic environment, the electrostatic repulsion between emulsion droplets is reduced (Taarji et al., 2018), and it is easy to aggregate. In alkaline environment, the formation of hydrogen bonds between tea saponin molecules and water molecules is hindered, the interfacial tension is reduced, and the emulsion formation ability is reduced.

Antioxidant and antibacterial activity of the nanoemulsions

Antioxidant activity is an important index to evaluate the biological activity of preservation materials. The DPPH free radical scavenging capacity and total free radical antioxidant activity of the nanoemulsion are shown in Fig. 5(1). The NE has a certain scavenging ability on DPPH free radical activity, which is attributed to the strong scavenging ability of tea saponin on hydroxyl free radicals and superoxide anion free radicals. These results indicate that tea saponin has strong antioxidant properties and could remove free radicals well. After added TTO and the main components, the DPPH scavenging activity and total free radical antioxidant capacity of the nanoemulsion are significantly enhanced. The DPPH free radical scavenging activity increases from 32% to 69–75%, and the total antioxidant capacity is 25.8–29.8 times that of NE. This is due to the ability of TTO to trap free radicals (P < 0.05). Among them, the antioxidant capacity of TTO-NEis the best, indicating the synergistic effect of the components of TTO.

Fig. 5.

Fig. 5

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DPPH removal capacity (1), total anti-oxidation capacity (ABTS) (1) and the antibacterial diameters (2) of the nanoemulsions. Different lowercase letters (a, b, c, etc.) in the figures indicate significant differences among these groups. (p < 0.05)

As seen from Fig. 5(2), NE has certain antibacterial properties because of the emulsifier tea saponin in the emulsion. With TTO and the main components as core materials, the antibacterial properties of the nanoemulsion on the dominant spoilage bacteria in fish fillets are significantly enhanced. The antibacterial activity of CN-NE is slightly higher than that of NE, and the antibacterial activity of T4O-NE is much higher. Meanwhile, the antibacterial property of the TTO-NE is the optimal, indicating that there is a synergistic effect among the antibacterial components in TTO. This result is similar to the previous research, complex essential oils showed synergistic inhibition of Staphylococcus aureus (Deng et al., 2024). The possible reason is that the antibacterial components in essential oils can break the integrity of the bacterial cell membrane, improve its permeability, and make a large number of substances in the cell to overflow, resulting in the death of the bacteria (Li et al., 2020).

Preservation properties of the pads to L. japonicus fillets

Effects of the nanoemulsion pads on the freshness index of L. japonicus fillets

The sensory score is the most intuitive evaluation index to evaluate the freshness of fillets. As seen in Fig. 6(a), the sensory scores of L. japonicas fillets show a downward trend during storage. Among them, the sensory scores of CK’s fillets reach the acceptable minimum limit at 8 d. The decline rate of sensory score of L. japonicas fillets treated with PAD is slightly reduced, indicating that PAD can postpone the quality worsening of fillets by absorbing the exudate of the fillets. After treated with the NE-P, the sensory scores of fillets decrease slowly, which further optimizes the preservation properties of the PAD due to the antibacterial properties of tea saponin. After being treated with a preservation pad containing TTO and the main components, the sensory scores of fillets decrease more slowly. Among them, the sensory scores of T4O-NE-P are higher than those of CN-NE-P, but lower than TTO-NE-P, indicating that T4O has better preservation properties than CN, meanwhile, T4O and CN have synergistic effects. On the 15th day, the sensory scores of the fillets treated with the TTO-P and the TTO-NE-P still do not reach the acceptable minimum limit. The sensory score of the fillets treated with TTO-NE-P was 0.47 points higher than that of TTO-P treated fillets. This is because the nanoemulsion has a slow-release effect on TTO, and during the whole storage period, the TTO is slowly released, which has a more long-term preservation effect on the fillet.

Fig. 6.

Fig. 6

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The preservation properties of the pads for L. japonicas fillets during cold storage a sensory scores; b TVC; c pH; d TBA; e TVB-N. f Antibacterial and antioxidant diagram of the pads

The change of TVC is an important index to measure the degree of fillet spoilage (Zhang et al., 2023). As shown in Fig. 6(b), at the beginning, the TVC of the fillet is 2.61 lg CFU/g, which is in the first degree of freshness. The fastest increase in TVC is in CK, which exceeds the acceptable limit of 7 lg CFU/g at 9 d. The TVC of the fillets treated with PAD is always lower than that of the CK fillets in the same period of storage. The reason is that the pad can isolate the juice from fillets, restrain the growth and propagation of microorganisms in the fillets. Meanwhile, the increase of TVC of the fillets treated with NE-P is slightly slower, which may be due to the antibacterial properties of tea saponin. The TVC of the fillets treated with T4O-NE-P is always lower than that treated with CN-NE-P, because the antibacterial property of T4O is stronger than that of CN. The antibacterial effect of TTO-P and TTO-NE-P is better than that of CN-NE-P and T4O-NE-P, because CN, T4O and other components have synergistic antibacterial effects. Compared with the TTO-P, the growth rate of microorganisms in the fillets after treated with TTO-NE-P is slower, which is caused by the slow release of TTO from the nanoemulsion during the whole storage period, which can restrain the growth and propagation of microorganisms in a long-term and efficient manner. This result is similar to the previous research, adding the slow-release thyme essential oil microcapsule composite film has better preservation effect (Wang et al., 2025).

After the fish dies, the glycogen in the body undergoes a glycolysis reaction to produce lactic acid and pyruvate, which lowers the pH of the fish. Under the combined action of enzymes and microorganisms, the protein breakdown in the fish body produces a gradual accumulation of volatile salt compounds, resulting in an increase in pH. As shown in Fig. 6(c), the overall trend of the pH of the fillets decreases and then increases with the extension of storage time. At the later stage of storage, the pH rising rate of the CK fillets is the fastest, followed by the PAD, and the pH rising rate of the fillets treated by the NE-P is slightly lower. After treated with CN-NE-P and T4O-NE-P, the pH rise rate of the fillets is significantly reduced. After treated with TTO-P and TTO-NE-P, pH rise rate of the fillets is the lowest, indicating that the multi-components of TTO have synergistic preservation properties. Treated by the TTO-NE-P, the pH of the fillets fell to the lowest at the 9 d, and it is lower than that of TTO-P, this is because the TTO-NE can inhibit the oxidative degradation of proteins in a long time, thus extending the shelf life of the fillets more efficiently.

TBA is an important index to detect the degree of fat oxidation in fillets. When the TBA value of the fish reaches 1–2 mg MDA/kg, it indicates that the fish has deteriorated (Chen et al., 2022). As shown in Fig. 6(d), the TBA of fresh fillets is 0.129 mg MDA/kg, and the TBA of fillets shows an increasing trend with the extension of storage time. Studies have shown that the reason is the multiple interactions between malondialdehyde (MDA) and glucose, and amino acids during storage (Jouki et al., 2014). The TBA of CK and those treated with PAD increases the fastest, transcending the acceptable limits on the 10th day and 13th day, respectively. By the 15th day, the TBA of the fillets treated with NE-P is still within the acceptable limit value, which is attributed to the antibacterial effect of tea saponin, which could delay the oxidation rate of fat. In the early storage stage, the TBA of fillets treated with TTO-P is lower than that of TTO-NE-P, but in the late storage stage, the TBA is significantly higher than that of TTO-NE-P. The reason is that in the early stage of storage, TTO-P releases a large amount of TTO, effectively inhibiting the reproduction of microorganisms and the activity of endogenous enzymes, and delaying the fat oxidation rate of fillets. At the end of storage, the content of TTO decreases, and the inhibitory effect on microorganisms and endogenous enzymes decreases. In the TTO-NE-P, the slow release of TTO can delay the fat oxidation of fillets in a long and efficient manner.

The TVB-N reflects the content of volatile base nitrogen substances produced by protein decomposition by endogenous enzymes and microorganisms during storage of aquatic products. According to the method of GB/T 18108–2019 “General rules of fresh marine fish”, superior products are TVB-N ≤ 15.00 mg/100 g, and qualified products are 15.00 mg/100 g ≤ TVB-N ≤ 30.00 mg/100 g. 30.00 mg/100 g is called an acceptable limit. As shown in Fig. 6(e), the TVB-N of fresh fillets is 7 mg/100 g, indicating that the fillets are superior quality. During storage, the TVB-N value of the fillets increases. Among them, the TVB-N of CK increases fastest, while the TVB-N of fillets treated with PAD increases slightly slower. The TVB-N of fillets treated with NE-P is significantly lower than that treated with PAD in the same period. The TVB-N of the above fillets reached the limit of qualified products before the 9 d. After treated with CN-NE-P and TTO-P, the TVB-N of the fillets reached the qualified product limit before the 15 d, because the essential oil can restrain the growth and propagation of microorganisms and slowed down the degradation of the protein. However, the samples treated with T4O-NE-P and TTO-NE-P are still superior on the 15 d. Among them, in the early storage period, the TVB-N of the fillets treated with the TTO-NE-P is higher than that treated with TTO-P, however, in the late storage period, that is lower than that treated with TTO-P, which is similar to the change of TBA, further verifying that TTO-NE-P has the best preservation effect.

Effect of the nanoemulsion pads on the structure of L. japonicus fillets

The change of fish tissue structure is an important factor affecting fish taste and fish quality, and also indirectly reflects the degradation and corruption of fish protein. Fig. S4 shows the structural changes of fillets during storage. The muscle fibers of fresh fillets are clearly defined, evenly distributed, compact and orderly, and have small internal gaps. After storage to the 6 d, the distribution of muscle fibers is more uniform, the degree of arrangement density changed in different degrees, and a small number of small holes appeared in different degrees. After 15 days of storage, the contours of muscle fibers in the CK are blurred, and most of the muscle fibers are broken and dissolved. After treated with PAD, the muscle fibers of the fillet are twisted to a certain extent and there are many internal holes. Treated with NE-P, the muscle fibers of the fillets are partially broken. Treatment with CN-NE-P and T4O-NE-P, the muscle fibers of the fillets are still closely arranged and there are small cracks in the interior. It shows that essential oil can inhibit the growth and reproduction of microorganisms in fillets, delay the degradation of their proteins, and then reduce the destruction of muscle fibers. After treated with TTO-P and TTO-NE-P, the muscle fibers of the fillets are more uniform, the fibers are regular and the integrity is higher. Among them, the effect of treated with TTO-NE-P is optimal.

Conclusion

The sustained release system of essential oil nanoemulsion with long-term and good stability was successfully constructed. As the main components of TTO, CN, T4O and others have synergistic antioxidant and antibacterial effects. The release time of essential oil in nanoemulsion can reach more than 24 h. Taking L. japonicus fillet as the object, the preservation performance of nanoemulsion pad was verified. The combination of a slow-release system and preservation pad extends the effectiveness of TTO, but also improves the preservation performance of the pad. TTO-NE-P has the best preservation effect and is better than TTO-P, and the shelf life of fillets is extended from 9 d to 14.5 d. This study provides a new direction for the construction and application of slow-release systems in the field of food packaging materials.

Supplementary Information

Below is the link to the electronic supplementary material.

10068_2024_1812_MOESM1_ESM.docx (11.9MB, docx)

Fig. S1: Thermal stability and rheological properties of nanoemulsions; Fig. S2: Release properties of essential oils in nanoemulsions; Fig. S3: Stability of the nanoemulsion under different conditions; Fig. S4: Change of muscle tissue structure of L. japonicus fillets; Table S1: Analysis of release kinetics model of nanoemulsions

Supplementary file1 (DOCX 12201 KB)

Funding

This study was supported by Application Basic Scientific Research of Liaoning Province (2023JH2/101300170) and Basic scientific research Project of Education Department of Liaoning Province (LJKMZ20221488).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

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Contributor Information

Yingchang Li, Email: liyingchangsy@126.com.

Tong Sun, Email: jzsuntong@sina.com.

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Associated Data

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Supplementary Materials

10068_2024_1812_MOESM1_ESM.docx (11.9MB, docx)

Fig. S1: Thermal stability and rheological properties of nanoemulsions; Fig. S2: Release properties of essential oils in nanoemulsions; Fig. S3: Stability of the nanoemulsion under different conditions; Fig. S4: Change of muscle tissue structure of L. japonicus fillets; Table S1: Analysis of release kinetics model of nanoemulsions

Supplementary file1 (DOCX 12201 KB)

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