Quality Changes in Fresh-Cut Lettuce When Subjected to Ultrasound Combined with Zinc Oxide Nanoparticle (ZnO NP) Treatment

Abstract

The effects of three preservation methods (ultrasound, ZnO NPs, and ultrasound combined with ZnO NPs) on the odor, microstructure, and edible quality of fresh-cut lettuce were investigated in this study. When stored for 8 days, significant improvements were observed in the following when using ultrasound combined with ZnO NP treatment to better preserve fresh-cut lettuce (and were reduced when compared with the control group): the color (L* value (34.53); a* value (−5.89); b* value (15.00); browning index (40.63); firmness (25.66); propectin (2.12%); chlorophyll (2.75 mg/100 g); cellulose (20.35%); total phenolic content (0.95 mg/100 g); PAL activity (54.91 U·h⁻¹·g⁻¹); CAT activity (41.78 U·min⁻¹·mg⁻¹); ABTS free-radical scavenging ability (137.62 µmol/L); FRAP total reducing ability (94.42 µmol/L) (p < 0.05), PPO activity (0.85 U·min⁻¹·g⁻¹); MDA (1.97 µmol/g); and H₂O₂ (54.26 µmol/g). In addition, the results of the volatile components indicated that the use of ultrasound combined with ZnO NP treatment decreased the production of adverse flavor compounds by inhibiting the generation of aldehydes and ketones, as well as by promoting the generation of olefins, nitriles, and quinolines, and the contents of nitriles and quinolines were 20.07% and 2.07% of the total components, respectively. The resultant microstructure indicated that the microchannels generated by ultrasound allowed for the ZnO NPs to enter the intracellular cavity of the fresh-cut lettuce more efficiently; such a finding could serve as a basis for a hypothesis on the mechanism of ultrasound combined with ZnO NP treatment. The results of fresh-cut lettuce preservation when using ultrasound combined with ZnO NPs were better than those that were obtained when using ultrasound and ZnO NP treatment alone. And, using ultrasound combined with ZnO NP treatment as a new preservation method for fresh-cut lettuce provides a promising preservation idea for other fresh-cut fruits and vegetables.

1. Introduction

Lettuce (Lactuca sativa) is a common leafy vegetable belonging to the genus Lactuca in the Compositae family and is an annual or biennial herb. It is popular with consumers because of its crisp texture and convenient preparation [1]. As lettuce is rich in nutrients and functional components, including protein, vitamins, cellulose, mineral, organic acid, phenolic, terpenoid, etc., it contributes to health by improving anti-inflammatory, anti-tumor, anti-cancer, and lipid-lowering functions, among others [2].

Due to consumers’ increasing demand for convenient foods, fresh-cut fruits and vegetables represent a broad sales market [3]. Fresh-cut fruits and vegetables are lightly processed foods that are prepared via a semi-processing method and then transported through a cold chain after a series of operations, including washing, peeling, cutting, and packaging. This preparation method is convenient, hygienic, and natural [4,5]. Lettuce must undergo mechanical operations before consumption, including peeling and slicing, which makes it one of the most suitable vegetable varieties for fresh cutting. However, fresh-cut lettuce is usually accompanied by enzymatic browning on the surface due to the oxidation of phenolic substances; thus, the cutting treatment could also cause tissue damage, induce an increase in polyphenol oxidase activity, exacerbate browning, and affect storage quality and shelf life [6,7]. The present preservation methods for fresh-cut lettuce are mainly focused on physical preservation, chemical preservation, and low-temperature storage, which can effectively extend the storage time. Good physical preservation is capable of inhibiting the browning of fresh-cut lettuce, and it can also work to delay decreases in nutrient content. In aiming to obtain better preservation effects, it is necessary to use high-efficiency physical preservation equipment. However, this results in high technology and labor costs, and there is also the concern that it is not conducive to large-scale applications [8]. Although chemical preservation is cheaper and suitable for large-scale production applications, their abuse could easily lead to excessive reagent residues and environmental pollution [9]. Low-temperature storage is safe for human health, cost-effective, and environmentally friendly. However, improper temperatures could cause undesirable water crystallization inside freshly cut lettuce, resulting in rapidly increasing weight loss rates and decreased firmness [10]. Therefore, it is urgent to find an efficient, non-toxic, and low-cost method for fresh-cut lettuce preservation.

Ultrasound (US) is widely used in fruit and vegetable preservation due to its safety, efficiency, and pollution-free characteristics [5]. The cavitation effect of ultrasound could cause regional high temperature and pressure within the lettuce, thus leading to enzyme and bacterial inactivation [11]. Previous studies have shown that ultrasound treatment (20 kHz + 23 W/L) can effectively inhibit the browning and weight loss of fresh-cut lettuce; delay the loss of vitamin C and chlorophyll content; and inactivate antioxidant-related enzyme activities. In addition, ZnO nanoparticles (ZnO NPs) are considered the most promising nanoscale materials due to their non-toxicity, high antibacterial ability, and good biocompatibility [12,13]. According to a regulation from the United States Food and Drug Administration (21CFR182.8991), ZnO NPs can be used as food additives and antibacterial agents [14]. In addition, their maximum addition amount as a food contact substance is 2% according to the European Food Safety Authority (EFSA). The antibacterial activity of ZnO NPs is related to the formation of free radicals. This is because lipids on bacterial cell membranes are destroyed by free radicals, thereby resulting in bacterial inactivation [13]. Yuliani et al. [15] showed that a composite coating treatment of 20 g/L of cassava starch, 6 g/L of stearic acid, and 2% ZnO NPs could maintain the quality and flavor of fresh-cut mangoes while also reducing water evaporation and bacterial infection. A chitosan/gum arabic + 0.5% ZnO NP composite coating treatment was found to maintain the titratable acid and the content of soluble sugar high in bananas. It could also reduce the rate of weight loss and inhibit fungus reproduction [16]. Although ultrasound and ZnO NPs have been applied to the preservation of fruits and vegetables, there is currently no study on the preservation of fresh-cut lettuce when it has been treated with ultrasound combined with ZnO NP treatment. Therefore, the use of ultrasound combined with ZnO NP treatment is expected to become a promising method for improving the storage quality of fresh-cut lettuce. Fresh-cut lettuce is popular among consumers, but there are issues such as storage quality and shelf life. Based on our previous research, ultrasound treatment has been found to be effective in addressing these problems. In this study, we examined the impact of three preservation methods (ultrasound, ZnO NPs, and a combination of ultrasound and ZnO NPs) on the odor, microstructure, and edible quality of fresh-cut lettuce in order to provide guidance and new approaches for extending its shelf life.

2. Materials and Methods

2.1. Materials

All reagents and standards were purchased from Sinopharm Chemical Reagent (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) or Merck Life Science (Merck & Co., Inc., Kenilworth, NJ, USA). The purple leaf “Jinnong” lettuce was provided by New City Central Vegetable Market, Wuhu, China.

2.2. Preparation of the ZnO NP Solution

The method of preparation of the ZnO NP Solution was used as per previous studies [17]. A total of 0.7 g of ZnO NP powder (<100 nm) and 7 mg of PEG (polyethylene glycol) 6000 (to improve the dispersive stability of solution) were dissolved in 1.0 L of sterilized deionized water. This was then continuously stirred for 24 h with a magnetic stirrer (JB-11, Magnetic Stirrer, Shanghai Precision Scientific Instrument Co., Ltd., Shanghai, China). The mixed solution was subjected to ultrasound (KS-500DE, Ultrasonic Cleaner, Kunshan Jielimei Ultrasonic Instrument Co., Ltd., Kunshan, China) treatment for 30 min (40 kHz, 200 W, and 30 °C). Finally, 0.7 g/L of a ZnO NP solution with good dispersion was prepared and used instantly.

2.3. Preparation of the Fresh-Cut Lettuce Samples

Flawless lettuces of uniform size and robust growth were selected as samples. After removing the surface stems and leaves, they were washed, dried, peeled, and cut into slices with a thickness of about 0.4 cm. These were then randomly divided into four groups for preservation treatment (with 600 g of fresh-cut lettuce used for each group):

(1) Ultrasound treatment group (US): fresh-cut lettuces were treated with ultrasound (300 W and 45 °C) for 10 min.

(2) ZnO NP treatment group (ZN): fresh-cut lettuces were immersed in 0.7 g/L ZnO NPs solution for 10 min at room temperature.

(3) Ultrasound combined with the ZnO NP treatment group (UZ): fresh-cut lettuces were immersed in 0.7 g/L of a ZnO NP solution, and they were then treated with ultrasound (300 W and 45 °C) for 10 min.

(4) Control treatment group (CK): fresh-cut lettuces were immersed in ultrapure water for 10 min at room temperature.

The optimal treatment conditions for different preservation methods were obtained from previous studies [17]. After four different treatments, the fresh-cut lettuces were drained and packed in 7 cm × 10 cm × 5 cm polyethylene bags and then refrigerated at 4 °C and 45%–75% humidity. All of the relevant quality indicators of fresh-cut lettuces were measured on the 0, 2, 4, 6, and 8 days of storage. Each experiment was repeated three times. All tools were disinfected before use.

2.4. Firmness and Propectin

The method of Xu et al. was used to evaluate firmness [18]. The firmness was measured at three different locations in the equatorial region of the fresh-cut lettuce; this was achieved via cutting the surfaces with a P5 cylindrical test probe of a food texture analyzer (TA.new plus, Texture Instrument, Shanghai Bosin Industrial Development Co., Ltd., Shanghai, China), and the scores were then averaged. We chose a compression ratio of 30%; a time interval of 3 s between two tests; a triggering force of 5 g; a speed of 3 mm/s before, during, and after testing; and a displacement of 10 mm.

The propectin content was determined following the method of Fan et al., with some modifications. Fresh-cut lettuces (1 g) were ground. Propectin samples were extracted using 25 mL of 95% ethanol at 100 °C for 30 min (which was repeated five times), and then the extraction contents were combined [8]. The speed and time of the centrifugal machine (TGL-16A, Centrifugal Separator, Changsha Ordinary Instrumentation Co., Ltd., Changsha, China) were set as 8000 r/min and 15 min, respectively. The sediment was determined using 20 mL of distilled water centrifuged at 50 °C for 30 min. After centrifuging, the sediment was added to 25 mL of 0.5 mol/L sulfuric acid solution at 100 °C for 1 h, which was then centrifuged. The supernatant (1 mL) was obtained with 6 mL of concentrated sulfuric acid at 100 °C for 20 min, and then 0.2 mL of 0.15% carbazole ethanol solution was added. This was then left in the dark for 30 min, and the absorbance was measured at 530 nm.

2.5. Colors and Browning Index

The L*, a*, and b* values of the fresh-cut lettuces were measured at three different positions using a colorimeter (CHINSPEC, Colorimeter, Hangzhou CHNSpec Technology Co., Ltd., Hangzhou, China), and the results were then averaged according to the method of Wang et al. [3]. Then, the browning index (BI) was calculated with Equation (1):

$$BI={\displaystyle \frac{100\times \left(\frac{a^{\ast }+1.75L^{\ast }}{5.645L^{\ast }+a^{\ast }-3.012b^{\ast }}-0.31\right)}{0.17}}.$$

(1)

2.6. Chlorophyll and Cellulose

The chlorophyll content was determined in accordance with Wang et al., with minor modifications. Extraction from fresh-cut lettuces (0.2 g) was carried out in 10 mL of acetone under dark conditions until they appeared colorless [3]. The supernatant was fixed to 25 mL with acetone, and the absorbance was then measured at 645 nm and 663 nm (L3S, Visible Spectrophotometer, Shanghai Yidian Analytical Instrument Co., Ltd., Shanghai, China). The absorbance was included in Equations (2) and (3) to calculate the chlorophyll content as follows:

$$C_t=8.02A_{663}+20.01A_{645},$$

(2)

$$\mathrm{c}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}/\left(\mathrm{m}\mathrm{g}/100 \mathrm{g}\right)={\displaystyle \frac{C_t\times V}{m\times {10}^5}},$$

(3)

where A₆₆₃ is the absorbance of the specimen at 663 nm, A₆₄₅ is the absorbance of the specimen at 645 nm, C_t is the chlorophyll mass concentration (mg/L), V is the volume of extraction solution (mL), and m is the sample mass (g).

The cellulose content was determined in accordance with Wang et al., with some modifications. Fresh-cut lettuces (0.5 g) were ground [3]. Extraction was performed with 5 mL of acetic acid–nitric acid solution at 100 °C for 25 min. After cooling and centrifuging, the sediment was added to 20 mL of sulfuric acid–potassium dichromate solution at 100 °C for 10 min (HH-4, Thermostat Water Bath Cauldron, Jiangsu Jierier Electric Appliance Co., Ltd., Jintan, China). The supernatant was added to a KI–starch solution, which was then titrated with sodium thiosulfate.

2.7. The Total Suspended Solids (TSSs)/the Titratable Acid (TA) Ratio and the Soluble Sugar Content (SSC)/TA Ratio

TA was determined in accordance with Shahkoomahally et al. [19], with some modifications. Fresh-cut lettuces (10 g) were mixed with 90 mL of distilled water and then ground thoroughly. The supernatant had two drops of phenolphthalein reagent added to it. The filtrate was titrated with a NaOH solution until the filtrate appeared pink, and this was maintained for more than 30 s.

TSSs were determined in accordance with Shahkoomahally et al. [19], with some modifications. The fresh-cut lettuces (5 g) were ground at room temperature. The speed and time of the centrifugal machine were set as 4000 rpm and 10 min, respectively. A small amount of liquid was aspirated for light determination with an Abbe refractometer (WYA-2D, Abbe Refractor, Shanghai Optical Instrument Co., Ltd., Shanghai, China).

SSC was determined in accordance with Wang et al. [20], with some modifications. The fresh-cut lettuces (1 g) were ground thoroughly with 10 mL of distilled water, and the SSC was extracted in boiling water for 30 min (repeated twice). The filtrate was fixed to 100 mL. The filtrate (0.5 mL) was also mixed with 1.5 mL of distilled water, 1 mL of 9% phenolic solution, and 5 mL of concentrated sulfuric acid, and it was then kept at room temperature for 30 min. The absorbance was measured at 485 nm.

2.8. Total Phenolic Content

The total phenolic content was determined in accordance with Nikzad et al. [21], with some modifications. The fresh-cut lettuces (0.5 g) were ground thoroughly in an ice bath. The extraction was performed with 7 mL of 70% ethanol at 70 °C for 1 h. The speed, temperature, and time of the centrifugal machine were set to 11,500 RPM, 25 °C, and 15 min, respectively. The supernatant (1 mL) was diluted 7 times with 70% ethanol. The diluted extract (1 mL) was obtained with 0.5 mL of Folin–Ciocalteu reagent for 3 min at room temperature, which then had 1.5 mL of 10% Na₂CO₃ solution added to it. The supernatant was fixed at a volume of 70% ethanol at 30 °C for 90 min, and the absorbance was measured at 770 nm.

2.9. Antioxidant Enzymes

The polyphenol oxidases (PPO) were determined in accordance with Terefe et al. [22], with some modifications. The fresh-cut lettuces (0.5 g) were mixed with 4 mL acetic acid buffer solution, and they were ground thoroughly at low temperatures. The speed, temperature, and time of the centrifugal machine were set to 12,000 RPM, 4 °C, and 30 min, respectively. After centrifuging, the supernatant (100 μL) was obtained sequentially using 4 mL of a 0.05 mol/L acetic acid buffer solution and 1 mL of a 0.05 mol/L catechol solution. The absorbance was measured continuously at 420 nm in 1 min intervals.

The amount of phenylalanine ammonia (PAL) was determined in accordance with Yeoh et al. [23], with some modifications. The fresh-cut lettuces (0.5 g) were mixed with 4 mL of boric acid buffer solution and then ground thoroughly at low temperatures. The speed, temperature, and time of the centrifugal machine were set to 12,000 RPM, 4 °C, and 30 min, respectively. After centrifuging, the supernatant (0.5 mL) was obtained sequentially using 3 mL of a pH 8.8 boric acid buffer solution and 500 µL of a 0.02 mol/L L-phenylalanine solution that was pre-warmed at 37 °C. The absorbance was measured rapidly at 290 nm (UV-5800, UV-VIS Spectrophotometer, Shanghai Metash Intruments Co., Ltd., Shanghai, China). After holding the solution at 37 °C for 60 min, the absorbance was measured rapidly at 290 nm.

The amount of catalase (CAT) was determined in accordance with Qiao et al. [24], with some modifications. The fresh-cut lettuces (0.5 g) were mixed with 4 mL of phosphate buffer solution and then ground thoroughly at low temperatures. The speed, temperature, and time of the centrifugal machine were set to 12,000 RPM, 4 °C, and 30 min, respectively. After centrifuging, the supernatant (100 µL) was obtained with 2.9 mL of a 0.02 mol/L H₂O₂ solution. The absorbance was measured continuously at 240 nm in 30 s intervals.

2.10. Antioxidant Capacity

The fresh-cut lettuces (1 g) were mixed with 8 mL of 80% ethanol and then ground thoroughly under ice bath conditions. The mixture was extracted using ultrasound at 60 °C for 40 min. The speed and time of the centrifugal machine were set to 10,000 r/min and 20 min, respectively. The supernatant was collected and stored at a low temperature.

The 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) free radical scavenging capacity was determined in accordance with Wang et al. [21], with some modifications. The supernatant (0.1 mL) was mixed with 3.9 mL of an ABTS diluent solution, and it was also kept at 30 °C for 20 min in darkness. The absorbance was measured at 734 nm and labeled A_t. The absorbance was measured at 734 nm and labeled A_b, and this was performed under the same conditions that were used for the anhydrous ethanol instead of the ABTS diluent solution. The absorbance was measured at 734 nm and labeled A_o, and this was performed under the same conditions as used for the distilled water instead of the supernatant. The ABTS free-radical-scavenging ability was calculated with Equation (4) as follows:

$${SC}_{ABTS}=\left(1-{\displaystyle \frac{A_t-A_b}{A_o}}\right)\times 100\mathrm{\%}.$$

(4)

The ferric-reducing antioxidant power (FRAP) total reducing capacity was determined in accordance with Mustapha et al. [25], with some modifications. The twice-diluted extraction (0.1 mL) was mixed with 3 mL of a FRAP working solution, which was then kept at 30 °C for 20 min in darkness. The absorbance was measured at 593 nm.

2.11. Malondialdehyde and H₂O₂

The amount of malondialdehyde (MDA) was determined in accordance with Xu et al. [26], with some modifications. The fresh-cut lettuces (0.8 g) were mixed and ground with 8 mL of a 10% trichloroacetic acid solution. The speed, temperature, and time of the centrifugal machine were set to 2650 r/min, 4 °C, and 10 min, respectively. After centrifuging, the supernatant (2 mL) was obtained using 2 mL of a 0.6% thiobarbituric acid solution at 100 °C for 30 min. The absorbance was measured at 450 nm, 532 nm, and 600 nm.

$$\mathrm{M}\mathrm{D}\mathrm{A}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\left({\mathrm{m}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{k}\mathrm{g}}^{-1}\right)=(6.45\ast (A532-A600)-0.56\ast A450)\ast V_0/V_1\ast W$$

(5)

where V₀ is the volume of the sample in mL; V₁ is the volume used for detection in mL; and W is the sample weight in g.

The amount of H₂O₂ was determined in accordance with Fan et al. [19], with some modifications. The fresh-cut lettuces (1 g) were mixed and ground with 2.5 mL of a phosphate buffer solution [10]. After centrifuging, the sediment was rinsed continuously until it was rendered colorless using pre-cooled acetone at −20 °C. The colorless supernatant was mixed thoroughly with 3 mL of a 2 mol/L sulfuric acid. The absorbance was measured at 412 nm.

2.12. GC-MS Analysis

Sample processing was conducted with fresh-cut lettuces (5 g) that were sealed in an extraction vial. The extraction vial was placed in a microextraction device to be preheated for 15 min. The extraction temperature was 80 °C. The extraction head was first aged at the GC-MS inlet at 250 °C (7890A-5975C, GC-MS, Agilent, Santa Clara, CA, USA), and it was then inserted into the extraction vial in the headspace position. The extraction head was at the upper surface of the sample for 1 cm for 40 min, and this was then pushed out and inserted into the inlet. The fiber head was pushed out, and this was then resolved at the original temperature for 3 min. After this, the sample analysis was commenced. And, the method of GC-MS was used as previous studies [17].

The GC conditions were as follows: The chromatographic column was HP-5MS (30.0 m × 250 μm × 0.25 μm). The temperature of the chromatographic column was kept at 40 °C for 5 min, heated to 240 °C at a speed of 5 °C/min, and then maintained at 240 °C for 5 min. The gasification chamber temperature was 250 °C. The transmission line temperature was 240 °C. He was used as the carrier gas, and the flow rate was 1 mL/min. The process did not involve tapping.

The MS conditions were as follows: an EI source was used, the ion source temperature was 230 °C, the electron energy was 70 eV, the quadrupole temperature was 150 °C, the mass spectrometry scan mode used was Scan, and the scan range was 30–600 m/z.

2.13. The Microstructure

Thin slices (5 mm × 5 mm × 3 mm) were cut from the fresh-cut lettuce blocks. The slices were soaked in 5 mL of a 2.5% (v/v) glutaraldehyde at a low temperature for 20 h, and these were then rinsed with a pH 6.8 phosphate buffer solution for 40 min. Then, the slices were dehydrated with 20%, 40%, 60%, 70%, 80%, and 90% ethanol for 15 min. Finally, the slices were dehydrated with pure ethanol for 90 min, and these were then freeze-dried (XHDGJ-18N, Freezing Drier, Xiaohan Industrial Development (Guangzhou) Co., Ltd., Guangzhou, China) for observation via electron microscopy (S-4800, Scanning Electron Microscope, Hitachi High tech Company, Tokyo, Japan) The freeze-dried, fresh-cut lettuce slice samples were gold-plated and then observed at an accelerating voltage of 5.0 kV, according to our previous studies [17].

2.14. Data Analysis

Excel software 2021 was used to organize the experimental data (which are expressed as mean ± error). Sigmaplot 14.0 software and Design Expert 8.0.6 software were used to analyze and plot the values. IBM SPSS 19.0 software was used to perform the significance tests, and significant differences between mean (p < 0.05) were analyzed with ANOVA.

3. Results and Analysis

3.1. The Effects of Ultrasound Combined with ZnO NP Treatment on the Firmness and Propectin of Fresh-Cut Lettuce

Decreased firmness leads to the softening of fresh-cut fruits and vegetables during storage, which affects their future sale and acceptability [27]. As is shown in Figure 1A, the firmness of the fresh-cut lettuces continuously decreased during storage; however, the firmness of the UZ treatment group was significantly higher than that of the control group during the 6th day of storage (i.e., at 1.04 times). On the 8th day of storage, the firmness of the UZ treatment group was the highest (25.66), and there was a significant difference found when compared to the control group (p < 0.05), which was followed by the US (25.20), ZN (24.91), and CK (24.72) treatment groups. Xu et al. showed that the use of ultrasound combined with 1-MCP (1-methylcyclopropene) treatment can more effectively reduce the activities of polygalacturonase and pectin methylesterase, thus inhibiting the degradation of pectin and improving the firmness, which is consistent with the results of this study [18]. In addition, ZnO NPs have strong bacteriostatic properties and can reduce microbial infestation [15]. Furthermore, their use will mitigate the decay of fresh-cut lettuces, maintaining firmness. In addition, UZ treatment demonstrated a positive effect on delaying the decrease in the firmness of the fresh-cut lettuces.

As it is the highest content of fruit and vegetable cell wall matrices when in a class of macromolecular substances, pectin combines with cellulose to form propectin to make fruits and vegetables harder. ZnO NP treatment can reduce the rate of gas exchange between the fresh-cut lettuces and the outside of the internal CO₂ and O₂ layers (which also renders fruits and vegetables hard). In contrast, in the second half of the storage, due to enhanced respiration, the propectin was found to be gradually hydrolyzed by pectinase in the water-soluble pectin, which reduces the firmness of fruits and vegetables [7,28]. As shown in Figure 1B, the propectin of the fresh-cut lettuces in different treatment groups showed a decreasing trend as time increased. In the period during the initial 6 days of storage, the propectin content of the fresh-cut lettuces in the US and UZ treatment groups was significantly higher than that in the CK treatment group (p < 0.05), thus indicating that the US and UZ treatments could effectively delay the hydrolysis of propectin and maintain the firmness of fresh-cut lettuces. The cavitation effect of ultrasound can delay the enzymatic hydrolysis of propectin on the cell wall of fresh-cut lettuces, thereby affecting the stability of the cell wall and the adhesion ability between cells and reducing firmness loss [11]. During the storage process, the fresh-cut lettuces from the UZ treatment group demonstrated the best firmness preservation, with the propectin content being found to be consistently maintained at the highest level and significantly different from that of the control group (p < 0.05). On the 8th day of storage, this decreased to 2.12%, which was still 0.32% higher than the control group. This could possibly be due to the UZ treatment increasing the inhibitory effect of pectinase, thereby reducing the hydrolysis of propectin and the control softening process [29], which is consistent with the previous conclusion on firmness.

3.2. Effects of Ultrasound Combined with ZnO NP Treatment on the Colors and BI of Fresh-Cut Lettuce

The colors and BI of the fresh-cut lettuces under different treatment conditions are shown in Figure 2. With the extension of storage time, the browning of the fresh-cut lettuces gradually became more severe. From Figure 2A, it can be seen that the L* values of the fresh-cut lettuces gradually decreased during storage. Furthermore, the L* values of the UZ treatment group were significantly higher than those of the other treatment groups from the 4th day of storage, and they also were found to be 1.02, 1.08, and 1.09 times higher than those of the US, ZN, and CK treatment groups, respectively, on the 8th day (p < 0.05). The a* and b* values of the different treatment groups were found to gradually increase (Figure 2B,C), among which the a* and b* values of the CK treatment group remained the highest during storage by reaching −1.95 and 16.48 on the 8th day, respectively. On the 8th day of storage, the a* (−5.89) and b* (15.00) values of the UZ treatment group were both significantly lower than those of the other treatment groups (p < 0.05). The browning index is an important indicator of the shelf life and market sales of fresh-cut lettuces (Figure 2D) [30]. Consistent with the trend of the a* and b* values, the browning index of the fresh-cut lettuces gradually increased during storage, and the three preservation treatment groups demonstrated significantly lower browning indices than the CK treatment group from the second day (p < 0.05). Among them, the UZ treatment maintained the lowest browning index throughout the entire storage period, with only 40.63 on the 8th day of storage. This may be due to the use of ultrasound promoting the entry of ZnO NPs into the cells, thus inhibiting antioxidant enzyme activity and bacterial growth.

3.3. Effects of Ultrasound Combined with ZnO NP Treatment on the Chlorophyll and Cellulose of Fresh-Cut Lettuce

As is shown in Figure 3A, the chlorophyll content of the fresh-cut lettuces gradually decreased during storage time, and the downward trend in the three preservation treatment groups was found to be smaller than that in the control group. The initial chlorophyll content of the fresh-cut lettuces was 4.90 mg/100 g. From the 4th day of storage, the chlorophyll content of the UZ treatment group was found to be higher than that of the other treatment groups, but the difference was not significant compared to the ZN treatment group. On the 8th day of storage, the chlorophyll retention rates of the US, ZN, UZ, and CK treatment groups were 52.24%, 52.80%, 56.04%, and 48.67%, respectively (p < 0.05). The results showed that UZ treatment can more effectively reduce the degradation of chlorophyll in fresh-cut lettuces. Wang et al. found that although ultrasound or acetic acid/erythromycin acid treatment alone has a positive effect on maintaining the chlorophyll content of green asparagus [3], a combined treatment of ultrasound and acetic acid/erythromycin acid produces the highest chlorophyll content (2.96 mg/g), which is consistent with the results of this study. In addition, chlorophyll degradation was inevitably accompanied by changes in the apparent color, such as an increase in the a* value (which is consistent with the experimental results of the a* value mentioned above).

The effects of four different treatments on the cellulose of fresh-cut lettuces are shown in Figure 3B. The cellulose content of the fresh-cut lettuces decreased continuously with time. During storage, the cellulose content of the UZ treatment group remained the highest, and there was a significant difference found when comparing it to the control group (p < 0.05). On the 8th day of storage, the cellulose content of the US, ZN, UZ, and CK treatment groups was 18.78%, 17.44%, 20.35%, and 14.86%, respectively, thus indicating that the UZ treatment group was significantly higher than the other treatment groups. Therefore, the UZ treatment significantly delayed cellulose loss in the fresh-cut lettuces. In the early stages of storage, cellulose was combined with propectin to keep the fruits and vegetables hard. Furthermore, in the later stages of storage, both were gradually decreased under the action of their respective degrading enzymes, leading to decreased firmness and damage to the quality of the fruits and vegetables [31]. The trend of cellulose changes in the fresh-cut lettuces was consistent with the gradual decrease in propectin content and firmness mentioned above.

3.4. Effects of Ultrasound Combined with ZnO NP Treatment on the TSS/TA Ratio and SSC/TA Ratio of Fresh-Cut Lettuce

The TSS/TA ratio is generally used to evaluate the maturity of fruits and vegetables [25]. For fresh-cut fruits and vegetables, the higher the solid acid ratio, the more mature they are and the poorer their storage quality. As is shown in Figure 4A, the TSS/TA ratio of the fresh-cut lettuces in the UZ treatment group remained the lowest throughout the storage process. In the first 2 days and 4 days of storage, this ratio was significantly higher in the UZ treatment group than it was in the control group (p < 0.05). However, on the 6th day of storage, the TSS/TA ratio was higher in the UZ treatment group (58.43) than in the control group (p < 0.05), but the difference was not significant compared to the control group (62.43) (p > 0.05). This phenomenon could be related to storage time; on this note, Zhao et al. found that the significance of the difference in the TSS/TA ratio under different preservation treatments was not the same at different storage times [32].

The SSC/TA ratio is the same as the TSS/TA ratio, which is also a vital indicator for evaluating the maturity of fruits and vegetables [33]. As is shown in Figure 4B, the SSC/TA ratio of fresh-cut lettuces first showed an increasing trend and then a decreasing trend with the extension of storage time. The initial value of the SSC/TA ratio in the fresh-cut lettuces was 12.94. On the 8th day of storage, the SSC/TA ratios of the US, ZN, UZ, and CK treatment groups were 18.65, 18.84, 17.63, and 17.18, respectively. Although the SSC/TA ratio was the lowest in the control group at this time, the change in the SSC/TA ratio in that group was the largest from the perspective of the entire storage time, as it had both the highest and lowest SSC/TA ratios. This indicated that the control group experienced the strongest physiological and biochemical reactions, as well as the fastest decrease in the storage quality, during storage [34].

3.5. Effects of Ultrasound Combined with ZnO NP Treatment on the Total Phenolic Content and Activities of the Antioxidant Enzymes of Fresh-Cut Lettuce

The browning system of fresh-cut fruits and vegetables is relatively complex, and it is influenced by various factors, including the degree of cutting injury, oxygen, substance content, and antioxidant enzyme activities [20]. The effects of using ultrasound combined with ZnO NP treatment on the PPO, PAL, CAT, and the total phenolic content of the fresh-cut lettuces during storage are shown in Figure 5.

The degree of the total phenolic content, i.e., the secondary metabolites produced by stress in fruits and vegetables, has antioxidant effects and can prevent oxidant damage to fruit and vegetable cells [35]. As can be seen in Figure 5A, the total phenolic content of the fresh-cut lettuces first showed an increasing and then decreasing trend during storage, with the UZ treatment group consistently maintaining the highest total phenolic content. On the 8th day of storage, the UZ treatment group had the highest total phenolic content, which was 1.04, 1.12, and 1.15 times that of the US, ZN, and CK treatment groups, respectively. Wang et al. found that the use of ultrasound combined with an acetic acid–erythromycin acid solution treatment increases the total phenolic content of asparagus [3], and the total phenolic content following this combined treatment was also found to be significantly higher than the single treatment group (p < 0.05). As such, it was no surprise when the UZ treatment increased the total phenolic content of the fresh-cut lettuce. When the storage time was extended, the total phenolic content decreased. This may have been induced by the continuous oxygen consumption of the total phenolic content, the decreased PAL activity, and the increased PPO activity [5].

PPO is an intracellular o-diphenol oxidase in fruits and vegetables that promotes the oxidation of the total phenolic content to form quinones. These are further oxidized to form final black substances, thus leading to the surface browning of fresh-cut fruits and vegetables [36]. As can be seen from Figure 5B, the PPO activity of the fresh-cut lettuces gradually increased during storage. The PPO activity of the CK group was found to be significantly higher, except on the 4th day, than that of the US, ZN, and UZ treatment groups (p < 0.05). Ultrasound treatment inhibits PPO activity by disrupting its structure [5]. However, it would be worthwhile to further study how ZnO NPs inhibit PPO activity. What can be speculated is that a ZnO NP film reduces the rate of gas exchange (O₂, CO₂), thus resulting in the excessive accumulation of the total phenolic content and inhibiting PPO activity. On the 8th day of storage, the PPO activity of the UZ treatment group (0.85 U·min⁻¹·g⁻¹) was found to be significantly lower than that of the US (1.05 U·min⁻¹·g⁻¹), ZN (1.06 U·min⁻¹·g⁻¹), and CK (1.28 U·min⁻¹·g⁻¹) treatment groups. As a liquid medium, ZnO NPs can enhance the cavitation effect of ultrasound while the pressure around the cells is compressed and expanded, thereby inhibiting PPO activity. On the other hand, ZnO NPs better penetrate lettuce cells, as well as enhance their antioxidant capacity [37], via ultrasound. This is in line with the results of Zhu et al., who found that the use of ultrasound combined with purine treatment can effectively inhibit the PPO activity of fresh-cut potatoes; in addition, they also demonstrated that the inhibitory effect is significantly better than that found in a single treatment group [7].

During phenylpropane metabolism, PAL mainly inhibits enzymatic browning by catalyzing the generation of the total phenolic content, while processing methods such as cutting further activate PAL activity. As the storage time increased, the PAL activity of the fresh-cut lettuces showed a trend of first increasing and then decreasing (Figure 5C). During the first two days of storage, the PAL activity of the US and UZ treatment groups was found to be significantly higher than that of the ZN and CK treatment groups (p < 0.05). This may have been due to the enhanced oxidative stress response that was caused by the cavitation effect of ultrasound that occurred in the fresh-cut lettuce cells, which thus resulted in increasing PAL activity [23]. The PAL activity of the US (94.15 U·h⁻¹·g⁻¹) and CK (79.62 U·h⁻¹·g⁻¹) treatment groups reached its peak on the 2nd day of storage, while the ZN (86.94 U·h⁻¹·g⁻¹) and UZ (101.32 U·h⁻¹·g⁻¹) treatment groups reached their peak on the 4th day of storage. During the later storage stages, the PAL activity continued to decrease due to the long-term refrigeration treatment and substrate consumption. On the 8th day of storage, the PAL activity of the UZ treatment group was 1.20 times that of the CK treatment group (p < 0.05). The above results showed that the UZ treatment increased the mass transfer rate of the ZnO NPs via ultrasound’s cavitation effect, thereby enhancing the non-biotic stress of the fresh-cut lettuces and improving PAL activity. However, the increase in PAL activity caused by the cutting operation in the CK treatment group gradually decreased with continuous extensions of the storage time [26].

CAT is one of the most important enzymes involved in scavenging the free radicals in fruits and vegetables [38,39]. As is seen in Figure 5D, the CAT activity of the fresh-cut lettuces peaked on the 4th day of storage and then gradually decreased. The most significant decrease was observed in the UZ treatment group, while only a slight decrease was observed in the CK treatment group. Therefore, it can be concluded that UZ treatment improves the peak CAT activity of fresh-cut lettuces. Throughout the storage process, the CAT activity of the US treatment group was consistently higher than that of the ZN treatment group (p < 0.05). This may be due to the fact that the US treatment increases CAT activity, while the ZN treatment produces more H₂O₂ [36]. On the 8th day of storage, the CAT activity of the UZ treatment group was 8.34%, 15.48%, and 24.97% higher than that of the US, ZN, and CK treatment groups, respectively. This indicates that a synergistic treatment has the strongest effect on the enhancement of CAT activity, which is consistent with the research results of Xu et al. [18]. Compared with a single treatment, the use of ultrasound combined with 1-MCP treatment was found to be more effective in improving CAT activity.

3.6. Effects of Ultrasound Combined with ZnO NP Treatment on the Antioxidant Capacity of Fresh-Cut Lettuce

The FRAP (ferric-reducing antioxidant power) detection method was used to determine the total reducing capacity of the fruits and vegetables via the SET (single-electron transfer) mechanism [23]. As is shown in Figure 6A, the FRAP total reducing capacity of the fresh-cut lettuces first showed an increasing and then decreasing trend. The FRAP total reducing capacity of the ZN treatment group (89.42 µmol/L) peaked on the 4th day of storage, while the FRAP total reducing capacity of the US (97.75 µmol/L), UZ (128.17 µmol/L), and CK (72.75 µmol/L) treatment group all peaked on the 6th day of storage. This indicates that the US and ZN treatments could effectively improve the FRAP total reducing capacity of fresh-cut lettuces, and the ZN treatment could also accelerate its peak arrival time. On the 8th day of storage, the FRAP total reducing capacity of the US and UZ treatment groups was found to be 1.23 and 1.76 times higher than that of the control group, respectively. This indicates that the UZ treatment improves the FRAP total reducing capacity of fresh-cut lettuces more effectively while also decreasing the tissue damage caused by reactive oxygen species and free radicals. Mustapha et al. also found the same result when they used ultrasound combined with peracetic acid–hydrogen peroxide treatment: they found that stronger non-biotic stress occurred in cherry tomatoes, thus inducing a higher level of the FRAP total reducing capacity [25].

As an important indicator of antioxidant capacity, the ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) free-radical-scavenging ability is based on a measurement of the scavenging ability of the antioxidants in fruits and vegetables to superoxide anion free radicals [23]. As can be seen in Figure 6B, the ABTS free-radical-scavenging ability of the fresh-cut lettuces gradually decreased during storage time. The ABTS free-radical-scavenging capacity of the US, ZN, and UZ treatment groups was always found to be significantly higher than that of the control group (p < 0.05). This may be due to non-biotic stress, which promotes the accumulation of phenolic content and the production of a higher antioxidant capacity [20], which could be observed while using the ultrasound and ZnO NP treatment on the fresh-cut lettuces. On the 8th day of storage, the ABTS free-radical-scavenging ability of the UZ treatment group (137.62 µmol/L) was significantly higher than that of the US (115.85 µmol/L), ZN (111.10 µmol/L), and CK (103.58 µmol/L) treatment groups. Previous studies have shown that bioactive substances such as the total phenolic content could effectively reduce the accumulation of reactive oxygen species and free radicals, thereby improving antioxidant capacity [25]. Therefore, the UZ treatment could effectively improve the ABTS free-radical-scavenging ability of fresh-cut lettuces, reduce the damage to the cell membrane, and increase the total phenolic content, which is consistent with the experimental results obtained for the total phenolic content mentioned above.

3.7. Effects of Ultrasound Combined with ZnO NP Treatment on the Membrane Integrity of Fresh-Cut Lettuce

Lipid peroxidation leads to direct contact between the enzymes and substrates in cells, thereby accelerating the browning of fruits and vegetables. MDA is the main secondary metabolite of lipid peroxidation, and its content can be used as a representative indicator of cell membrane integrity [26]. As is shown in Figure 7A, the MDA content of the fresh-cut lettuces showed a gradually increasing trend during the storage period, with the MDA content in the UZ treatment group found to be consistently lower than that in the CK treatment group. On the 8th day of storage, the CK treatment group had the highest MDA content (2.72 µmol/g), which was followed by the ZN treatment group (2.03 µmol/g) and the US treatment group (2.24 µmol/g). Both groups had an inhibitory effect on the MDA content, but there was no significant difference between them (p > 0.05). Meanwhile, the UZ treatment group had the lowest MDA content (1.97 µmol/g). Under appropriate conditions, ultrasound not only prevents damaging the cell structure of fresh-cut lettuces, but it also inhibits the lipid peroxidation of cell membranes. The interfacial tension and high humidity of the ZnO NP solution increase the cavitation effect of the ultrasound, thereby more effectively inhibiting lipid peroxidation [5]. Therefore, the UZ treatment effectively reduces the production of MDA in the fresh-cut lettuces, thus delaying its enzymatic browning.

H₂O₂ content is a key indicator of cell membrane integrity, as well as of the accumulation of products that are caused via the oxidative stress reactions generated by cutting operations [39]. As is shown in Figure 7B, the H₂O₂ content of the fresh-cut lettuces continued to increase, and the three preservation methods reduced the H₂O₂ content to varying degrees. On the 8th day of storage, the H₂O₂ content of the UZ treatment group was the lowest (54.26 µmol/L), thus indicating that the UZ treatment better maintains the cell membrane integrity of the fresh-cut lettuces. Notably, the H₂O₂ content of the US treatment group (53.51 µmol/L) was not significantly different from that of the UZ treatment group (53.57 µmol/L) on the 6th day of storage. However, on the 8th day, there was a rapid increase that surpassed the increase in the UZ treatment group observed on the 6th day (p > 0.05). This may be due to the increasing severity of cell damage caused by ultrasound over time, and it could be that the UZ treatment can effectively alleviate the damage caused by ultrasound [40]. The results show that the UZ treatment effectively reduced the cell membrane damage and lipid peroxidation, increased CAT activity, and decreased the H₂O₂ content of the fresh-cut lettuces, which is consistent with the CAT activity results mentioned above. ZnO sensitivity to ultrasonic waves and its easy permeability make it incredibly easy for ultrasonic waves to act on fresh-cut lettuces when they are subjected to pulsed ultrasonic stimulation. This reduces the rate of gas exchange between the fresh-cut lettuces and the outside of the internal CO₂ and O₂ layers and slows down the physiological metabolic activities of the fresh-cut lettuces.

3.8. Effects of Ultrasound Combined with ZnO NP Treatment on the Volatile Organic Compounds of Fresh-Cut Lettuce

Volatile organic compounds determine the quality and flavor of fresh-cut lettuces, and these factors are closely related to the consumer market’s love for this vegetable. Fresh-cut lettuces contain a variety of aroma components. The differences in the contents and types of volatile organic compounds caused by the four different treatments are shown in

Table 1. The volatile organic compounds of fresh-cut lettuces under different preservation treatments.

Classification	The Aroma Components	Area/%
		US	ZnO NPs	US + ZnO NPs	CK
Benzenes	Toluene	1.82 ± 0.11 a	1.19 ± 0.09 b	1.11 ± 0.08 b	0.58 ± 0.03 c
	p-Xylene	2.00 ± 0.15 a
	o-Xylene	1.01 ± 0.08 a
	1,4-Bis(trimethylsilyl)benzene	16.29 ± 1.04 a
	Benzene, 1,2,4,5-tetramethy	0.59 ± 0.02 a
	Benzene, 1,2,3,5-tetramethyl	0.56 ± 0.03 a
	Ethylbenzene		0.21 ± 0.02 a
	Benzene, 1,3-dimethyl		0.05 ± 0.00 a
	Benzene, 1-ethyl-2,4-dimethyl		1.79 ± 0.09 a
	Benzene, 1-methyl-3-(1-methylethyl)		0.24 ± 0.00 a
	p-Cymene		0.06 ± 0.00 a
	Benzene, 2-ethyl-1,3-dimethyl			0.24 ± 0.02 a
	Benzene, 1,2,3,4-tetramethyl			0.36 ± 0.02 b	1.59 ± 0.11 a
Alkanes	Ethylene oxide	10.08 ± 0.64 a
	Heptane, 2,2,4,6,6-pentamethyl	1.62 ± 0.10 a	1.34 ± 0.07 b	1.41 ± 0.07 ab	1.6 ± 0.09 a
	Silane, [[4-[1,2-bis[(trimethylsilyl)oxy]ethyl]-1,2-phenylene]bis(oxy)]bis[trimethyl		0.71 ± 0.06 a
	Silane, trimethyl[5-methyl-2-(1-methylethyl)phenoxy]			13.71 ± 0.86 a	13.16 ± 0.84 a
	3-Amino-2-phenazinol ditms			0.39 ± 0.01 a
Amines	Formamide, N-ethyl-N-phenyl	24.16 ± 1.53 a
	Chlorodifluoroacetamide	11.15 ± 0.68 a
	4-Amino-5-imidazole carboxamide,N,N,O- tris(trimethylsilyl)	0.54 ± 0.03 a
	1H-Indole-3-ethanamine, 6-fluoro-.beta.-methyl		21.83 ± 1.39 b	26.24 ± 1.64 ab	27.28 ± 1.68 a
	2-Methylamino-N-phenyl-acetamide		15.39 ± 0.93 a
	N-Benzyl-N-ethyl-p-isopropylbenzamide			0.14 ± 0.00 a
	Acetamide, N-[(4.alpha.,5.alpha.)-cholestan-4-yl]				0.21 ± 0.01 a
Aldehydes	1-Cyclohexene-1-carboxaldehyde, 4-(1-methylethenyl)-, (S)				0.3 ± 0.02 a
Ethers	2′,6′-Dihydroxyacetophenone, bis(trimethylsilyl) ether		11.52 ± 0.70 a	0.33 ± 0.01 b
Phenolics	1,2-Benzenediol, 3,5-bis(1,1-dimethylethyl)		12.91 ± 0.82 a
Alkenes	2-Propen-1-amine, 2-bromo-N-methyl		5.29 ± 0.32 b	20.44 ± 1.27 a
Nitriles	2-Amino-4-dimethylaminomethylenepentanedinitrile	3.34 ± 0.17 c	13.01 ± 0.85 b	20.07 ± 1.21 a
Ureas	1-(6-Methyl-benzothiazole-2-yl)-3-(4-methyl-benzoyl)-thiourea	1.06 ± 0.04 a	0.32 ± 0.01 c	0.38 ± 0.02 c	0.64 ± 0.02 b
Ketones	7-Methoxy-2,3-diphenyl-4H-chrome-4-one		0.31 ± 0.01 a
Hydrazines	N-Phenyl-N’-(4-N,N-diethylaminobenzylidene) hydrazine	0.12 ± 0.00 a
Acids	Arsenous acid, tris(trimethylsilyl) ester				3.33 ± 0.16 a
Azoles	5H-Naphtho[2,3-c]carbazole, 5-methyl	0.47 ± 0.03 a
	1,2,4-Oxadiazole, 5-(4-nitrophenyl)-3-phenyl	0.29 ± 0.01 a
Quinolines	Benzo[h]quinoline, 2,4-dimethyl		1.46 ± 0.08 b	2.07 ± 0.13 a
	6-Chloro-3-ethyl-2-methyl-4-phenylquinoline		0.27 ± 0.01 a
Thiadiazines	5-Methyl-2-phenylindolizine		0.76 ± 0.03 a
Silicon	Silanediamine, 1-chloro-N,N,N’,N’,1-pentamethyl	24.9 ± 1.47 a
Others	Carbon dioxide		11.33 ± 0.69 b	13.09 ± 0.86 b	51.3 ± 3.11 a
Benzenes		22.27	3.54	1.71	2.17
Alkanes		11.7	2.05	15.51	14.76
Amines		35.85	37.22	26.38	27.49
Aldehydes					0.3
Ethers			11.52	0.33
Phenolics			12.91
Alkenes			5.29	20.44
Nitriles		3.34	13.01	20.07
Ureas		1.06	0.32	0.38	0.64
Ketones			0.31
Hydrazines		0.12
Acids					3.33
Azoles		0.76
Quinolines			1.73	2.07
Thiadiazines			0.76
Silicon		24.9
Carbon dioxide			11.33	13.09	51.3

Different letters indicate statistical differences between different treatments (p < 0.05).

. There are 41 different main volatile organic compounds in fresh-cut lettuces, including benzenes (13), alkanes (5), amines (7), azoles (2), and quinolines (2), as well as various other volatile organic compounds. The content of 2,2,4,6,6-pentamethylheptane in the fresh-cut lettuces remained essentially the same or did not significantly differ under different treatments. Usually, esters, aldehydes, ketones, etc., are the main volatile organic compounds that produce the aroma of fruits and vegetables [41]. However, no matter which treatment was used, lipids were not detected in the fresh-cut lettuces. The aldehydes only detected (S)-4-(1-methyl vinyl)-1-cyclohexen-1-formaldehyde, which was only present in the control group. This may be due to the fact that three of the preservation treatments were able to effectively inhibit the generation of unpleasant odors in the fresh-cut lettuces. The ketones also only detected 7-methoxy-2,3-biphenyl-4H-phenylpyran-4-one, which was found to only exist in the ZN treatment group. This may be due to the fact that the ZN treatment is able to induce the production of new volatile organic compounds in fresh-cut lettuces but that the US treatment is not capable of avoiding the production of such volatile organic compounds [42]; therefore, they were not detected in the US and UZ treatment groups. Hydrocarbons generally have a fragrant odor, although they are not readily detectable by humans due to their high threshold value. However, they may enhance or reduce the overall odor quality of fresh-cut lettuces [11]. For example, the UZ treatment contained the highest amount of olefin, accounting for 20.44% of the total compositions. The unique volatile organic compounds provided a new flavor to the fresh-cut lettuces. The US and UZ treatments also contained the highest levels of nitriles and quinolines, accounting for 20.07% and 2.07% of the total components, respectively. These two substances also gave a unique aroma to the fresh-cut lettuces. Therefore, it can also be speculated that the UZ treatment reduces the generation of unpleasant odors during storage by inhibiting the generation of aldehydes and ketones. On the other hand, fresh-cut lettuces are endowed with a more unique and pleasant aroma when the generation of olefins, nitriles, and quinolines is promoted, as these reduce the adverse odors generated during storage [43].

3.9. Effects of Ultrasound Combined with ZnO NP Treatment on the Microstructure of Fresh-Cut Lettuce

The microstructures of the fresh-cut lettuces in the US, ZN, UZ, and CK treatment groups are shown in Figure 8. From Figure 8, it can be seen that the tissue structure of the CK treatment group was closely arranged due to the complete, regular cells that were roughly similar in shape. Each cell was tightly connected, with some cell membranes showing signs of disconnection (which was likely due to the cutting operation). The tightness of the intercellular connections in the US treatment group was found to change, and the cell shape was no longer regular. Some of the folds even appeared on the cell wall. Using ultrasound not only enlarged the cell lumen, but it also generated a small amount of cell debris to deposit within the cell lumen. The tissue structure and cell shape of the ZN treatment group were roughly similar to those of the control group. The only difference was that there was a small number of ZnO NPs attached to the cell lumen of the former, which adhered together in a filamentous manner. At the same time, the lettuce had a certain biosorption capacity, consistent with similar results reported by Nagdalian et al. [44]. The cellular changes in the UZ treatment group were similar to those in the US treatment group, with the intercellular adhesion reduced and shape changed. A small amount of the structure was damaged, and some cell fragments were deposited in the lumen. However, due to the ultrasound effect, some microchannels and larger cavities may have arisen [45], thus allowing more ZnO NPs to enter the lettuce cells. There were more ZnO NP filamentous attachments in the cell lumen compared to the ZN treatment group. The reason the cells were tightly connected could have been mainly due to the mixture of pectin and polysaccharides between the connected cell walls. Although the cavitation effect of the ultrasound inactivates some of the pectinase activities, the pectin in the cells was degraded due to the thermal effect generated by the use of ultrasound in the early stages of treatment, thus resulting in a decrease in the intercellular adhesion and the appearance of wrinkles in the intercellular space and cell wall [46]. This would also explain the decrease in the firmness of the fresh-cut lettuces during the initial stages of ultrasound treatment, which is also consistent with the other research conclusions on firmness mentioned above.

Although ultrasound treatment damages the tissue and structure of fresh-cut lettuces in the early stages, the shear force generated by cavitation also damages the integrity of the cell membrane to a certain extent. From a long-term storage perspective, the use of ultrasound inactivates pectinase activity, thereby delaying the decrease in the firmness of fresh-cut lettuces [47]. Previous studies have shown similar results, where the cavitation effect generated by ultrasound inhibits the cell wall. This phenomenon degrades the enzymes of the fresh-cut cucumbers, which results in better cell integrity than those found in other treatments. In addition, it was also found to improve the firmness of fresh-cut cucumbers [11]. Compared with the US treatment, the UZ treatment not only exhibited all the characteristics of the US treatment, but it also promoted the entry of ZnO NPs into the lumen of the fresh-cut lettuce cell microchannels that were generated using ultrasound.

4. Conclusions

In aiming to obtain an efficient, non-toxic, and low-cost method for fresh-cut lettuce preservation. The effects of three preservation methods (ultrasound, ZnO NPs, and ultrasound combined with ZnO NPs) on the odor, microstructure, and edible quality of fresh-cut lettuce were investigated in this study. The results showed that the use of ultrasound combined with ZnO NP treatment was found to significantly inhibit PPO activity, increase PAL activity, and increase the total phenolic content. Collaborative treatments demonstrated higher chlorophyll contents and maintained the appearance of fresh-cut lettuces. The use of ultrasound combined with ZnO NP treatment effectively reduced the TSS/TA and SSC/TA ratios and delayed the occurrence of physiological and biochemical reactions in the fresh-cut lettuces. The collaborative treatments also increased the CAT activity, as well as the propectin and cellulose contents of the fresh-cut lettuce samples, and prevented decreases in firmness. However, the content of H₂O₂ and MDA remained the lowest, while the antioxidant capacity remained the highest. At the same time, the ZnO NP treatment reduced the water loss and dry matter consumption of the fresh-cut lettuces. In addition, the use of ultrasound combined with ZnO NP treatment was found to reduce the production of the adverse flavor compounds that develop during storage, which was achieved by inhibiting the generation of aldehydes and ketones. On the other hand, these pleasant odors could cover the undesirable ones that arise during storage by promoting the generation of olefins, nitriles, and quinolines. The microstructure of the fresh-cut lettuce indicated that in the presence of microchannels generated by the use of ultrasound, ZnO NPs could enter into the intracellular cavity of fresh-cut lettuce with more ease. This finding could serve as a basis for a hypothesis on the mechanism of ultrasound when combined with ZnO NP treatment. In summary, the use of ultrasound combined with ZnO NP treatment effectively improves the storage quality and extends the storage time of fresh-cut lettuces; thus, these findings can also offer a new research direction for new preservation technologies for fresh-cut fruits and vegetables. In future studies, we will explore this mechanism at the molecular level.