Response of Fresh-Cut Iceberg Lettuce to Fumigation with Botanical Essential Oils

Iceberg lettuce (Lactuca sativa L. var. capitata) ‘Ice Wave F₁’ was purchased from a commercial farm near Skierniewice (Poland). After being transported to the laboratory, the lettuce was placed at 0–1 °C for one night. The following EOs were used for the fumigation of fresh-cut iceberg lettuce: rosemary (Avicenna Oil, Wroclaw), peppermint and basil (Pranarom, Ghislenghien), thyme and lemon (Etja, Elblag), oregano (Hepatica, Nasutow), and marjoram (Farmvit, Szczecin). The oils were extracted by steam distillation without the use of chemicals. It involves passing steam through the plant elements (rosemary – flowering tops and leaves; peppermint – shoots and leaves; basil – leaves and flowers; thyme – flowering plants; marjoram – leaves and flowers; lemon – fruits; oregano – shoots and leaves) and then condensing the steam containing volatile fragrance molecules. The water-insoluble fragrance oil is then separated from the condensed water.

The storage experiments were set up two days after the iceberg lettuce harvest. The outer yellowed and damaged leaves were removed, and the lettuce was cut into strips 0.5–1.0 cm in width. Immediately after cutting, the strips were packed into polypropylene boxes with external dimensions of 355 mm × 175 mm × 105 mm (L × W × H) and a capacity of 6 L (Plast Team Margarit 6L, no 3629). Two portions of sliced lettuce (600 g in one box) were prepared for each experimental object. The selected EO was pipetted on two cotton disks (4 cm diameter) taped to the bottom of each box lid. Then, the boxes were tightly closed and left at room temperature for two hours. The EO doses were 100 or 200 μL per box (i.e., 16.7 μL·L⁻¹ or 33.3 μL·L⁻¹). Fresh-cut lettuce, not treated with EOs but also tightly closed in a box, was used as the control. After fumigation, the boxes were opened, and the plant material was divided into 100 g samples. Transparent polyethylene boxes, recommended for food products, measuring 154 mm × 98 mm × 70 mm (L × W × H) of 1.0 L capacity (GUILLIN W1/059C) were used to pack the samples. After packing, the boxes were covered with lids (GUILLIN W2/001), which had 77 holes for air exchange. The storage experiment was set up as four replicates. Three additional samples were prepared for microbiological, and two for sensory analyses. All samples were stored at 5 °C for six days. The experiments were repeated twice.

Quality assessment was carried out every two days during storage of fresh-cut plant material. The following quality traits were assessed visually: wilting, browning (darkening) of the cut surface, and rotting. The evaluation was based on a nine-point scale, in which 1 meant no sign of change in a given trait, while 9 meant maximum severity of a given change, i.e., very severe wilting, darkening, and rotting.

The market value was also assessed on a nine-point scale as follows: 1 – no market value, 3 – limited, 5 – fair (market value threshold), 7 – good, 9 – excellent.

Weight loss was calculated as the percentage difference between the initial weight of a plant sample and the weight after a specified storage period.

Sensory analyses of sliced lettuce treated with EO were carried out after four days of storage at 5 °C. Quantitative descriptive analysis (QDA) following ISO 13299:2016 was used to evaluate the following quality attributes: color, crispness, lettuce flavor, herbal aroma, sweet flavor, overall quality, and consumer acceptance, which were selected based on preliminary tests. The intensity of perception for each attribute was assessed on an unstructured graphical scale marked with the appropriate boundary terms. The scores marked on the scale were converted to numerical values ranging from 0 to 10 contract units, where 0 means no intensity, and 10 means the high intensity of the attribute. Sensory evaluation was performed in the sensory laboratory, meeting all the requirements in PN-EN ISO 8589:2010/A1:2014-07. The profile evaluation was performed by a team of 10 people with verified sensory sensitivity trained in PN-EN ISO 8586:2014-3 sensory evaluation techniques. Each object was assessed in duplicate, in random order. Individual samples of the test product were placed in coded plastic containers (250 mL) covered with lids. Noncarbonated water was used as a flavor neutralizer between the evaluated samples. For initial data processing, ANALSENS data preprocessing software was used. The obtained results were illustrated as a principal component analysis (PCA) graph.

Microbiological analyses were carried out on samples fumigated with EOs at a concentration of 16.7 μL·L⁻¹ after four days of storage at 5 °C. They were performed according to the following standard methodologies: PN-EN ISO 4833-2:2013-12 and PN-ISO 21527-1:2009. A portion of 25 g of cut lettuce was transferred to 225 ml of peptone water in sterile stomacher-type filter bags (400 ml). Samples were homogenized in a stomacher BagMixer^® 400 P at a constant speed of 8 strokes per second for 10 minutes. Decimal dilutions were then made using the same diluent and analyzed for microorganisms (aerobic mesophilic bacteria, yeast, and mold). Aerobic mesophilic bacteria were determined on plate agar (PCA, Merck) after incubation at 30 °C. Yeast and molds were counted on glucose-chloramphenicol agar with yeast extract (YGC agar, Merck) after incubation at 25 °C. Results were expressed as colony-forming units per gram of plant material (CFU per g) and converted to decimal logarithm (log10) for statistical calculations.

Storage experiments were set up in a two-factor design in a completely randomized system. The differentiating factors were the type of EO and EO concentrations used for lettuce fumigation. In this study, seven EOs (rosemary, peppermint, basil, thyme, marjoram, lemon, and oregano) were used, and fumigation was carried out at two concentrations (16.7 μL·L⁻¹, 33.3 μL·L⁻¹). Two-factor analysis of variance (ANOVA) was used to statistically calculate the results of the visual quality of plant material. Microbial contamination was analyzed only on samples fumigated with one EO concentration (16.7 μL·L⁻¹), so the results were statistical calculated in a one-factor system. The results of individual experiments did not differ significantly, therefore all means of the two tests (eight for browning and market value and six for microbiological parameters) were analyzed together. The means of storage quality and microbial contamination were compared using the Tukey HSD (Honestly Significant Difference) procedure at p = 0.05. Sensory results were described using PCA based on a correlation matrix. Calculations were performed in the STATISTICA 13 statistical package (Dell).

Fresh-cut iceberg lettuce fumigated with EOs and nonfumigated showed no signs of rot or wilting during six days of storage. The natural weight loss was very low and after six days of storage at 5 °C was approximately 0.1% for fresh-cut, fumigated and untreated iceberg lettuce. Fumigation with EOs had varying effects on the storage life of the lettuce (Tables 1, 2). After two days of storage, the browning of cut surfaces was the lowest compared with the storage for four and six days. The lowest browning was observed in the samples fumigated with rosemary and thyme, and the highest with marjoram and oregano. After four days of storage, the highest browning notes were obtained in samples fumigated with marjoram, with a higher concentration of oregano, and the lowest with rosemary. After six days of storage, rosemary caused the least intense browning, and marjoram and oregano caused the most intense browning, comparable with the nonfumigated control. Generally, the concentration of EOs did not influence browning. Fumigation with rosemary and thyme EOs was always significantly lower than without fumigation, ensuring better lettuce quality during six-day storage. The market value decreased with storage time (Table 2). After two days, the market value was higher than in the control. After six days of storage, the lowest value was found in lettuce fumigated with basil and marjoram. Lettuce treated with rosemary and a higher thyme concentration received the highest notes. Generally, the concentration of EOs had no effect on market values. Lettuce fumigated with rosemary EO and thyme EO with a higher concentration received the highest notes. Generally, the concentration of EOs did not affect market values. The market value notes mostly agreed with those regarding browning of cut surfaces.

The PCA graphic projection (Fig. 1) shows the sensory quality of fresh-cut EO-treated iceberg lettuce in the two principal components system (as coordinates). The evaluated lettuce treatments are scattered on the surface of Figure 1, indicating substantial differences in their sensory scoring. The overall quality was closely related to product acceptance and sweet flavor (vectors have the same direction). High notes of sensory quality were obtained by the lettuce fumigated with marjoram (33.3 μL·L⁻¹) and thyme (16.7 μL·L⁻¹). These objects are located in places closest to the features that distinguish overall quality and consumer acceptability. Thyme oil-fumigated lettuce (33.3 μL·L⁻¹), oregano (33.3 μL·L⁻¹), and lemon (33.3 μL·L⁻¹) were highly scored due to their high intensity of sweet flavor. High intensity of aroma and herbal flavor characterized the objects of lettuce fumigated with basil, rosemary, and peppermint EOs. These objects are located in the closest places to these distinguishing features, adversely affecting the lowest overall quality and product acceptance (on the opposite side of the evaluation vectors of overall quality and consumer acceptance). The results indicate that the type of EO used to fumigate fresh-cut iceberg lettuce significantly affects the sensory quality. On average, there were no statistical differences in the quality of lettuce treated with different concentrations of EO (16.7 and 33.3 μL·L⁻¹). However, in the case of marjoram EO, better acceptance was obtained for lettuce treated with the higher concentration (33.3 μL·L⁻¹). In contrast, in the case of peppermint EO, a higher score was obtained for lettuce fumigated with a lower concentration (17.7 μL·L⁻¹).

Figure 1.

Fumigation of fresh-cut lettuce with EOs did not significantly change the number of microorganisms in stored plant material. After four days of incubation at 5 °C, the number of bacteria reached an average of 7.4 log10 cfu·g⁻¹ (from 7.3 to 7.6). Mean number of yeasts was 6.7 log10 cfu·g⁻¹ (from 6.4 to 7.1). The density of molds in plant material was generally low and ranged from 0 to 1.25 log10 cfu·g⁻¹. The relatively high standard deviations (0.4 to 0.7) indicate significant differences between samples from the same object.