Influence of the Application of Rosemary Essential Oil (Salvia rosmarinus) on the Sensory Characteristics and Microbiological Quality of Minimally Processed Pumpkin (Cucurbita moschata) ^†

Abstract

Pumpkin (P) production in Santiago del Estero, Argentina, is of high quality. The product is sold whole and fresh, and is traditionally consumed after being cooked in water. Due to its pleasant flavor (semi-sweet), it is widely accepted among consumers, being a potentially versatile product to be consumed fresh or minimally processed (MP). Processing increases food’s susceptibility to microbial spoilage, making it necessary to use sanitizers. In this work, the effect of different application methodologies at different concentrations of rosemary essential oil (REO) on the sensory characteristics and microbiological quality of grated P was evaluated. The product was washed, sanitized, cut, peeled, grated and centrifuged, and then REO was superficially incorporated in two concentrations (4 and 8 μL/mL), applied in three ways: sprayed (TA), immersion (TI) and by strips embedded in EO being adhered to the storage container (TV). The product was subsequently stored at 5 °C in sealed polypropylene bags of 35 μm. In addition, an immersion treatment with NaClO-100 ppm-3 min and a control without any additive were included. Samples were taken at 24 h and 8 days of storage, evaluating mesophilic aerobic microorganisms (MA), psychrophilic (Psy), enterobacteria (E) and mold and yeasts (MY). Sensory evaluation was carried out by trained judges, determining that aroma and flavor were the critical attributes for acceptability. At the end of storage, microbiological analysis showed that REO treatments presented levels of 10⁷ CFU/g, similar to NaCLO treatment, except TI with both concentrations, which was more effective in inhibiting the development of Psy and MY. Regarding aroma evaluation, at 8 days all treatments were acceptable, with the exception of TI and TV (8 μL/mL), while for flavor all treatments were acceptable with the exception of TI for both concentrations. Therefore, the application of TA and TV treatments at the lowest concentration could be considered for further studies to increase the shelf life of MP P.

1. Introduction

In Argentina, pumpkin (Curcubita moschata) production in 2022 was 259,562 tons [1]. Santiago del Estero is one of the main producers and suppliers of the local or internal market through its commercialization in the Central Market of Buenos Aires (MCBA). This vegetable is mainly destined for the domestic market, where approximately 95% of the national production is sold. Pumpkin has been part of people’s diet since pre-Columbian times. In Argentina, the consumption of this curcubit is 22 kg per capita per year. The fruits are an important source of carbohydrates, vitamins A and C and essential amino acids. It is easy to digest and provides few calories, which is why it is included in most hospital diets, community canteens and infant and child feeding programs [2].

Fruits and vegetables can be subjected to different processes to improve their conservation. In this sense, the global market for minimally processed vegetables is constantly expanding due to lifestyle changes that imply less time to prepare food. In Argentina, there is a very important market niche for these foods, which has not yet been exploited, even though there is a significant percentage of the population interested in this type of product [3]. In addition, it is important to note that, in recent years, there has been a significant increase in consumer interest in the quality and safety of the foods they consume [4,5]. For this reason, consumers demand that foods, in addition to being safe, promote health and physical well-being [6].

In response, food researchers and producers are faced with the need to find natural and safe alternatives to replace synthetic compounds for preservation products, such as natural antimicrobials and antioxidants agents [7,8]. The new trend of minimally processed and ready-to-eat foods has brought with it the emergence of new forms of microbial development, partly responsible for the global problem of food waste [9]. The use of antimicrobial compounds in this type of food would not only help to preserve the food and ensure food safety by preventing or controlling microbial growth, but would also reduce food waste [9,10]. These natural preservatives can be obtained from plants, animals or microorganisms, since they are part of the natural defense system of these organisms [10].

One of these natural preservatives are essential oils (EOs), and their components have been recognized as GRAS or approved as food additives by the US FDA [11]. One type of EO is rosemary essential oil (REO), and several studies have indicated that REO has significant inhibitory activity against a wide range of common foodborne bacteria. It has been proven that the use of 10 mg/mL free REO substantially inhibited the growth of Salmonella typhimurium and Listeria monocytogenes. However, the use of EOs is often limited because they can negatively affect sensory properties [12].

It is important to mention that in activities prior to this work, our research group carried out various tests to evaluate the effect of different OEAs (tea tree, oregano and rosemary) on the sensory quality (general appearance, smell and flavor) of minimally processed grated pumpkin. The results obtained indicated that the samples treated with REO were the most accepted by the evaluators. For this reason, based on these results, it was decided to further the research with rosemary oil.

The objective of the present work was to evaluate the effect of REO application on the sensory characteristics and microbiological quality of grated pumpkin produced in Santiago del Estero, Argentina.

2. Materials and Methods

2.1. Sample Preparation and Treatments

The pumpkins were obtained from producers from Santiago del Estero, Argentina, at a state of commercial maturity. They were washed and disinfected with chlorinated water (150 ppm—3 min). After draining, the pumpkins were peeled manually with a sharp knife, cut into slices and then into smaller pieces, removing the seeds and always respecting good manufacturing practices. Subsequently, they were grated using a food processor. The samples were subjected to different treatments: TC (control), grated pumpkin; TH, grated pumpkin immersed in NaClO at 100 ppm for 3 min; TS4, spraying with commercial REO (Rivadavia Homeopathic Pharmacy, Córdoba, Argentina) at 4 μL/mL on grated pumpkin; TS8, spraying of REO at 8 μL/mL on grated pumpkin; TI4, pumpkin soaked in REO at 4 μL/mL; TI8, pumpkin soaked in REO at 8 μL/mL; TV4, absorbent paper strips soaked in REO at 4 μL/mL in the storage container; and TV8, absorbent paper strips soaked in REO at 8 μL/mL in the storage container. Finally, the samples were stored at 5 °C in sealed 35 μm polypropylene bags and extractions were performed twice for the different determinations.

2.2. Methodology Used for Microbiology

All treatments were analyzed at 24 h and 8 days after storage. The procedure for determining the microbiological load was carried out in a laminar flow chamber. For the 10⁻¹ dilution of each treatment, sterile scissors were used to cut the packaging. Then, exact quantites of each sample were weighed and transferred to a sterile 90 μm bag. Subsequently, 90 mL of sterile water was added to the same bag. Each of the bags was subjected to a shaking process for 3 min in a stomacher (IUL Instruments, Barcelona, Spain). To carry out the 10⁻² dilution, 1 mL of the 10⁻¹ dilution was taken and diluted in 10 mL of sterile water in a test tube. This procedure was replicated for the following dilutions, ensuring the homogeneity of the solution by using a vortex before each dilution. To carry out the inoculation, 1 mL of the selected dilution was taken and placed in a sterilized Petri dish. The already-corresponding melted culture medium was then poured in, ensuring thorough manual homogenization. The counts of mesophilic aerobic bacteria (MA), psychrophilic aerobic bacteria (Psy), enterobacteria (E) and molds and yeasts (MY) were performed [13]. For the growth of MA and Psy, PCA (plate count agar) medium at 37 °C for 48 h and 5 °C for 7 days, respectively, was used. For E, EMB (eosin methylene blue) medium at 37 °C for 48 h was used. Finally, for the cultivation of MY, PDA (potato dextrose agar) medium at 28 °C for 7 days was used. All culture media were purchased from Merck (Darmstad, Germany). Microbial counts were determined from three samples of each treatment, with the sowings performed in triplicate, in two independent experimental runs. The results were expressed in log CFU/g.

2.3. Sensory Analysis

Sensory evaluation was carried out at 24 h and 8 days of storage by a panel of 10 trained judges, ranging in age from 25 to 58 years. Samples were coded with three-digit random numbers and presented to judges who performed independent evaluations. Each attribute evaluated was rated on a scale of 1 to 9, with a score of 9 being considered optimal and the lowest score being 1, with 5 being the marketable threshold [4,14,15]. Six attributes were evaluated: overall visual appearance, color, aroma, presence of exudate, moisture to the touch and flavor, of which aroma and flavor were critical from the judges’ acceptance point of view.

2.4. Statistical Analysis

Results were analyzed by Analysis of Variance (ANOVA), using Infostat 2011 software (UNC-Argentina). All experiments were performed in triplicate. Means were compared by the least significant difference (LSD) test at a significance level of 0.05.

3. Results and Discussion

3.1. Effect of REO on the Native Microflora of Grated Pumpkin

The mesophilic aerobes in grated pumpkin with different treatment are shown in Figure 1.

The TH, TS4, TS8, TI4 and TV4 treatments presented results that oscillated between 10⁵ and 10⁶ CFU/g after 24 h of storage for MA. However, treatments TI4, TI8 and TV8 presented a significantly lower count, with values around 10³ CFU/g. After 8 days of storage, an increase in the growth of microorganisms was observed, presenting levels of 10⁶ CFU/g in all treatments, including TH. On the other hand, Kvapil [16] reported the initial counts of MA in fresh pumpkin cut into cubes, indicating values of 2.5 and 7 log CFU/g at 8 days of storage. In minimally processed vegetables, high counts are expected, of 10⁶–10⁸ CFU/g, which gives an idea of endogenous microflora and contamination suffered by the material. The initial results are determinants of shelf life, as microbial deterioration can occur quickly and normally becomes visible, negatively affecting the fresh appearance [17,18].

With respect to the E count, after 24 h of storage, the treatments TH, TS4, TS8, TI4, TV4 and TC displayed values close to 10³ CFU/g. Significant differences were observed between the TI8 and TV8 treatments in comparison with the TC treatment, reaching a difference of 1 log. After 8 days of storage, the TS4, TS8 and TC treatments presents results exceeding 10⁶ CFU/g, while the other treatments were below this value, with results of approximately 10⁵ CFU/g. TH treatment showed the lowest level, reaching 10⁴ CFU/g.

Regarding the growth of Psy bacteria, it was notable that after 24 h of storage, TC and the samples treated with REO presented values of approximately 10³ CFU/g, except for TH, which presented values of 1 log CFU/g below the rest. After 8 days of storage, all treatments showed counts greater than 10⁶ CFU/g, except treatments TI4 and TI8, which had values close to 10⁵ CFU/g. Similar results were obtained for minimally processed broccoli treated with REO by spraying, in which all concentrations studied (5, 10 and 15 μL/mL) presented results greater than 10⁶ CFU/g after 7 days of refrigerated storage [18].

Regarding MY, at 24 h, it was observed that the TC treatment presented values of approximately 10³ CFU/g, while all treatments with REO and TH presented values of approximately 10² CFU/g. When considering storage for 8 days, a significant increase in the growth of these microorganisms was evident, where TC and treatments TS4, TS8, TV4 and TV8 showed values of approximately 10⁷ CFU/g. However, treatments TI4 and TI8 presented counts of approximately 10⁴ CFU/g.

3.2. Sensory Evaluation

The aroma and flavor attributes are shown in Figure 2. When analyzing the evolution of the aroma, it was observed that, after 24 h, the immersion treatments for both REO concentrations presented values below the acceptability limit (<5) for both aroma and flavor, so they were discarded. The other REO treatments, except TS8 and TV8, showed initial scores between 8 and 9, indicating good initial acceptance, as observed in Figure 2A.

The acceptability of REO aroma in vegetables for the 24 h storage time was also reported for broccoli. Rosemary and tea tree essential oils applied in the form of a spray on minimally processed broccoli transmitted their characteristic odor, a product of the volatile components that make up each oil. Immediately after their application, the volatiles were perceived as strange odors, although the scores indicated that the product was acceptable on a scale of 1 to 5, taking the extreme 5 as an optimal score that represents the fresh vegetable [18].

Then, after 8 days, these treatments showed a significant decrease, although they presented similar values and were close to the established limit. Regarding the flavor, all the treatments with REO presented initial values higher than the acceptance limit as observed in Figure 2B, and they decreased after 8 days, but remained above it.

From the sensory point of view, the application of the TA4, TA8 and TV4 treatments could be considered to carry out further studies analyzing the microbiological quality in order to increase the shelf life of fresh grated squash.

4. Conclusions

In this study, the influence of different REO application methodologies and different concentrations on the sensorial characteristics and microbiological quality of minimally processed pumpkin cultivated under edafoclimatic conditions from Santiago del Estero-Argentina was reported. Future investigations should focus on exploring the applications of REO in this product, particularly in relation to TA and TV4 treatment, with the aim of increasing the shelf life of the minimally processed pumpkin. Likewise, it is suggested to combine this technique with other emerging technologies, such as UV-C radiation, ultrasound and modified atmospheres. Another relevant aspect would be to evaluate the effect of the treatment by immersion, using reduced concentrations to optimize results. Furthermore, the evaluation of the effect of the incorporation of REO on the antioxidant capacity and phenolic compounds of pumpkin would be essential, with the purpose of obtaining a food with improved functional properties.