Changes in microbial population and physicochemical properties of fresh-cut cucumbers subjected to near-freezing temperature at −2 °C

Abstract

In this study, fresh-cut cucumbers were subjected to near-freezing temperature storage at −2 °C (NF) for 12 d, and microbial population (aerobic bacteria and coliforms), physicochemical properties (weight loss, color, firmness, and pH), and O₂ consumption were compared with samples stored at 5 °C (C). Observation indicated that no sample showed any critical freezing phenomena during near-freezing temperature storage. Results showed that the microbial population (aerobic bacteria and coliforms) was suppressed in NF samples but increased significantly in C samples compared with the stored samples on day 0. Results of physicochemical properties showed that weight loss and total color difference of NF samples were less than those of C samples. Moreover, near-freezing temperature storage at −2 °C inhibited O₂ consumption compared with storage at 5 °C. These results provide new insights into the application of near-freezing temperature storage for fruits and vegetables in the food cold chain.

Introduction

Sub-zero temperature storage is considered to be the most efficient technology for quality maintenance of fresh foods (Liu et al., 2020). Recently, supercooled storage, in which fresh foods are stored at sub-zero temperatures, has been studied to demonstrate the potential of long-term storage to maintain the freshness of, for example, fruit (Osuga et al., 2021a; Osuga et al., 2021b), vegetables (Koide et al., 2019; Quang et al., 2017), meat (You et al., 2020), seafood (Park et al., 2022), and fermented food (Lee et al., 2021), etc. Although supercooling is a thermodynamically metastable state (Bilbao-Sainz et al., 2022) and the supercooling state of fresh-cut vegetable is affected by various factors (storage period, storage temperature, and sample size) (Koide et al., 2022), supercooled storage of fresh foods is considered to be an innovative method to prolong their shelf-life (Stonehouse and Evans, 2015). Our previous research showed that supercooled storage (−5 °C for 10 d) of fresh-cut apples was successful in 95 % of samples, in which freezing phenomena were not observed during storage (Osuga et al., 2021b).

Here, we propose near-freezing temperature storage (Liu et al., 2020; Xiao et al., 2022; Zhao et al., 2018) at −2 °C. Near-freezing temperature, between the supercooling point and the freezing point of an individual material, is within the range of minimal non-freezing temperatures, and has been used for the storage of animal organs and fresh fish (Zhao et al., 2018). We propose that if the storage temperature was higher than the temperature performed in previous supercooled storage (−5 °C), but lower than the freezing temperature, the possibility of near-freezing temperature storage of fresh foods without critical freezing would be close to 100 %.

In this study, we used fresh-cut cucumber as a model sample for fresh-cut vegetables, and the samples were placed in hermetically sealed plastic boxes and subjected to near-freezing temperature storage (−2 °C) or storage at 5 °C for 12 d. Herein, 5 °C was used as the control temperature, as some reports evaluated the changes in quality and/or microbial level of fresh-cut cucumber at about 4 °C (Fan et al., 2019; Hou et al., 2023; Wei et al., 2020).

The objectives of this study were to evaluate the potential of near-freezing temperature storage of fresh-cut vegetables without critical freezing phenomena, identify changes in microbial populations and physicochemical properties after near-freezing temperature storage, and demonstrate changes in O₂ and CO₂ compositions in the plastic boxes during near-freezing temperature storage at −2 °C and storage at 5 °C.

Materials and Methods

Material and storage conditions Cucumbers (Cucumis sativus L.) were purchased from a local supermarket in Morioka, Japan. The sample design is shown in Figure 1. The cucumbers were cut with a knife into a half-circle shape about 6.5 cm long and weighing 20 g, then immersed in tap water for 5 min and dried on a clean bench (MCV-710ATS, Sanyo, Osaka) for 10 min. Next, the samples were placed in a plastic box (230 × 170 × 120 mm; A-110, Mitsubishi Gas Chemical Company, Inc. Tokyo, Japan), and then enclosed with a lid on a clean bench. In the above process, the knife, plastic box, and plastic cutting board were sprayed with 75 % ethanol, dried, and subjected to UV-C irradiation (254 nm) on a clean bench prior to use for measurements. Each box contained 4 samples. The boxes were stored in an incubator (LHU-114, Espec, Osaka, Japan) at −2 °C for 12 d or in an incubator (MIR-153, Sanyo, Gunma, Japan) at 5 °C for 12 d. The samples stored at 5 °C were used as the control. Hereafter samples subjected to near-freezing temperature storage and control are expressed NF and C, respectively. During storage, the relative humidity of the NF and C samples in plastic boxes was approximately 97 % and 99 %, respectively. The microbial populations (aerobic bacteria and coliforms) and physicochemical parameters (weight loss, color, firmness, and pH) were measured in the fresh-cut cucumber samples on day zero and after storage (12 d).

Fig. 1

Experimental sample designing used in this study.

Determination of freezing point and supercooling point The freezing point and supercooling point of fresh-cut cucumbers were measured according to the procedure described by Koide et al. (2020). A T-type thermocouple was inserted into each sample, which were then they were placed in a plastic box and cooled to 0 °C in an incubator (LHU-114, Espec). After the temperature of each sample reached 0 °C, they were cooled to −20 °C at 0.2 °C/min by decreasing the temperature of the incubator (n = 16). The temperature was recorded at 5 s intervals using a datalogger (midi Logger GL220, Graphtec Corp., Yokohama, Japan). The temperature at which the low temperature exothermic reaction occurred in the time-temperature profile was taken as the supercooling point, and the temperature where the measured temperature is almost constant after rising from that of the supercooled state was determined as the freezing point (Fig. 2) (Koide et al., 2020; Meng et al., 2007).

Fig. 2

A representative time-temperature profile of a fresh-cut cucumber sample during the experiment of determination the freezing point and supercooling point.

Microbiological analysis Microbiological examinations were conducted as described by Koide et al. (2011). Twenty grams of fresh-cut cucumber sample was mixed with 180 mL of sterile 0.85 % sodium chloride solution in a sterile polyethylene bag, and pummeled using a stomacher (Seward Stomacher 400, West Sussex, UK) for 2 min at high speed. The aliquot was then used for various serial dilutions. The diluted samples were analyzed for the populations of aerobic bacteria and coliforms. All of the microbiological analysis were conducted under sterile conditions. Total aerobic bacterial counts were enumerated on plate count agar with the following composition (g/L): yeast extract (Difco Laboratories Inc., Detroit, MI, USA), 2.5; tryptone (Difco Laboratories Inc.), 5.0; glucose (Wako Pure Chemical Industries, Osaka, Japan), 1.0; and agar (Difco Laboratories Inc.), 15.0. The plates were incubated at 35 °C for 48 h, and then the colonies were counted. The detection limit of this method was 1 log₁₀ CFU/g. Coliforms were enumerated on X-GAL agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). The plates were incubated at 35 °C for 20 h prior to counting of colonies. The detection limit of this method was 1 log₁₀ CFU/g. In this study, the bacterial number at an undetectable level was considered to be zero (Koide and Shi, 2007; Sun and Griffiths, 2000). The microbial analysis was performed in quadruplicate.

Physicochemical properties

Weight loss The weight loss of the fresh-cut cucumbers was measured using an electronic balance on day zero and after storage. The weight loss was expressed as the percentage loss from the initial weight. Weight loss was calculated according to the following equation:

  

where W_i and W_a are the initial weight (g) and the weight after storage (g), respectively.

Color The color of the flesh of the cucumbers was measured using a Minolta Chromameter (CR-20, Minolta, Tokyo, Japan). The color variables L* (whiteness/darkness), a* (redness/greenness), and b* (yellowness/blueness) were measured at three points on each sample. The mean value from three points was used as the color of each fresh-cut cucumber. The total color difference (ΔE) was calculated according to the following equation:

  

where ΔL*, Δa*, and Δb* are the changes in the ratios of each color parameter.

Firmness The firmness of fresh-cut cucumbers was measured using a TPU-2C texture analyzer (Yamaden Co., Ltd., Tokyo, Japan). The sample was positioned onto the plate, then a cylindrical plunger (3 mm diameter) was pressed into the cucumber sample at a speed of 5 mm/s, according to Osuga et al. (2021b). The mean value of breaking stress at three different points on the sample surface was defined as the firmness of the sample (Orikasa et al., 2014), and firmness was expressed in N.

pH The fresh-cut cucumber samples were homogenized with a double weight of distilled water using a blender (CM-100, AS ONE Corp., Osaka, Japan). The pH of the juice was measured using a D-72 pH meter (Horiba Ltd., Kyoto, Japan).

The above measurements of physicochemical properties (weight loss, color, firmness, and pH) were performed in triplicate.

Gas composition in the storage box The O₂ and CO₂ concentrations in the plastic boxes were determined using a gas chromatograph (Shimadzu GC-8A, Tokyo, Japan) equipped with a molecular sieve 5A column, a Porapak Q column, a Shimalite Q, and a thermal conductivity detector (TCD). Column and INJ/DET temperatures were determined as 75 °C and 80 °C, respectively. Helium was used as the carrier gas at a flow rate of 50 ml/min. The measurement was conducted in triplicate.

Statistical analysis The quantitative data were presented as mean ± standard deviation and then analyzed using a one-way analysis of variance. The t-test (for two groups) and the Tukey-Kramer test (for three groups) was used to assess the significance of differences among mean values at a level of p < 0.05 using an Excel statistical software package (BellCurve for Excel version 2.11, Social Survey Research Information Co., Ltd., Tokyo, Japan). For principal component analysis (PCA), biplots were constructed using R (v.4.2.2) and RStudio (v. 2022.07.1 Build 554) software.

Results and Discussion

Freezing point and supercooling point The mean values and standard deviations of the freezing and supercooling points were −0.79 ± 0.27 °C (minimum temperature was −1.3 °C) and −3.34 ± 0.52 °C (maximum temperature was −2.1 °C), respectively. Figure 3 shows a representative sample temperature of fresh-cut cucumber during near-freezing temperature storage at −2 °C for 12 d. During near-freezing temperature storage at −2 °C, the temperature in the incubator and samples was −2.01 ± 0.08 °C and −2.05 ± 0.06 °C, respectively. It is known that when ice crystallization occurs, an increase in temperature is observed due to the release of latent heat (James et al., 2009). There was no obvious change in the temperature profile of fresh-cut cucumbers (n = 41) during near-freezing temperature storage; thus, all samples could be kept at −2 °C for 12 d. Because −2 °C was in the range of partial freezing, we should consider the possibility of a non-injurious freezing state in samples (Lu et al., 2015). Thus, we cannot address that the samples subjected to near-freezing temperature were in a supercooled state. However, since there was no critical freezing in the vegetable samples at a temperature of −2 °C, which showed little temperature fluctuation (standard deviation of sample was 0.06 °C), this will give new insights into the application of supercooled storage for practical use.

Fig. 3

A representative time-temperature profile of a fresh-cut cucumber sample during near-freezing temperature storage at −2 °C.

Microbiological analysis Figure 4(a) shows the population of aerobic bacteria on day zero and after storage (12 d). The number of aerobic bacteria of C samples was increased significantly compared to that of F samples. Meanwhile, the number of aerobic bacteria of NF decreased slightly by 0.25 log₁₀ CFU/g. Recently, there have been a few reports which mentioned a slight decrease in bacterial population in fresh food during sub-zero temperature storage. Osuga et al. (2021b) reported that the number of aerobic bacteria was slightly decreased in fresh-cut apples after supercooled storage at −5 °C for 10 d, and Kim et al. (2021) mentioned a microbial reduction in kimchi following supercooled storage at −2.5 °C for 12 d, which support our results of a slight decrease in bacterial population during near-freezing temperature storage at −2 °C for 12 d.

Fig. 4

Changes in the number of aerobic bacteria (a) and coliforms (b) of fresh-cut cucumbers during near-freezing temperature storage.

Error bars show the standard deviation. F: Fresh (day zero), NF: Near-freezing (−2 °C for 12 d), C: Control (5 °C for 12 d). Mean values with different letters are significantly different (p < 0.05).

Izumi (2005) reported that the bacterial flora of cucumber is mainly composed of gram-negative bacteria, including some psychrotrophic bacteria, such as Pseudomonas, Enterobacter, Pantoea, and Citrobacter. In this study, we measured the number of Pseudomonas spp. of F, NF, and C samples using the same diluted samples used for the populations of aerobic bacteria and coliforms, and 4.72 ± 0.68, 4.47 ± 0.26, and 7.12 ± 0.54 log₁₀ CFU/g were found in F, NF, and C, respectively. These results implied that changes in populations of psychrotrophic bacteria such as Pseudomonas spp. showed a similar trend to that of aerobic bacteria, and would be one of the important bacteria for understanding significant differences in microbial populations between NF and C. Based on the above results, psychrotrophic bacteria showed increased numbers in C samples at 5 °C. Meanwhile, in NF samples at −2 °C, it is suggested that some psychrotrophic bacteria were injured or in a dormant state, resulting in a decrease in their microbial population during storage.

Figure 4(b) shows the population of coliforms on day zero and after storage (12 d). The number of coliforms of C samples was increased by approximately 4 log₁₀ CFU/g. On the other hand, that of NF samples was decreased by approximately 1 log₁₀ CFU/g. Overall, our results indicated that near-freezing temperature at −2 °C would be an effective method for maintaining the microbiological quality of fresh-cut vegetables during storage.

Physicochemical properties Weight loss of NF and C samples is shown in Table 1. Weight loss of NF samples was significantly less than that of C samples. In addition, weight loss of C samples was close to 5 %, which is an index of whether fruits and vegetables reach retail value or not (Koide and Shi, 2007). In previous studies, weight loss of fresh-cut vegetables and fruits is considered to be mainly due to water loss, indicating that water may have been lost through respiration and transpiration, and their rates increase with increasing temperature (Kang et al., 2021; Mahajan et al., 2008). Thus, it is considered that NF samples at a temperature of −2 °C showed significantly lower weight loss compared to C samples.

Table 1. Changes in physicochemical properties (weight loss, ΔE, firmness, and pH) of fresh-cut cucumbers during near-freezing temperature storage.

Sample	Weight loss (%)	ΔE (-)	Firmness (N)	pH (-)
F	-	-	14.6 ± 1.9ab	5.61 ± 0.18b
NF	1.91 ± 0.56b	2.21 ± 1.02b	15.8 ± 1.8a	6.09 ± 0.42a
C	4.62 ± 4.16a	4.87 ± 2.50a	13.8 ± 1.4b	6.39 ± 0.22a

The values are shown as mean ± standard deviation. F: Fresh (day zero), NF: Near-freezing (−2 °C for 12 d), C: Control (5 °C for 12 d). Mean values with different letters are significantly different (p < 0.05).

Regarding color change, results indicated that samples subjected to sub-zero temperature storage for 12 d showed significantly inhibited color change (ΔE) compared to samples stored at 5 °C (Table 1). As shown in Figure 5, leakage, browning, and yellowing were observed on the flesh of C samples, while the flesh of NF samples was visually maintained. Browning and yellowing found in C samples were considered to be a result of polyphenol oxidase (PPO) reaction of polyphenols and chlorophyll degradation (Toivonen and Brummell, 2008). In this study, there might be an increase of PPO reactions due to tissue or cell damage from chilling injury (Li et al., 2023; Ohkawa et al., 2003) (Fig. 5); however, there were no symptoms of chilling injury on the surface of NF samples. These results are similar to the previous study, in which near-freezing temperature storage showed significant inhibitory effects on the occurrence of guava chilling injury and on increases in cell membrane permeability, and contributed to a compact and well-organized cell structure at the end of storage compared to cold storage treatments (6 °C and 10 °C) (Xiao et al., 2022). Further study on why C samples but not NF samples showed chilling injury is required in the future.

Fig. 5

Representative color images of fresh-cut cucumbers on day zero and after storage. F: Fresh (day zero), NF: Near-freezing (−2 °C for 12 d), C: Control (5 °C for 12 d).

As for firmness, there was a significant difference between NF and C samples. The pH values of NF and C samples increased significantly compared to F samples, suggesting that respiration of fresh-cut cucumber, which requires carbohydrates and organic acids, might affect the pH change. Overall, it is suggested that near-freezing temperature storage of fresh-cut cucumber at −2 °C for 12 d can maintain the physicochemical properties of fresh-cut cucumber.

Principal component analysis (PCA) was used to objectively interpret and compare the physicochemical properties obtained in this study (Fig. 6). The cumulative contribution was 74.3 % (PC1, 48.6 %; PC2, 25.7 %); thus, the data of PC1 and PC2 were suggested to be sufficient to explain the physicochemical properties. Factor loadings of weight loss, ΔE, and pH to PC1 were 0.726, 0.878, and 0.762, respectively, and that of firmness to PC2 was 0.912. Regarding the PC1 axis, F samples were located in the negative area, C samples were located in the positive area, and NF samples were located close to the neutral area between F samples and C samples. PC1 was positively correlated with weight loss, color change (ΔE), and pH, and PC2 was positively correlated with firmness. As shown in Figure 6, near-freezing temperature storage could suppress changes in physiological properties, and slightly increased firmness during storage compared to storage at 5 °C. PCA clearly showed the results of physicochemical properties obtained in this study.

Fig. 6

The PCA biplot of physicochemical properties of fresh-cut cucumber.

F: Fresh (day zero), NF: Near-freezing (−2 °C for 12 d), C: Control (5 °C for 12 d).

Gas composition Figure 7 shows the changes in O₂ and CO₂ concentrations in the plastic boxes stored at −2 °C (NF) and 5 °C (C) for 12 d. The O₂ and CO₂ concentrations at 12 d were 18.6 ± 0.5 % and 2.1 ± 0.7 % for NF, and 13.1 ± 0.6 % and 6.4 ± 0.2 % for C, respectively. The RQ values of NF and C samples were 1.07 and 0.93, respectively, which suggests that the samples respirated aerobically. In near-freezing temperature storage, O₂ consumption from samples was significantly lower compared to samples stored at 5 °C. Kang et al. (2021) showed that the respiration rate increases with increasing temperature. Thus, a lower storage temperature, even at near-freezing temperature, is likely to be the cause of the difference in O₂ consumption between the NF and C samples. This is the first report to address O₂ consumption and CO₂ accumulation during near-freezing temperature storage of fresh fruits and vegetables.

Fig. 7

Effect of near-freezing temperature storage on changes in gas composition.

(●) O₂ concentration in a plastic box stored at −2 °C (NF); (■) O₂ concentration in a plastic box stored at 5 °C (C); (○) CO₂ concentration in a plastic box stored at −2 °C (NF); (□) CO₂ concentration in a plastic box stored at 5 °C (C). Points and error bars represent the mean ± standard deviation.

Conclusions

In this study, we evaluated changes in microbiological populations and physicochemical properties of fresh-cut cucumbers, and gas composition in the plastic storage boxes during near-freezing temperature storage at −2 °C for 12 d compared to samples stored at 5 °C for 12 d. Observations showed that no samples exhibited any critical freezing phenomena during near-freezing temperature storage. Results indicated that the microbial populations were suppressed slightly in NF samples but increased significantly in C samples compared to stored samples on day zero. The physicochemical properties of NF samples were maintained compared to those of C samples. Results also showed that near-freezing temperature storage was capable of decreasing O₂ consumption from NF samples compared to that from C samples stored at 5 °C.

Acknowledgements This research was supported by JSPS KAKENHI [grant number 21K05843], and Fuji Science and Technology Promotion Foundation [research grant FY 2023].

Conflict of interest There are no conflicts of interest to declare.

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