3.1. MIC and MBC values of FEO
The MIC and MBC values of FEO are provided in Table 1. The results depicted that in concentrations between 8 and 20 mg/mL, FEO was active against L. monocytogenes, Escherichia coli O157:H7, P. fluorescens, and S. putrefaciens, respectively. Our results lent support to those of Roby et al. (2013) in evaluating the antimicrobial properties of FEO[20]. Our study also corroborated the study of Ojeda-Sana AM, et al (2013) on rosemary essential oil in that L. monocytogenes was the most sensitive bacterium to essential oils [21].
Table 1
The MIC and MBC values of FEO
MBC (mg/ml)
|
MIC (mg/ml)
|
Bacteria
|
10.00
|
8.00
|
Listeria monocytogenes
|
14.00
|
10.00
|
Escherichia coli O157:H7
|
20.00
|
16.00
|
Shewanella putrefaciens
|
16.00
|
14.00
|
Pseudomonas fluorescens
|
3.2. Antimicrobial activity of films determined by disc diffusion method
The antimicrobial activities of films were determined by disk diffusion method. Growth inhibition as the zone of inhibition diameter is given in Table 2. The CMC/PG film showed no antimicrobial activity against the tested bacteria. Moreover, based on the results, CMC/PG/FEO/TiO2 films had the greatest effect on the studied bacteria. As Table 2 shows, CMC/PG/FEO/TiO2 and CMC/PG/FEO treatments had the highest and the lowest antimicrobial effects, especially on L. monocytogenes and S. putrefaciens, respectively. Sani et al. (2017) obtained similar results for TiO2 and rosemary essential oils[18]. Our results also showed that the effect of FEO on Gram positive bacteria was greater than that on Gram negative bacteria; this greater effect is ascribed to the structure of the outer membrane of Gram (-) bacteria, which limits the diffusion of hydrophobic components of FEO to the lipopolysaccharide layer [22]. In general, the antimicrobial effects of TiO2 and FEO are associated with microbial cell components, destroying the cytoplasmic membrane, affecting cell wall permeability, and affecting biological molecules such as DNA and protein [18]. The antimicrobial effect of FEO is also related to the phenolic compounds called terpenoids [23].
Table 2
Growth inhibition zone of film disks (mm)
CMC/PG/F/TiO2
|
CMC/PG/TiO2
|
CMC/PG/F
|
CMC/PG
|
Bacteria
|
19.00±0.67
|
16.00±0.23
|
15.00±0.34
|
-
|
Listeria monocytogenes
|
16.00±0.34
|
15.00±0.16
|
14.00±0.12
|
-
|
Escherichia coli O157:H7
|
10.40±0.17
|
9.50±0.07
|
9.00±0.09
|
-
|
Shewanella putrefaciens
|
14.00±0.25
|
13.00±0.11
|
12.50±0.13
|
-
|
Pseudomonas fluorescens
|
3.3. Microbial tests
3.3.1. Total viable count
Total viable count (TVC) indicator for rainbow trout fillets kept in refrigerator during storage is shown in Fig. 1. The initial TVC was 3.01 log CFU/g, which according to the standards, this level of microbial load in fish fillets indicates the good quality of fish fillets; this result substantiated those of Ojagh SM, et al (2010) [24] and Arashisar Ş, et al (2004) [25]. Control and CMC/PG treatments had very high microbial loads due to the lack of antimicrobial agents during refrigeration, and the lower microbial load of CMC/PG films compared to the control samples was probably due to the mere film, preventing secondary contamination, reducing available oxygen, and reducing the water available to microorganisms. Compared to the control samples, CMC/PG/FEO, CMC/PG/TiO2, and CMC/PG/FEO/TiO2 treatments exerted a significant effect (P <0.05) in reducing the growth rate of bacteria. According to the International Commission on Microbiological Specifications for Foods (ICMSF), the standard log level of microbial load in fish fillets is 7 log CFU/g. Accordingly, CMC/PG/FEO treatment can be stored for 6 days, CMC/PG/TiO2 treatment for 9 days, and CMC/PG/FEO/TiO2 treatment for 12 days in the refrigerator. The lowest bacterial growth was observed in CMC/PG/FEO/TiO2 treatment, which is due to the synergistic effect of these two compounds (FEO and TiO2). This result lends support to that of Farshchi E, et al (2019) for the antimicrobial effect of TiO2-Ag [14], and Maghami M, et al (2019) for FEO [26].
3.3.2. Pseudomonas spp. count
Pseudomonas spp. are Gram (-), highly aerobic, and CO2-sensitive bacteria [27]. As shown in Fig. 2, compared to the control group, the significant effect of treatments with antimicrobial compounds was observed on the growth rate of Pseudomonas spp. (P <0.05). The initial Pseudomonas spp. value for all treatments was 3.02 log CFU/g on the first day and 7.87 log CFU/g on the 12th day for the control samples. CMC/PG treatment was not a good barrier to the growth of these microorganisms due to the lack of antimicrobial compounds, but treatments containing FEO and TiO2 significantly reduced the growth rate of these microorganisms (P <0.05); this result substantiates the results of Sani MA, et al (2017) [18] For TiO2, Maghami M, et al (2019) [26] and Kivanç M, Akgül A (1986) [28] for FEO. A decrease in the antimicrobial effects of CMC/PG/FEO, CMC/PG/TiO2, and CMC/PG/FEO/TiO2 treatments in the last days was attributed to the decrease in the release of these substances.
3.3.3. Lactic Acid Bacteria
LAB are optional anaerobic bacteria that grow in low concentrations of O2. They make up a significant portion of the meat's natural microflora. As Fig. 3 displays, the LAB content for the treatments on the first day was 2.0 log CFU/g, which increased during the maintenance period and for the control sample reached 5.67 log CFU/g. CMC/PG/FEO, CMC/PG/TiO2, and CMC/PG/FEO/TiO2 treatments significantly reduced the growth rate of LAB compared to the control samples (P<0.05), Maghami M, et al (2019) for FEO [26] and Sani MA, et al (2017) for TiO2 [18] achieved the same results on LAB. On the last day, the lowest level of LAB was related to CMC/PG/FEO/TiO2 treatment, which was due to the synergistic effect of two antimicrobial agents.
3.3.4. Enterobacteriaceae count
Enterobacteriaceae microorganisms are health indicators and part of the natural microbial flora of rainbow trout [29]. Based on the results, the initial level of Enterobacteriaceae was 2.12 log CFU/g, which indicates the good quality of the meat (Fig. 4) [18]. During the storage period, the number of Enterobacteriaceae increased and reached 6.21 log CFU/g on day 15 for the control sample. On the 15th day, the number of Enterobacteriaceae for CMC/PG/FEO/TiO2 treatment was 3.99 log CFU/g which showed the favorable effect of antimicrobial agents. FEO and TiO2 reduced the growth of Enterobacteriaceae compared to the control sample (P <0.05); this shares a number of similarities with the results of Österblad M, et al (1999) [30], Kačániová M, et al (2019) [31] and Sani MA, et al (2017) [18]. Furthermore, the synergistic effect of FEO and TiO2 was quite evident in reducing the growth of Enterobacteriaceae.
3.3.5. Psychrotrophic bacteria
Changes in psychrotrophic bacteria for treatments during refrigeration are shown in Fig. 5. The initial number of psychrotrophic bacteria at the beginning of the period was 3.42 log CFU/g. Over time, the number of these bacteria increased in all treatments, and in the control samples reached 8.77 log CFU/g in the end of the period, which was very high. The difference between the control samples and CMC/PG/FEO/TiO2 treatment in the end of the period was about 2 log CFU/g, which indicates the favorable antimicrobial effect of TiO2 and FEO (P<0.05). These results concurred well with the results of Mazandrani HA, Javadian S, Bahram S (2016) [32] and Bagheri R, et al (2016) [33] for the antimicrobial effect of FEO on silver carp and common kilka, respectively, and the results of Alizadeh-Sani M, et al (2020) [34] for TiO2.
3.4. pH
The pH changes of fish fillets during the storage period are shown in Fig. 6. The initial pH of the fish fillet was 6.33, which was consistent with the pH levels in the studies of Arashisar Ş, et al (2004) [25] and Gimenez B, et al (2002) [35] for rainbow trout. The pH level decreased until the third day due to the glycolysis process of fish [36]. During this period, the pH increased and bacterial metabolites, and microbial enzymes produced in the fillet tissue [37]. By increasing the pH, the quality characteristics of the fish fillets decreased. Treatments with antimicrobial agents significantly kept the pH at lower levels than that in the control samples; this was consistent with the results of the microbial part.
3.5. Thiobarbituric acid (TBA)
TBA changes of different treatments of fish fillets during refrigeration are shown in Fig. 7. The initial TBA level of fish fillets was determined to be 0.13 mgMDA/Kg, which was in the same level of that in the study of Ojaq et al. (2010) for rainbow trout. In all treatments, the amount of TBA increased over time (P<0.05). Perumalla AVS, Hettiarachchy NS (2011) [38] showed that essential oil reduces the amount of TBA in samples by various mechanisms such as preventing the onset of radical formation, and reducing the transfer of metal ion catalysts, as well as peroxide decomposition and reaction with free radicals [39]. TBA level is about 1-2 mgMDA/Kg in fish fillets, which is the starting point for an unpleasant odor. However, in general, less than 5 mgMDA/Kg is desirable for consumption [40]. The effect of TiO2 on the reduction of TBA was much less than that of FEO, because TiO2 has antimicrobial properties and FEO has high antioxidant properties in general, and as a result, these further reduce the TBA level. The film around the fish fillets also reduced the TBA level by reducing the amount of oxygen available. The trend of change is consistent with the results of Ojagh SM, et al (2010) [24] obtained for rainbow trout.
3.6. Sensory evaluation
As depicted in Fig. 8, the quality of sensory indicators decreased over time (P<0.05). Odor index under the influence of TiO2 and FEO treatments scored higher than the control sample because odor is one of the most sensitive indicators in fish fillets that is strongly influenced by storage conditions, microbial load, and chemical reactions. The color index was not significantly affected during the storage and all samples had a relatively high score. Microbial activity and chemical reactions that occur during fillet storage have less effect on the color index. The tissue of fish fillets was severely affected by the storage time and treatments, and in the control samples, due to the bacterial growth and chemical reactions, the surface of the fish fillets became viscous and soft, and had an undesirable texture. TiO2 and FEO treatments were significantly lower (P <0.05) due to the reduction of microbial growth and chemical reactions. The overall acceptance index decreased during the storage; and considering the acceptance limit of 5, the samples treated with TiO2 and FEO could be easily used up to day 6. This result is completely consistent with the results obtained in the microbial stage.