Carboxymethyl Cellulose Film Optimized with Persian Gum, Titanium Dioxide Nanoparticles, and Fennel Essential Oil: Investigation of Chemical, Antimicrobial, and Sensory Properties on Rainbow Trout Fillet

Today, the use of biodegradable packaging containing antimicrobial agents has been approved by the people and industry. In this study, the effect of carboxymethyl cellulose (CMC) lm optimized with Persian gum (PG) containing titanium dioxide nanoparticles (TiO 2 ) and fennel essential oil (FEO) was inspected and the sensory, chemical, and microbial properties were evaluated on refrigerated rainbow trout llets during the storage period (0, 3, 6, 9, and 12 days). The lowest values for the minimum inhibitory concentration (MIC) (8 mg/mL) and the minimum bactericidal concentration (MBC) (10 mg/mL), and the highest diameter of the inhibitory zone in the disk diffusion (19 mm) for FEO were observed against Listeria monocytogenes, Escherichia coli.O157:H7, Pseudomonas uorescens, and Shewanella putrefaciens, respectively. Moreover, as a microbiological analysis, total viable count (TVC), Pseudomonas spp, lactic acid bacteria (LAB), Enterobacteriaceae, and psychrotrophic bacteria were determined on rainbow trout llets covered with different treatments (CMC/PG, CMC/PG/FEO, CMC/PG/TiO 2 , and CMC/PG/FEO/TiO 2 ) during the storage at 4°C. The results showed that three treatments of CMC/PG/FEO, CMC/PG/TiO 2 , and CMC/PG/FEO/TiO 2 had a signicant effect on growth control in the studied bacteria (P <0.05). In all treatments, the amount of thiobarbituric acid increased over time, but in the treatment containing FEO, this amount was lower. Sensory scores of the samples were very high until day 6. In general, the results of this study showed that the use of FEO and TiO 2 in biodegradable lms improves the antimicrobial, chemical, and sensory properties of rainbow trout llets. psychrotrophic bacteria were quantied. The results showed that lms containing antimicrobial of FEO and TiO 2 signicantly reduced the bacterial growth during the refrigeration. When FEO and TiO 2 were used simultaneously in the lm, the growth of microorganisms was severely reduced due to their synergistic properties. Samples treated up to day 6 received acceptable scores in terms of sensory evaluation. Based on the results, in general, the use of FEO and TiO 2 in biodegradable lms improves the antimicrobial, chemical, and sensory properties of rainbow trout llets.


Introduction
Foods are always exposed to chemical, microbial, and physical spoilage and food preservation plays an essential role in controlling changes during the storage of different products such as fresh vegetables, fruits, and meats [1]. This would lead to the increased shelf life through inhibiting the oxidative changes and growth of microorganisms [2] [3].
Films are thin layers around food that prevent the transfer of moisture and oxygen, as well as solutes to the food. In this regard, active packaging is designed to preserve the packaged material in the best possible manner according to the storage conditions of the package so that the food reaches the consumer in appropriate conditions. In general, biodegradable lms are made from three main components: protein, lipid, or polysaccharide; these materials can be used alone or in combination with each other. The physical and chemical properties of these materials remarkably in uence the properties of the lms [4].
Carboxymethyl cellulose (CMC) is known as a water-soluble cellulose derivative owing good lm-forming properties [5]. It is capable of forming colorless, water soluble, non-toxic, stable, and uniform lm-forming solution albeit with poor mechanical properties [6]. Persian gum (PG) is obtained from the wild almond tree (Amygdalu scoparia Spach) and is available in white to dark colors, in different shapes and sizes. This gum dissolves easily in hot water [7][8].
Films used in the food industry may contain a variety of substances, including antioxidants, antimicrobials, and avorings. This type of packaging prevents the activity of pathogenic and spoilage microorganisms during the storage and increases the shelf life [9].
Biodegradable lms are supplemented with essential oils to improve the antimicrobial-antioxidant effects, the aroma of the lm, and the permeability properties of hydrophilic lms [10]. Fennel (Foeniculum vulgare Mill) is a biennial or perennial herbaceous plant of the Umbelliferae family, and fennel essential oil (FEO) has antiseptic, antifungal, anti-atulence, and appetizing properties [11].
Silver, zinc oxide, copper, and titanium dioxide nanoparticles (TiO 2 ) are the most important nanoparticles used in the production of nanocomposites, with many applications, and antimicrobial and optical properties [12]. TiO 2 , used in the structure of nanocomposites, prevents the growth of pathogenic microorganisms in addition to improving their properties [13]. It is widely used in the lm in the range of 0.5 to 1 w/w, based on dry matter weight [14].
CMC lm with PG, TiO 2 , and FEO has desirable physical and mechanical properties [15]. Packaging the refrigerated rainbow trout llet with the biodegradable lm of CMC/PG, containing TiO 2 and FEO, and subsequently, the study of microbial, chemical, and sensory properties of the lm on rainbow trout llet at refrigeration temperatures were the objectives of this study.

Preparation of biodegradable lm
To prepare the treatments (CMC/PG, CMC/PG/FEO, CMC/PG/TiO 2 , and CMC/PG/FEO/TiO 2 ), 1.25 g of CMC was added to 100 mL of water at 60°C and mixed with a mixer for 15 min. Then PG solution (0.65% w/w), glycerol (40% w/w), FEO (2% w/v), and TiO 2 (0.5% w/w) were added to the lm solution and mixed with a stirrer for 15 min. To remove the air bubbles, an ultrasound bath (Schaper Unique USC 25 KHz) with a frequency of 25 KHz and a eld strength of 100 W was used for 15 min. Then it was dried at 37°C for 24 h in a hot air dryer [15].

Determination of MIC and MBC of FEO
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were measured in vitro on pathogenic bacteria including Listeria monocytogenes ATCC 7644 and Escherichia coli O157:H7 NCTC 12900, and the causative agents including Shewanella putrefaciens NCTC 10762 and Pseudomonas uorescens NCTC 10038. Bacterial strains were cultured for 24 h at 37°C and 22°C in Müller-Hinton broth, respectively. Speci c concentrations of FEO (in DMSO) were prepared and 20 µL of it was added to 96-well microplates containing 20 µL bacterial suspension (0.5 McFarland) and 160 µL sterile broth. Similar tests were performed for both positive control (culture medium containing bacteria without antimicrobial agent) and negative control (culture medium without bacteria). The microplates were kept in an incubator at 37°C and 22°C (Pseudomonas spp.) for 24 h. The lowest concentration of FEO in which no turbidity was observed was reported as the MIC. Ten microliters were taken from all wells and cultured on a plate containing brain heart infusion (BHI) agar. The plates were stored in the incubator at 37°C for 24 h. The lowest concentration of FEO, which was associated with the absence of bacterial colony, was considered as MBC. All these tests were performed in triplicate [16].

Antimicrobial activity of lms by disc diffusion method
Antimicrobial activity of the lm was analyzed on S. putrefaciens, P. uorescens, E. coli O157: H7, and L. monocytogenes. To this end, the lms were cut into discs (1 cm in diameter) and placed on Müller Hinton agar plates. First, 0.1 mL of the 24-hour bacterial suspension containing the desired bacteria (10 6 -10 5 CFU/mL) was cultured freshly. The plates were inoculated at 37°C for 24 h. The diameter of the growth inhibition halo was measured by an accurate caliper and its area was reported in terms of square millimeters [17].

Preparation of rainbow trout samples and treatments
Rainbow trout were obtained from reputable shopping centers in Mashhad and transferred to the laboratory in cold conditions. Cutting, abdominal emptying, and washing operations were performed. The sh meat was divided into the pieces of about 50 g with an average thickness of 2 cm. The sh pieces were covered with lms. The coated samples and controls were placed in sterile plastics and stored at refrigeration temperature (4 to 5°C). All samples were evaluated at intervals of 0, 3, 6, 9, 12, and 15 days.

Microbial analysis
Ten grams of sh samples were transferred to 90 mL of peptone water 0.1% and mixed with a mixer (Lab Blender 400, Stomacher, USA) for 1 min at room temperature. Peptone water 0.1% was used to prepare serial dilutions and count the desired bacteria by placing them in a speci c culture medium and the speci c temperature of each bacterium [16].
For total viable count (TVC), nutrient agar medium was used by surface culture at 37°C for 48 h. Pseudomonas spp. was determined on cetrimide fusidin cephaloridine agar (supplemented with selective supplements) at 22°C for 48 h. Lactic acid bacteria (LAB) were cultured in deMan Rogosa Sharpe (MRS) anaerobic medium at 25°C for 5 days under anaerobic conditions. For Enterobacteriaceae count, 0.1 mg of sample was inoculated in 10 mL of Violet Red Bile Glucose agar (VRBGA) culture medium. Then 10 mL of molten culture medium was poured on them and VRBGA medium was kept in a 30°C oven for 24 h. To determine and count the psychrotrophic bacteria, 0.1 mL of the desired dilution was cultured completely and super cially on Tryptic Soy Agar (TSA). Samples were kept at 7°C for 10 days and then the colonies were counted [14][18] 2.6. pH Ten grams of each sample were homogenized in 100 mL of distilled water and then ltered. A digital pH meter was employed to measure the pH of the solution (Hanna pH meter, HI 221, Romania).  [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]. The antimicrobial activities of lms were determined by disk diffusion method. Growth inhibition as the zone of inhibition diameter is given in Table 2. The CMC/PG lm showed no antimicrobial activity against the tested bacteria. Moreover, based on the results, CMC/PG/FEO/TiO 2 lms had the greatest effect on the studied bacteria. As Table 2  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 TiO 2 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].  [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 lms compared to the control samples was probably due to the mere lm, preventing secondary contamination, reducing available oxygen, and reducing the water available to microorganisms.

Pseudomonas spp. count
Pseudomonas spp. are Gram (-), highly aerobic, and CO 2 -sensitive bacteria [27]. As shown in Fig. 2, compared to the control group, the signi cant 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 rst 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 TiO 2 signi cantly reduced the growth rate of these microorganisms (P <0.

Enterobacteriaceae count
Enterobacteriaceae microorganisms are health indicators and part of the natural microbial ora 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)

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/TiO 2 treatment in the end of the period was about 2 log CFU/g, which indicates the favorable antimicrobial effect of TiO 2 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 TiO 2 .

pH
The pH changes of sh llets during the storage period are shown in Fig. 6. The initial pH of the sh llet 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 sh [36]. During this period, the pH increased and bacterial metabolites, and microbial enzymes produced in the llet tissue [37]. By increasing the pH, the quality characteristics of the sh llets decreased. Treatments with antimicrobial agents signi cantly kept the pH at lower levels than that in the control samples; this was consistent with the results of the microbial part.

Thiobarbituric acid (TBA)
TBA changes of different treatments of sh llets during refrigeration are shown in Fig. 7. The initial TBA level of sh llets 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 sh llets, 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 TiO 2 on the reduction of TBA was much less than that of FEO, because TiO 2 has antimicrobial properties and FEO has high antioxidant properties in general, and as a result, these further reduce the TBA level. The lm around the sh llets 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.

Sensory evaluation
As depicted in Fig. 8, the quality of sensory indicators decreased over time (P<0.05). Odor index under the in uence of TiO 2 and FEO treatments scored higher than the control sample because odor is one of the most sensitive indicators in sh llets that is strongly in uenced by storage conditions, microbial load, and chemical reactions. The color index was not signi cantly affected during the storage and all samples had a relatively high score. Microbial activity and chemical reactions that occur during llet storage have less effect on the color index. The tissue of sh llets 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 sh llets became viscous and soft, and had an undesirable texture. TiO 2 and FEO treatments were signi cantly 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 TiO 2 and FEO could be easily used up to day 6. This result is completely consistent with the results obtained in the microbial stage.

Conclusion
To determine the microbial properties of the lms, disk diffusion test was performed, which showed that the lms with TiO 2 and FEO had a signi cant antimicrobial effect. To determine the antimicrobial effect of the lms on food models, 4 lm treatments were prepared and rainbow trout were covered with the produced lms. During the refrigeration, the TVC, LAB, Enterobacteriaceae, Pseudomonas spp., and psychrotrophic bacteria were quanti ed. The results showed that lms containing antimicrobial compounds of FEO and TiO 2 signi cantly reduced the bacterial growth during the refrigeration. When FEO and TiO 2 were used simultaneously in the lm, the growth of microorganisms was severely reduced due to their synergistic properties. Samples treated up to day 6 received acceptable scores in terms of sensory evaluation. Based on the results, in general, the use of FEO and TiO 2 in biodegradable lms improves the antimicrobial, chemical, and sensory properties of rainbow trout llets.

Declarations
Con ict of interest: Changes in Pseudomonas spp. Count of different treatments during storage at 4°C for 12 days Changes in pH of different treatments during storage at 4°C for 12 days Figure 7 Changes in TBA of different treatments during storage at 4°C for 12 days