Effect of nanocomposite alginate‐based film incorporated with cumin essential oil and TiO2 nanoparticles on chemical, microbial, and sensory properties of fresh meat/beef

Abstract The environmental problems of synthetic plastics in food packaging have led researchers to synthesize biodegradable films. In this study, nanocomposite alginate‐based films containing TiO2 nanoparticles (1%) and cumin essential oil (CEO, 2%) were fabricated and the potential of these films to protect beef from chemical [pH, total volatile base nitrogen (TVBN), peroxide value, and thiobarbituric acid reactive substances (TBA)] and microbial [total viable count, Enterobacteriaceae, lactic acid bacteria, Listeria monocytogenes, and Pseudomonas spp.] spoilage was evaluated during 24 days of storage (4°C). The active films significantly induced the reduction in lipid oxidation, microbial growth, and TVBN values, improved the sensory attributes of treated samples, maintained the redness of meats for a longer time, and increased the shelf life of beef from 4 to 16 days. The results of this study showed that TiO2/CEO alginate‐based nanocomposite film has a great potential for application in meat and meat products.


| INTRODUC TI ON
In general, meat-based foods have very low microbial and oxidative stability and are easily exposed to microbial and chemical spoilage during the production and storage chain . Therefore, improving the storage life of fresh meat is one of the important challenges of the meat industry which has attracted the attention of many researchers in recent years (Junior et al., 2020;Sepahvand et al., 2021). In this regard, the use of biopolymeric-based coatings and films is proposed as an important solution due to their barrier, mechanical, optical, and biodegradability characteristics (Bagheri et al., 2019a;Molayi et al., 2018). Sodium alginate is a biopolymer with a high potential for being used as the basic component of films and coatings due to its unique properties such as biodegradability, low cost, availability, ease of use, nontoxicity, stability, and gel formation in the presence of polyvalent cations (Bagheri et al., 2019b;Karimi Khorrami et al., 2021;Nehchiri et al., 2021). The use of biopolymers in the forms of coating and film can restrict oxygen availability and prevent moisture loss, and thereby can increase the shelf life of many products in many researches (Bagheri et al., 2020;Martiny et al., 2020;Radi, Akhavan-Darabi, et al., 2017;Radi, Firouzi, et al., 2017). The effectiveness of such coatings can be remarkably increased by incorporating antimicrobial and antioxidant compounds in their matrix to maintain high concentrations of these substances on the surface of coated products which are more susceptible to bacterial infestation . Different compounds like green and black tea extracts Radi, Firouzi, et al., 2017), Thymus vulgaris EO , polylactic acid (Guo et al., 2014), orange peel EO microemulsion (Radi, Akhavan-Darabi, et al., 2017), (hot) acetic acid (Hosseini-Farahi et al., 2018;Radi et al., 2010), calcium sulfate (Hosseini-Farahi et al., 2016), and salycilic acid Hosseinifarahi et al., 2020), and treatments like UV irradiation (Abdipour et al., 2020) have been incorporated into the biopolymeric-active packagings to enhance the antimicrobial activity of the films. In this regard, the use of essential oils (EOs) has received much attention due to their naturality and high antimicrobial activity (Najjaa et al., 2020;Radi, Akhavan-Darabi, et al., 2017).
Cumin is known for its antimicrobial, nutritional value, antioxidant, and pharmaceutical (antihypertensive, anticancer, etc.) properties, which are mainly associated with its EO (CEO) (Haghiroalsadat et al., 2010). The main components of CEO are cuminol, cumin aldehyde, menthon derivatives, and γ-terpinene, which are responsible for its odor and biological effects. The antimicrobial effects of CEO on foodborne pathogens in different food products have been well demonstrated (Hyldgaard et al., 2012;Sharafati Chaleshtori et al., 2016;Taheri et al., 2018).
Titanium dioxide (TiO 2 ), also known as titanium oxide, has many applications in today's modern world, and is mainly used as a pigment in different industries (Hur et al., 2005). The US Food and Drug Administration has approved the use of TiO 2 as a harmless color additive in food, medicine, and cosmetic products (Alizadeh- Sani et al., 2018). Extensive researches have been performed on the antimicrobial effect of TiO 2 on a wide range of living organs including viruses, bacteria, fungi, algae, and cancer cells (Paspaltsis et al., 2006). The photocatalytic reaction of TiO 2 is one of its known natural effects in reducing fungal contamination. Improving the application properties of biopolymer films by metal nanoparticles of TiO 2 has been considered by researchers in recent years (Alizadeh- Sani et al., 2018;Azizi-Lalabadi et al., 2020;Marcous et al., 2017). In this study, the potential application of alginate-based film containing TiO 2 nanoparticles and CEO as functional ingredients on chemical, microbial, and physical properties of beef was evaluated during cold storage.

| Materials
TiO 2 nanoparticles (anatase) with purity of more than 99% and particle size of 10-25 nm were obtained from US Research Nanomaterials, Inc. Na-alginate was obtained from Behin Azma Co.

| The extraction of CEO
The CEO was extracted by the steam distillation method with the seed/water ratio of 1:5 (w/v) for 4 h by using a Clevenger apparatus.

| Chemical composition of CEO
A capillary gas chromatography (GC-MS; Hewlett Packard 6890) and a mass spectroscopy (Hewlett Packard 6890) were used to analyze the chemical composition of CEO. A capillary column with 0.32 mm diameter and length of 30 mm was used for the analysis.
The analysis conditions were defined as follows: oven temperature (60°C), the column temperature (60°C-for the first 3 min after the injection, which was raised to 220°C with a rate of 6°C/min), the injection temperature (250°C), the flow rate of helium as the carrier gas (1 ml/min), and the MS ionization voltage (70 electronvolt; Amiri et al., 2013).

| Alginate film preparation
To prepare the alginate film, 4 g of Na-alginate was dissolved in 200 ml distilled water and stirred (200 rpm, for half an hour) at 60°C [to prevent lump formation, Na-alginate powder was added gradually to the stirring water, and time was given for the Na-alginate to be dissolved during the addition of powder (Radi & Amiri, 2013)].
Thereafter, 1 g glycerol was added to the film solution as a plasticizer. One percent of TiO 2 nanoparticles (Al + TiO 2 ), 2% CEO (Al + CEO), and a mixture of 1% TiO 2 and 2% CEO (Al + TiO 2 + CEO) were added, separately, as antimicrobial and antioxidant agents to prepare different treatments. Then, an ULTRA-TURRAX ® homogenizer (T18; IKA) was used to homogenize the solution (6000 rpm, 10 min). The prepared solutions were deaerated (at 40°C, 5 min) by a rotary vacuum evaporator (RV-10 control; IKA). Then, 20 g of the solutions was poured into polystyrene Petri dishes (11 cm diameter) and dried in an incubator at 25°C with 40% relative humidity for 24 h (Nehchiri et al., 2021).

| Samples preparation
After purchasing the beef from a local slaughterhouse, the meat was transferred to the laboratory under aseptic conditions (pH = 5.70 ± 0.04), cut into pieces with a thickness of 2 cm and a length of 20 cm, and then wrapped with the preprepared films.
The treatments were defined as follows: Al, Al + TiO 2 , Al + CEO, and Al + TiO 2 + CEO, as well as a control sample which was not wrapped within an Al-based film. The wrapped meat samples were transferred to the polyethylene plastic bags and stored in a refrigerator (4 ± 1°C) for 24 days (Almasi et al., 2021). The sampling was performed at 4-day intervals for microbiological, physical, chemical, and sensory experiments. Five Al-based wraps containing meat samples were placed in a tray to be used as a replicate for each measurement time. Regarding the presence of seven measurement times (throughout the storage time), 35 plastic wraps were considered for the total measurement time of 24 days for one replicate and a total of three replicates were considered for each treatment.

| Microbial analyses
To count the microbial population, 10 g of each sample was asepti-

| The measurement of pH
Ten grams of meat samples was added to 90 ml of deionized water and homogenized (1000 rpm) for 1 min. After 10 min, the pH of the homogenate was measured using a pH meter (CG824, Schott pH meter; AOAC, 2000).

| Chemical properties
The macrodistillation method was used to measure the total volatile base nitrogen (TVBN) content of the meat samples (Kirk & Sawyer, 1991). A colorimetric method was used for the measurement of thiobarbituric acid (TBA; Barbin, 1975

| Sensory evaluation
The sensory evaluation was conducted according to the method de-

| Statistical analysis
Statistical analyses were conducted by running one-way analysis of variance (ANOVA), using MSTAT-C. The comparison of means was performed by Duncan's multiple-test range at p < .05.

| Microbial analyses
The meat samples wrapped with Al films were evaluated for total mesophilic bacteria, Enterobacteriaceae, LAB, pseudomonas spp.,  and Al + TiO 2 + CEO, respectively, while the increase in L. monocytogenes population was from 3.93 to 7.13 log CFU/g in the control sample (p < .05). Results showed that L. monocytogenes was the most sensitive bacteria against the used treatments, as the used films were able to inhibit the growth of L. monocytogenes remarkably.
According to the results, all treated samples, especially those

| pH
The initial pH of beef samples was 5.70 which increased significantly

| Total volatile base nitrogen
The activity of the endogenous enzymes of the meat as well as the bacteria enzymes produces nitrogenous compounds which are measured in the TVBN test. Therefore, the higher values of

| Peroxide value
The PV is usually measured to evaluate the chemical health status of product fat. After the formation of peroxides, the formation of volatile compounds (ketones, aldehydes, and alcohols) is accelerated, resulting in rancidity of fat and production of off-flavor (Khezrian But the PVs increased steadily and reached the highest value until the 12th day (3.94 meq peroxide/1000 g lipid for the control), and then decreased until the end of the 24th day (Figure 4). An increase in the formation of PV caused an increasing trend in the PV curve up to day 12 and the decomposition of peroxide compounds from day 12 onward caused the curve to start a declining trend. This reduction was accompanied by a sudden increase in secondary products.
The PV of the control was significantly higher than those of treated samples during the storage time. Up to the 12th day, the Al samples showed the highest PV after control, followed by Al-TiO 2 or Al-CEO, and Al-TiO 2 -CEO-treated meats, respectively. But from day 16 until the end of the storage time, there was no statistically significant difference among the treated samples. It is noteworthy that the changes in PV of Al-TiO 2 -CEO sample were very small and it increased from 1.00 to 1.23 meq peroxide/1000 g lipid (until the 12th day). However, this increase was from 1.00 to 1.76 meq peroxide/1000 g lipid for Al-TiO 2 and Al-CEO samples.
According to the obtained results, the treatments of this study could effectively control the lipid oxidation of meat during 24 days of storage. The antioxidant activity of sodium alginate compound has been reported before (Sellimi et al., 2015). Sellimi et al. (2015) reported that sodium alginate extracted from Tunisian brown seaweed exerted 74% free radical scavenging activity at 0.5 mg/ml in DPPH radical scavenging activity test and demonstrated significant reducing activity (OD at 700 nm = 2) at 1.2 mg/ml in Ferric-reducing activity test. These researchers showed that sodium alginate displayed a moderate ability to prevent bleaching of β-carotene according to ß-carotene-linoleic acid assay. In addition, sodium alginate, at 4 and 5 mg/ml, exerted potent scavenging activities (80% and 82%, respectively) according to hydroxyl radical scavenging activity test.
Meanwhile, Al may act as an oxygen barrier (Nehchiri et al., 2021) between meat and its surroundings, resulting in the reduction in the lipid oxidation rate. On the other hand, it has been confirmed that CEO can reduce Fe 3 ions effectively and scavenge the superoxide anion. This property comes from its polyphenolic compounds and good antioxidant activity of CEO (El-Ghorab et al., 2010). The strong antioxidant activity of cuminal and γ-terpinene has been reported. Meanwhile, monoterpene alcohols like linalool, terpineol, and pinocarveol have been indicated for CEO antioxidant activity (Abbdellaoui et al., 2019).
The antioxidant activity of TiO 2 nanoparticles is confirmed by researchers (Alizadeh- Sani et al., 2018). Alizadeh- Sani et al. (2020) reported that TiO 2 nanoparticles could turn the purple color of DPPH radical into yellow color of DPPH-H, indicating that TiO 2 has the ability to scavenge the free radical of DPPH, and therefore, can scavenge the produced free radicals during the oxidation reaction and therefore decrease the rate of oxidation. In our study, the synergistic effect of Al, CEO, and TiO 2 in the prevention of lipid oxidation was evident. Similar results were also obtained on chilled meat coated with whey protein nanofibrils containing TiO 2 nanotubes (Feng et al., 2019), fresh chicken packaged with gelatin film containing TiO 2 nanoparticles and CEO , turkey breast meat coated with chitosan film containing 1% CEO (Taheri et al., 2018), and chicken breast fillet on application of silver nanoparticles in polyvinyl chloride films (Azlin-Hasim et al., 2016).

| Thiobarbituric acid
In the TBA test, MDA as an aldehyde compound is measured.
Aldehydes are the secondary products of lipid oxidation, and their increase is an indication of lipid rancidity (Heydari-Majd et al., 2019).
The TBA of all samples increased significantly throughout 24 days of storage ( Figure 5). The initial TBA value of beef was 0.45 mg MDA/ kg, which was low and showed the freshness of the initial meat. The wrapped with citric acid, cornstarch, and linear LDPE active films (Júnior et al., 2015). Table 2 shows the color coordinates (L*, a*, and b*) of beef samples.

| Color analysis
According to Table 2, lightness (L*) decreased throughout the storage time which is related to the oxidation phenomenon that turns the color of meat from red to dark brown. Therefore, the increase in

| Sensory properties
All sensory attributes of the meat samples (color, odor, and overall acceptability) declined throughout the storage time (Table 2). This reduction trend was much faster in the control sample than in the treated samples during the storage time, and hence, the obtained score for color, odor, and overall acceptance was zero for the control sample on day 20.
Regarding the color parameter, at the beginning of storage, there was no significant difference among the treated samples.
However, by moving toward the end of the storage time, the Al sample obtained lower scores than the other treated samples and the Al + TiO 2 + CEO sample received the highest score. Regarding the odor parameter, it seems that the treated samples without CEO obtained a higher score than the samples containing CEO. This can be due to the effect of the CEO smell. It is noteworthy that although the odor parameter score was significantly lower in the CEO-containing samples over time, this difference was small. Another important point was that by moving toward the end of the storage time, this difference disappeared, which could be due to the decrease in CEO odor over time, and finally, the Al + TiO 2 + CEO samples obtained the highest score. In terms of overall acceptance, it was observed that the score obtained by the treated samples throughout the storage time was significantly higher than that obtained by the control sample. Meanwhile, the scores obtained at the beginning of storage were not much different for all treatments, but toward the end of the storage time, the Al + TiO 2 + CEO sample obtained the highest scores. The antimicrobial effect of Al, CEO, TiO 2 , and Al + TiO 2 + CEO induced the enhancement of sensory attributes in the treated meats compared to the control. Our results are consistent with those of Alizadeh- Sani et al. (2017) and Azizi-Lalabadi et al. (2020). In these studies, it was demonstrated that the application of TiO 2 nanoparticles effectively improved the sensory attributes of white shrimp and lamb, respectively. Sharafati Chaleshtori   Taheri et al. (2018) declared that the application of chitosan containing CEO resulted in good overall acceptance in chicken meat and turkey breast meat, respectively. Similar results were obtained by other researchers (Chen et al., 2016;Diao et al., 2020;Sayadi, Amiri, et al., 2021).

| The estimation of shelf life for beef samples
The shelf life of beef samples was estimated according to the results of microbial and chemical tests (Table 3). In terms of TVC, TBA, PV, a*, and TVBN results, all treated samples were acceptable until day 24, while this number was 8 for the control. However, the pH and sensory evaluation tests limited the shelf life of control to 4 days.
Although the treated samples were sensually acceptable up to day 16, the pH test reduced the shelf life of the Al sample to 8 days and Al + CEO and Al + TiO 2 samples to 12 days. However, the pH of Al + TiO 2 + CEO sample did not exceed the upper limit until the 24th day. Overall, the shelf life of control, Al, and composite films was around 4, 8, and 16 days, respectively.

| CON CLUS ION
In this study, nanocomposite alginate-based films containing TiO 2 nanoparticles and CEO (as antioxidant and antimicrobial agents), alone or in combination, were fabricated. The produced active packaging films could remarkably reduce the lipid oxidation and microbial spoilage, improve the color quality and sensory attributes, and increase the shelf life of fresh beef. In this regard, the combination use of TiO 2 and CEO resulted in better results compared to the sole use of TiO 2 and CEO. The results of this study showed that the use of Al + TiO 2 + CEO as a novel nanocomposite film is greatly beneficial in preserving the quality parameters of fresh beef.

ACK N OWLED G M ENT
The research was financially supported by the Fasa University of Medical Sciences.

E TH I C A L A PPROVA L
This article does not contain any studies with human participants or animals performed by any of the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be made available upon reasonable request. a According to Iran National Standards Organization, the upper limit value for TVC in fresh beef is 5 × 10 5 log CFU/g, 5 meq/kg fat is the upper limit value for peroxide value, 25 mg N/100 g is the upper limit value for TVBN, 5.8 is the upper limit value for pH, and no upper limit value is defined for TBA, but according to literature [Alizadeh Sani et al. (2020)], 2 mg MDA/1000 g is the upper limit value for TBA. b According to the color analysis results.

Mehran
c According to the sensory evaluation results and overall acceptance score more than 4.