Physicochemical, microbial, and functional attributes of processed Cheddar cheese fortified with olive oil–whey protein isolate emulsion

Abstract Olive (Olea europaea L.) has triacylglycerols, phenolics, and other antioxidants in its composition playing significant roles in maintaining health and reducing the onset of diseases. This study aimed to analyze the quality, antioxidant, textural profile, and sensory properties of processed Cheddar cheese fortified with 0%, 5%, 10%, 15%, and 20% (v/w) olive oil–whey protein isolate emulsion during 60 days of storage period. The results showed that processed cheese had significantly higher (p < .05) antioxidant activity, and total phenolic and flavonoids contents, whereas nonsignificant increase (p > .05) in moisture and acidity while decreasing tendencies in pH, fat, protein, and ash contents. Sensory analysis showed that processed Cheddar cheese with 5% emulsion had higher taste, aroma, texture/appearance, overall acceptability scores, and hardness. Conclusively, results indicated that olive oil–whey protein isolate emulsion could be beneficial for manufacturing and commercializing processed cheeses, analogs, or spreads with improved nutritional value and sensory characteristics.


| INTRODUC TI ON
Processed cheese is a homogenous blend of cheese manufactured from several ingredients, such as the same or different natural cheese varieties, vegetable oils, butter oil, milk solids, emulsifying salts, and other dairy or nondairy ingredients with extended shelf life (Gulzar et al., 2020). Processed cheese has low functionality because of its low nutritional profile, which can be enhanced by incorporating valuable ingredients with high content of bioactive and functional components (Shaukat et al., 2022). Several studies have been conducted to evaluate the influence of different functional materials, such as vegetable powders (El-Loly et al., 2022), fruits (Abbas et al., 2021), grain flour, and cheese fat substitution through the addition of vegetable oils (Hamdy et al., 2021) on processed cheese.
Moreover, olive oil provides phenolic compounds that are wellknown for their health-beneficial biological properties. In particular, olive oil's phenolic compounds possess anti-inflammatory, antioxidant, and antimicrobial activities (Melguizo-Rodríguez et al., 2021).
Currently, the food industry has a great demand for emulsions (Ranjha et al., 2021). The emulsion is thermodynamically unfavorable (breakdown over time) due to physiochemical mechanisms and can be stabilized using emulsifiers as stabilizers specifically (Weiss et al., 2020). Proteins exhibit amphiphilic character, therefore widely used as emulsifying/stabilizing ingredient in food emulsions. Proteins have the ability to create electrostatic and steric repulsive forces by forming an interfacial layer between oil droplets. During long-term storage, these forces stabilize the droplets as opposed to coalescence and flocculation (Ding et al., 2021;Marhamati et al., 2021).
Whey protein isolates (WPI), an emulsifier, enhance the formation and stability of oil-in-water emulsions (Hwang et al., 2017). These also have the capacity to prevent prooxidants to access the droplets, and consequently inhibit lipid oxidation (Nooshkam & Varidi, 2021).
So far, the characteristics and importance of processed cheese fortified with olive oil-whey protein isolate emulsion have not been studied. Therefore, the proposed study was designed to prepare and optimize the emulsion from olive oil with whey protein, improve the functional properties, and assess the physicochemical composition, antioxidant potential, microbiological characteristics, texture, and sensory acceptability of processed cheese fortified with olive oilwhey protein isolate emulsion.

| Materials
Natural Cheddar cheese (3 months ripened) was procured from Noon Pakistan Ltd. Bhalwal, Sargodha, Pakistan. Extra virgin olive oil was purchased from the Pansari Premium Herbal Store, Lahore, Pakistan.

| Preparation of olive oil emulsion (OOE)
Extra virgin olive oil and whey protein isolate (WPI) (oil-in-water) emulsion was prepared by following the procedure described by Kuhn and Cunha (2012) with some modifications. To prepare WPI stock solution, WPI was dissolved into deionized water at room temperature for 90 min with magnetic stirring and was subjected to overnight storage to achieve complete dissolution at room temperature (25 ± 2°C). The pH of WPI stock solution was adjusted to neutral by using 2.0 M buffer solution (NaOH) and stored at 10°C for a night.
To prepare emulsions, WPI stock solution and olive oil were used in 0.5%:5% (v/v) ratio, respectively. For the preparation of extra virgin olive oil and whey protein isolate emulsion, required amount of olive oil was poured drop-wise into the required amount of encapsulating WPI solution. The mixture was homogenized at 14000 rpm for 4 min. Sodium azide (0.02% w/v) was mixed into the emulsion. The pH of the emulsions was adjusted to neutral by using 2.0 M buffer solution (NaOH).

| Preparation of processed Cheddar cheese
Processed Cheddar cheese was prepared using ripened natural Cheddar cheese, olive oil emulsion (OOE) at 0% (control), 5%, 10%, 15%, and 20% concentrations, and 2% emulsifying salt by following the process described by Kapoor and Metzger (2008), with some modifications. Shredding of the Cheddar cheese was carried out to prepare processed Cheddar cheese. All the ingredients mentioned above were cumulatively poured in a steam-jacked cooker followed by mixing aided with thermal treatment at a temperature range = 65 to 75°C for a time interval of 15 min. From the cooker, hot-processed Cheddar cheese was taken out of the molds made with stainless steel having a depth of 10.16 cm. After that, processed cheese samples were cooled to bring their temperature to normal room temperature followed by slicing and packaging. Small rectangular shape blocks were made of processed cheese by slicing and were subjected to packaging under vacuum in polythene bags. Samples were then transferred to the storage facility and processed Cheddar cheese was stored at 2°C for 60 days.

| Physicochemical analyses
AOAC (2016) methods were employed for analyzing moisture (AOAC Method No. 948.12), protein (AOAC Method No. 2011.04), ash (AOAC Method No. 942.05), and acidity (AOAC Method No. 942.15) (%lactic acid) contents of processed Cheddar cheese. The fat content of processed Cheddar cheese was evaluated by following the Gerber method with some modifications as given by Marshall (1993). The Gerber method is a volumetric method that employs chemical reagents, such as sulfuric acid to carry out the breakdown of emulsion and fat separation. A special flask was utilized for the measurement of fat content known as butyrometer. Briefly, 10 ml of the pipette was used for transferring sulfuric acid (10 ml) to the butyrometer.
The samples were placed in the butyrometer and 1 ml of the amyl alcohol was added using the 1 ml pipette. Then, the butyrometer was placed in the water bath for a time interval of 5 min. After centrifugation, the butyrometers stoppers were oriented downward in the water bath for 3 to 10 min. The difference in the readings was denoted as fat mass in terms of percentage in cheese samples.
The pH of processed Cheddar cheese was evaluated for pH by following the method described by Ong et al. (2007) using an Electronic Digital pH Meter. All analyses were carried out thrice (n = 3) at the interval of 0, 30, and 60 days during storage.

| Preparation of water-soluble extracts (WSEs)
WSEs of processed Cheddar cheese were prepared by following the method developed by Gupta et al. (2013) with some modifications.
Briefly, WSEs were prepared by first mixing 15 g of grated cheese in water (50 ml) followed by mixture placement in the water bath and thermal treatment at a temperature constraint of 40°C for a time interval of 5 min. Then, the mixture was homogenized using Omni-Mixer homogenizer (Omni International, Waterburg, CT) for a period of 2 min. Furthermore, HCl (2 M) was employed for pH adjustment at 4.6 and then the mixture was added with distilled water until reaching the sample weight (grated cheese sample mixtures) equivalent to 100 g. Then, the placement of the samples in water bath was again carried out at 40°C temperature for 1 h in order to allow the melting fat of cheese samples followed by centrifugation of samples at 4500 rpm (3000 × g) at 40°C temperature for 1 h. After centrifugation, Whatman filter paper No. 1 was used for filtration. WSEs of processed Cheddar cheese were collected in a round-bottom flask and then subjected to freeze-drying. Powdered freeze-dried samples were weighed, transferred to plastic tubes, and subjected to storage at −20°C.

| Determination of total antioxidant activity
For assessing the ascorbic acid equivalent (AAE) antioxidant capacity, the WSEs were evaluated by following the method described by Prieto et al. (1999) with some modifications at the interval of 0, 30, and 60 days during storage. Briefly, 1 ml of WSEs of processed Cheddar cheese was mixed with 4 ml of phosphomolybdate reagent [28 mM/L NaOH +0.6 M/L H 2 SO 4 + 4 mM/L (NH 4 ) 2 MoO 4 ]. The mixture was vortexed for 30 s. Then, it was incubated in the water bath at 95°C for 90 min. The incubated mixture was cooled to room temperature and again vortexed for 30 s. The absorbance of samples was measured at 695 nm through a spectrophotometer (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA). The ascorbic acidequivalent antioxidant capacity of processed Cheddar cheese was recalculated using the ascorbic acid standard curve as mg/100 ml AAE. All the replications and experiments were carried out thrice (n = 3).

| Determination of total phenolic content
For assessing the total phenolic content (TPC) in terms of gallic acid equivalent (GAE), the WSEs of processed Cheddar cheese were evaluated by following the method of Reis et al. (2012) with some modifications at the interval of 0, 30 and 60 days during storage. Briefly, 1 ml of WSEs of processed Cheddar cheese was mixed with 1 ml of 10% (v/v) Folin-Ciocalteu reagent, vortexed for 30 s and left for 10 min at room temperature. Then, 2 ml of 20% (w/v) sodium carbonate (Na 2 CO 3 ) was added and again vortexed for 30 s.
The mixture was incubated in dark at 30°C for 60 min. After incubation, the absorbance of samples was measured at 760 nm through a spectrophotometer. Gallic acid was dissolved in ethanol and used as a standard. The Gallic acid equivalent (GAE) phenolic content of processed Cheddar cheese was recalculated using the Gallic acid standard curve as mg GAE/100 ml. All the replications and experiments were carried out thrice.

| Determination of total flavonoid content
For assessing the total flavonoids content (TFC) in terms of Quercetin equivalent (QE), the WSEs of processed Cheddar cheese were evaluated by following the method described by Zhishen et al. (1999) with some modifications at the interval of 0, 30, and 60 days during storage. Briefly, 1.5 ml of WSEs of processed Cheddar cheese was mixed with 75 μl of 5% (w/v) sodium nitrite (NaNO 3 ) and vortexed for 1 min.
Then, 150 μl of 10% (wt/v) aluminum chloride (AlCl 3 ) solution was added, again vortexed, and left for 5 min. Later, 0.5 ml of 1 M/L sodium hydroxide (NaOH) was added and vortexed again. Afterward, samples were incubated in dark for 45 min. After incubation, the absorbance of samples was measured at 510 nm through a spectrophotometer. Quercetin was dissolved in ethanol and used as a standard.
TFC of processed Cheddar cheese was recalculated using Quercetin standard curve as mg QE/100 ml. All the replications and experiments were carried out thrice (n = 3).

| Microbiological analysis
The total plate count of processed Cheddar cheese was evaluated by following the method of APHA (1984). Bismuth sulfite agar (Hi Media Ltd.) was used for the analysis.
Briefly, 10 g of cheese sample was taken in a presterilized pestle and mortar and properly mixed with 90 ml of 0.1% sterile peptone water. Furthermore, 0.1% peptone water was used to prepare 10-fold serial dilutions. To observe the aseptic conditions, the preparation of samples and serial dilutions were carried out inside the laminar flow cabinet.

| Textural profile analysis
Processed Cheddar cheese was evaluated for the texture profile analysis to assess the effect of olive oil-whey protein isolate emulsion and emulsifying salt on the texture of processed cheese by using TA-XT plus Texture Analyzer and P-75 compression plate probe by following the method described by O'Mahony et al. (2005). Cheese samples were packed in air-tight plastic bags and equilibrated for 18 h at 8°C. Samples were cut into cubes of 25 mm height, length, and width through a stainless-steel wire cutter; before analysis, again equilibrated for 30 min at 8°C. Samples were taken out from the incubator and straightaway compressed in two consecutive cycles at 1 mm/s rate to 30% of the original cheese height.

| Sensory evaluation
Sensory quality properties in terms of sensory characteristics of processed Cheddar cheese, such as color, texture, taste, flavor, appearance, and overall acceptability, were evaluated during stor-

| Statistical analysis
Data were statistically analyzed using Statistix 8.1 software (analytical software, Tallahassee, Florida, USA). The two-way Analysis of variance (ANOVA) technique was used to compare the means. A probability of p < .05 was used to establish statistical significance.
Data were expressed as means ± SD.

| Physicochemical parameters
The moisture, fat, protein, and ash contents of processed Cheddar cheese samples were increased with the corresponding increase in the concentration of OOE. The change in the composition of processed cheese samples was highly significant (p < .05) with the addition of OOE. However, nonsignificant (p > .05) change was observed in the moisture, fat, protein, and ash contents of processed Cheddar cheese during 60 days of storage because each sample was air-tightly sealed separately (Table 1). Gab-Allah (2018) reported that during storage, moisture content of cheese was slightly decreased (p > .05) from different treatments that resulted in increased fat and moisture content of the cheese fortified with olive oil and sunflower oil wax. Khaliq et al. (2021) and Shekhar et al. (2015) reported a similar trend in the contents of moisture, fat, and protein of cheese fortified with olive oil during storage. Khaliq et al. (2021) employed extra virgin olive oil (EVOO) for the fortification of cream cottage cheese and concluded that a rise in total volatile fatty acids may be ascribed to protein breakage which leads to the enhanced flavor of cheese product. This enhanced flavor effect was related to moisture content replacement and high discharge of whey protein from the cottage cheese matrix.
The pH of the processed Cheddar cheese treatments observed a significant reduction (p < .05) with the addition of OOE, which could be due to the carboxylic group of the emulsion. However, the pH insignificantly (p > .05) changed during the storage period of 60 days (Table 1). Khaliq et al. (2021) found that the decrease in pH of cream cottage cheese fortified with extra virgin is associated with high acidity. Abbas et al. (2015) reported a decrease in the pH of cheese yogurt fortified with EVOO during cold storage. Shan et al. (2011) reported that the hindrance in pH increase of processed during storage is associated with the higher quantity of phenolic compounds available in the herbal extracts. The acidity of the processed Cheddar cheese increased with increasing the OOE but acidity had a slight decrease (p > .05) during storage ( Table 1). Shan et al. (2011) reported pH at the initial stage in the range 5.42-5.58 during 9 days of storage, and the authors reported a significantly rising tendency in the pH of control samples.

| Antioxidant activity
The results of WSEs of processed cheese are given in Table 2 which ranged from 88.98 mg GAE/100 ml to 107.92 mg GAE/100 ml. However, olive oil provides no less than 30 phenolic compounds (Musumeci et al., 2013). WSEs of processed Cheddar cheese showed a reduction in phenolic content (p < .05) during storage. However, phenolic content was higher in WSEs of processed Cheddar cheese as compared to control directly after the production ( Table 2). During storage, WSEs of control processed Cheddar cheese did not show a significant change. Peptides that are naturally produced in cheese (Murtaza et al., 2022), due to the activity of starter and rennet, may also act as phenolic compounds. On other hand, some of these peptides react with phe- nolic compounds present in the cheese to neutralize as well as inhibit their activity (Fox et al., 2004). These peptides may have  Emulsions stabilized with whey protein isolate (WPI) play the role of the antioxidant system, as α-lactalbumin and β-lactoglobulin are its major constituents having thiol function, disulfide bonds, and cysteyl residues, which are able to inhibit lipid oxidation by scavenging free radicals (Cayot & Lorient, 1997).

The total antioxidant capacity (TAC) of produced processed
Cheddar cheese' WSEs samples are given in  . In another study on cheese supplemented with extract of dehydrated cranberry fruit, Khalifa and Wahdan (2015) found a decrease in lipolysis, proteolysis, and acid value along with an increase in oxidation stability of the cheese.
The TFC of produced Cheddar processed cheese WSEs samples, given in showed the presence of TFC (43.46 mg QE/100 ml) but content was much less. In dairy products, the antioxidant activity of flavonoids is little known, however, flavonoids' antioxidant activity has been reported (Nadeem et al., 2013). Fruit or plant extracts/ oils may be the source of a higher quantity of flavonoids in any dairy products.  Note: Values are expressed as mean ± SD; means with different letter superscripts are significantly different (p < .05).

TA B L E 2
Effect of olive oil emulsion on the antioxidant potential of processed cheese

| Total plate count
Processed Cheddar cheese samples showed a significant (p < .05) increase in total plate count during 30 days of storage in control as well as in processed Cheddar cheese containing OOE. Singh et al. (2015) reported a similar decrease in the results of total plate count for the chevon cutlets treated with clove oil. Processed cheese samples having OOE showed a significant (p < .05) increase in total plate count throughout 30 days of storage period, but results were significantly (p < .05) less compared to the control during storage study.
Comparatively, a slow increase in the total plate count in processed Cheddar cheese containing OOE may be credited to the antimicrobial properties of extra virgin olive oil in OOE, as shown in Table 3.

3.4
| Textural properties of processed cheese Quigley et al. (2011) reported that texture, a critical characteristic, is the resultant of chemical and physical properties that define the attributes and identity of any food product certainly for the cheese.
Processing and compositional parameters greatly affect the textures of cheeses. Hardness has a critical role in the development of cheese texture as Meullenet and Gross (1999) have defined hardness as the required force to bite a sample, positioned in the middle of molar teeth, completely through it.

| Sensorial attributes of processed cheese
Sensory evaluation of different attributes revealed that processed Cheddar cheese treatments having OOE had higher sensory scores of for taste, aroma, texture/appearance, and overall acceptability in comparison to the control ( showed that 5% and 10% OOE improved the texture/appearance of processed Cheddar cheese as compared to the control. The results were similar to the study conducted by Prinsloo (2007). The quality, taste, aroma, texture, appearance, and likeness or dislikeness of judges decide the products' overall acceptance. Judges preferred T 1 and T 2 having better taste, aroma, and texture/appearance keeping the typical characteristics of Cheddar cheese.
These two treatments are even preferred over processed control (T 0 ) due to better sensory scoring.

| CON CLUS IONS
It was concluded that processed Cheddar cheese fortified with different concentrations of olive oil-whey protein isolate emulsion is nutritionally excellent as compared to the control. Antioxidant potential was significantly (p < .05) higher in processed Cheddar cheeses fortified with olive oil emulsion in comparison with the control.
In addition, processed Cheddar cheese with OOE showed better antimicrobial activity compared to the control. Processed Cheddar cheese with 5% emulsion showed excellent sensory perception.
Olive oil-whey protein isolate emulsion could be used to manufacture and commercialize processed cheese, analogs, or spreads with improved nutritional value and sensory characteristics. It is recommended that quantification of bioactive compounds (exopolysaccharides, organic acids, peptides, fatty acids, γ-aminobutyric acid, and vitamins) extracted from processed Cheddar cheese should be done for a better understanding of biological activities.

ACK N OWLED G M ENT
There was no funding received from any organization to complete this research.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the conclusions of this article are included in the manuscript.