# Effect of encapsulated olive leaf extract on the physicochemical, rheological and antioxidant properties of Cantal-type cheese

Ines Tarchi, Mohamed Bouaziz, Zuhaibf Bhat, Abderrahmane Aït-Kaddour

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Ines Tarchi, Mohamed Bouaziz, Zuhaibf Bhat, Abderrahmane Aït-Kaddour. Effect of encapsulated olive leaf extract on the physicochemical, rheological and antioxidant properties of Cantal-type cheese. Applied Food Research, 2025, 5 (1), pp.100925. 10.1016/j.afres.2025.100925. hal-05201328

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# Effect of encapsulated olive leaf extract on the physicochemical, rheological and antioxidant properties of Cantal-type cheese

Ines Tarchi<sup>a,d</sup>, Mohamed Bouaziz<sup>a,b</sup>, ZuhaibF. Bhat<sup>c</sup>, Abderrahmane Aït-Kaddour<sup>d,e,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Olive leaf
Polyphenols
Encapsulation
Cantal-type cheese
Storage quality

## ABSTRACT

This research evaluated how encapsulated olive leaf extract (E-OLE) influences the quality characteristics of Cantal-type cheese during 63 days of refrigerated storage at 2 °C. Cheeses were enriched with varying levels (1.0 %, 2.0 %, and 3.0 % w/w) of either free (OLE) or encapsulated (E-OLE) olive leaf extract. Multiple quality parameters were systematically assessed, including composition, physicochemical properties, antioxidant capacity, textural profile, microbial counts, rheological behaviour, and colour.

The results indicated that E-OLE supplementation led to significant modifications in cheese composition. Protein content and dry matter increased proportionally with extract concentration, whereas fat content and chloride levels showed an inverse relationship. Both OLE and E-OLE demonstrated strong antioxidant effects, as measured by enhanced total phenolic content and improved ABTS radical scavenging capacity. Microbiological analysis revealed reduced total aerobic mesophilic counts in treated samples.

Textural evaluation showed that E-OLE incorporation resulted in firmer, more cohesive cheeses with reduced elasticity. Colour measurements indicated a progressive darkening (decreased L\* value) accompanied by increased red (a\*) and yellow (b\*) colour intensity. Viscoelastic assessment demonstrated temperature-dependent changes, with notable modifications in storage and loss moduli.

The study concludes that E-OLE enrichment, particularly at the 3 % concentration, effectively enhances the functional properties of Cantal-type cheese while maintaining acceptable quality standards. These findings position E-OLE as a valuable natural additive for improving both the nutritional profile and shelf stability of cheese products.

## 1. Introduction

The olive tree is considered a reservoir of natural compounds with bioactive and functional properties (Boudhioua et al., 2008). Several phenolic compounds with antioxidant and antimicrobial activities, such as oleuropein and hydroxytyrosol, have been identified in olive leaves (Markhali et al., 2020). However, the practical applications of these compounds for product development are often limited due to their sensitivity to various environmental factors, such as heat, light and oxygen, which can cause their degradation and reduce their efficacy (Cao et al., 2021; Shi et al., 2022). To overcome these challenges, encapsulation has been proposed as a promising technique for protecting and

preserving the bioactive components of olive leaves (Culas et al., 2024). This process involves enclosing these compounds in a protective matrix, usually in the form of microcapsules or nanoparticles. Encapsulation protects sensitive ingredients against external factors and offers some additional advantages such as avoiding any deteriorative effect on the sensory quality of food products, enhanced stability, and improved digestibility and bioaccessibility (Gowda, 2023; Rokkam & Kumar 2024). It also allows for the controlled release of the phenolic compounds, ensuring their efficacy over time and expanding their potential applications (Xu et al., 2024). Encapsulated olive leaf extracts (E-OLE) can be incorporated into various food products for developing functional foods and for enhanced storage stability (El-Messery et al., 2021; Farrag et al.,

<footnote><sup>a</sup> Laboratory of Electrochemistry and Environment, National School of Engineers of Sfax (ENIS), University of Sfax, BP 1175, 3038, Sfax, Tunisia</footnote>

<footnote><sup>b</sup> Institut Supérieur de Biotechnologie de Sfax, Université de Sfax, BP1175, 3038, Sfax, Tunisia</footnote>

<footnote><sup>c</sup> Division of Livestock Products Technology, SKUAST-J, Jammu, India</footnote>

<footnote><sup>d</sup> Université Clermont-Auvergne, INRAE, VetAgro Sup, Unité Mixte de Recherche sur le Fromage UMRF, 89 Avenue de l'Europe, France</footnote>

<footnote><sup>e</sup> Departement of Food Technology, Faculty of Agroindustrial Technology, University of Padjadjaran, Sumedang, Jawa Barat 45363, Indonesia</footnote>

<footnote>* Corresponding author at: Université Clermont-Auvergne, INRAE, VetAgro Sup, Unité Mixte de Recherche sur le Fromage UMRF, 89 Avenue de l'Europe, Lempdes, France.</footnote>

<footnote>E-mail address: abderrahmane.aitkaddour@vetagro-sup.fr (A. Aït-Kaddour).</footnote>
2020; Ganje et al., 2016; Tavakoli et al., 2018). Due to its strong anti-oxidant and antimicrobial properties, E-OLE can enhance the storage stability of dairy products rich in fat and protein.

Milk and dairy products, such as cheese and fermented milk, are considered among the most prized functional foods due to their large market share and regular consumption by health-conscious consumers (Khan et al., 2019). In addition, these products contain various components with antioxidant activities, such as casein, whey protein, conjugated linoleic acid, vitamins A, E, and C, and enzymes such as catalase which can act as promoters of cellular antioxidant pathways and help restore the body's antioxidant system (Khan et al., 2019; Stobiecka et al., 2022). The antioxidant activities of dairy products can be further increased through various means such as by feeding specific feeds and antioxidant-rich additives to the dairy animals (Stobiecka et al., 2022). In addition, the ripening of dairy products and the addition of polyphenol-rich plant extracts, such as OLE, during processing are some common ways of promoting overall antioxidant activity (Granato et al., 2018; Khan et al., 2019). While studies have evaluated the effect of the incorporation of OLE on the quality characteristics of milk and dairy products (Cho et al., 2020a; El-Messery et al., 2021; Faraloni C, 2017; Marhamatizadeh et al., 2013; Palmeri et al., 2019; Peker & Arslan, 2017; Tavakoli et al., 2018; Zoidou et al., 2014, 2017), only a few studies have investigated the effect of the addition of encapsulated OLE (E-OLE) in dairy products, especially cheese. Among the few studies on this subject, Farrag et al. (2020) evaluated the effect of encapsulated olive phenolic compounds on the characteristics of soft white cheese. Recently, our research group studied the effect of the addition of OLE on Cantal-type cheese during cold storage and found a significant positive impact of OLE on the quality characteristics and antioxidant properties of the cheese (Tarchi et al., 2024). However, the study did not examine the effect of encapsulation and whether the encapsulated OLE will exert the same positive impact on the product characteristics.

The present study aims to address these gaps by evaluating the influence of incorporating E-OLE on the quality and functional characteristics of cheese during cold storage. Specifically, this research investigates and compares the texture, colour, rheological behavior, biochemical composition, physicochemical attributes, and antioxidant properties of Cantal-type cheese samples containing E-OLE, non-encapsulated OLE, and a control sample without OLE. By doing so, this study provides a comprehensive assessment of how encapsulation affects OLE's functionality in cheese matrices, while also contributing to the broader understanding of incorporating functional bioactive compounds into dairy products.

# 2. Materials and methods

## 2.1. OLE preparation

Fresh leaves of the Chemlali olive tree, a variety grown in Tunisia, were collected from the Sfax region between February and April 2023. After harvesting, the leaves were transported to the laboratory and air-dried at room temperature for a week. The phenolic compounds were extracted using the autoclave method elaborated by Bouaziz et al. (2016). In brief, 50 g of finely ground leaves were processed for 2 min using a Nima NM-8300 mill (150 W, Japan) and placed in a vial containing 750 mL of water. The flask was autoclaved at 121 °C for 20 min and the mixture was cooled and filtered. The resulting filtrate was subsequently dried using a Buchi Mini Spray Dryer B-290 (Switzerland) at a flow rate of 0.5 L/h. Spray drying conditions were set to an inlet temperature of 135 °C, an outlet temperature of 70 °C, an aspirator flow rate of 90 m³/h and a pump speed of 20 %. The dried samples were then stored in darkness at 4 °C for future analysis. Before microencapsulation, the encapsulating agents maltodextrin (ASIN, B08W23N8RJ, Nutripure, France) and sodium caseinate (Sigma Life Science, C8654–1KG) were added to the extract at a ratio of 3:3:1 (w:w:w) and homogenized with an ultra-turrax® homogenizer (IKA T25, Baden-Württemberg, Germany).

This preparation was then spray-dried using a Buchi Mini Spray Dryer B-290 (Switzerland) with a 0.7 mm diameter nozzle. A peristaltic pump fed the mixture into the drying chamber at a flow rate of 0.5 L/h, with constant stirring for uniform mixing before and during drying. The drying parameters were adjusted to an inlet temperature of 145 °C, an outlet temperature of 85±5 °C, an aspirator flow rate of 85 m³/h, and a pump speed of 20 % (Durmus et al., 2022). The final dried samples were stored in darkness at 4 °C till further analysis.

## 2.2. Preparation of Cantal-type cheese samples

Young Cantal cheese was sourced from a local supermarket in Clermont-Ferrand, France. Before the incorporation of OLE, the cheese was ground for 1 min using a domestic blender (DITO Electrolux K55, France). Four batches of cheese samples (500 g each) were prepared by mixing the ground cheese with different concentrations of OLE i.e., 0.0 % (control), 1.0 %, 2.0 %, and 3.0 % (w/w). Each 500 g batch was divided into two equal portions (250 g each) and placed in moulds (12×5.5 cm). The samples were then pressed using a pneumatic cheese press (1-cylinder ht 2250, Sopra, Italy) at a pressure of 3 bar. Pressing was performed in three stages: an initial 1-hour press, followed by a thorough mixing to ensure even distribution of the OLE, and a final 1-hour pressing on both sides of the cheese. The cheese samples were then vacuum-sealed in polyethylene bags and stored at 2 °C for 63 days. The obtained samples were called Cantal-type cheese. The samples containing E-OLE were developed using the same process. In order to ensure comparability between the samples, the quantities of E-OLE were adjusted to contain the same total phenolic content (TPC, expressed as mg GAE – gallic acid equivalents) as the OLE samples (1%, 2%, and 3%). These amounts corresponded to 13.55 g, 27.10 g, and 40.65 g per 100 g of cheese, respectively. The biochemical composition, physicochemical properties, texture, molecular structure, microbiology, colour, rheology, and antioxidant activity of the cheese samples were determined on days 1 and 63 of the storage.

## 2.3. Cheese composition and pH

The dry matter was determined by oven drying the samples (3 g) at 105 °C (Memmert, 854 Schwabach, Germany) following the method of ISO 5534 standard (ISO, 2004). Fat content was determined using the van Gulik method (NF V04-287, 2019). The pH was measured with a pH meter (Testo 205, Testo, Germany) after proper calibration (pH 4.0 and 7.0, HANNA instruments). The chloride (Cl) content in cheese samples was measured by potentiometric titration (Titroline Easy Potentiometer, SI Analytics, Weilheim, Germany) using 0.1 N silver nitrate (VWR International, Fontenay-sous-Bois, France) (NF ISO 5943 standard) (ISO, 2007). All physicochemical measurements were performed in duplicate. Protein content was determined using the FoodScan Lab (type 78,810, FOSS, Denmark) NIR (Near Infrared) instrument. Around 15 g of mixed cheese was placed in a Petri dish and analyzed using a FoodScan device. All experimental tests were performed in triplicate.

## 2.4. Preparation of cheese extract

The extract of the cheese samples was obtained to determine the total phenolic contents and antioxidant activity following the method described by Zandona et al. (2024). The method involved homogenization of 2.50 g of cheese with 5 mL of distilled water which was followed by centrifugation of the suspension at 5000 rpm for 10 min at 4 °C using a Hettich centrifuge. The supernatant obtained was recovered and stored at 4 °C for analysis. This procedure was repeated twice.

## 2.5. Total phenolic content and antioxidant activity of cheese

The total phenolic content (TPC) of the cheese extract was measured by the Folin-Ciocalteu method as described in our previous study (Tarchi
et al., 2024). Samples of 0.2–1.0 mL were transferred to a 10 mL flask and mixed with 0.5 mL of Folin-Ciocalteu reagent (Sigma-Aldrich) for 3 min. Next, 1 mL of saturated sodium carbonate solution was added and the total volume was made using distilled water. After one hour in the dark, the solutions were centrifuged for 15 min using a Hettich centrifuge, and their absorbance was measured at 765 nm against a blank using the Shimadzu UV-1280 UV-Vis spectrophotometer. Results were expressed as mg of gallic acid per 100 g of sample and the assays were performed in triplicate.

The antioxidant activity was determined following the ABTS spectrophotometric method described in detail in our previous study (Tarchi et al., 2024). Briefly, a volume of 0.05–0.5 mL of each sample was mixed with 2.45–2.0 mL of ABTS⁻ solution, incubated at 40 °C for 1 min and the absorbance was measured at 734 nm against a blank. Results were expressed as µmol Trolox equivalents (TE) per 100 g sample and the assays were performed in triplicate.

## 2.6. Microbiological analysis

Various counts were enumerated for evaluating the microbiological quality of cheese samples during storage. The cheese samples (10 g) were homogenized with 90 mL of peptone water for 1.5 min in a stomacher bag (BagMixer CC, France). Total aerobic mesophilic microflora (NF EN ISO 21149, 2017) and yeast and moulds were quantified (NF V08-059, 2002). Leuconostocs were determined using Mayeux-Sandine-Elliker (MSE) agar, lactococcus on M17 agar (ISO 17792:2006, 2006), and lactobacillus on MRS agar (ISO 20128, 2006). Enterococci were detected on Slanetz and Bartley agar (NF EN ISO 11133, 2014) and Staphylococcus spp. on Baird-Parker (BP) agar (NF EN ISO 6888-1, 2021).

The presence of Salmonella, Listeria monocytogenes, and spore-forming bacteria, such as Bacillus or Clostridium, were determined in the cheese samples by applying specific methods following ISO standards. The detection of Salmonella was carried out by mixing 25 g of sample in a pre-enrichment broth, incubation and transfer to selective media, followed by further incubation and confirmation tests (NF EN ISO 6579-1, 2017). Detection of Listeria monocytogenes began with a 25 g sample, enriched in selective broth Fraser 1/2 and plated on Listeria selective agar (Palcam) (NF EN ISO 11290-1, 2017). For spore-forming bacteria, the 25 g of sample mixed with peptone water was heat-treated to remove the vegetative cells and leaving only the spores which were then grown on selective media and incubated (NF V08-602, 2011). All microbiological measurements were done in triplicate.

## 2.7. Colour measurement

The colour of the cheese was measured using a VideometerLab2® (Videometer A/S, Denmark) equipped with a hollow sphere and a multispectral camera (Point Grey Research, model Scorpion SCOR-20SOM, 1200×1200 pixels). This system measures light intensity in the VIS-NIR range using 20 LEDs emitting light from 405 to 1050 nm. Before acquiring the multispectral images, the camera was successively calibrated for white reflectance correction, background correction and pixel position calibration. The lighting was adjusted to avoid pixel saturation. Each sample was illuminated to record three images per cheese. Colour values were extracted using the D65 illuminant. L* values correspond to brightness, a* values to red or green hue and b* values to blue or yellow hue (Boukria et al., 2023; Tarchi et al., 2024).

## 2.8. Texture profile analysis

Textural profile analysis (TPA) measurements were carried out using the Kinexus pro⁺ (Malvern Instruments, Malvern, UK) with a cylindrical acrylic probe measuring 12.7 mm in diameter and 14 mm in height (reference: 04178C74E0). The samples were compressed to 15 % of their initial height using two successive compression cycles. The mechanical

characteristics of hardness, elasticity, and cohesion were measured from the two successive uniaxial compression cycles of the TPA. Each test was carried out in triplicate for each formulation. The different TPA parameters were extracted automatically using a homemade code in Matlab® (2012b) (Tarchi et al., 2025).

## 2.9. Dynamic oscillatory experiment

Dynamic oscillatory parameters were measured under the same conditions as described by Loudiyi et al. (2017). A rheometer (Kinexus pro⁺, rSpace software for Kinexus 1.61, Malvern Instruments, Malvern, Worcs, UK) equipped with a 20 mm diameter plate geometry and a notched probe was used for the measurements. Prior to the analysis, the cheese samples were sliced at the center into thin discs (2 mm ± 0.05 mm thick and 20 mm in diameter) using a cheese slicer fitted with a millimeter guide and a 20 mm diameter cutter (TA Instruments, Leatherhead, UK). The oscillation experiments were performed in the linear viscoelastic region, determined before the actual experiment, by applying a shear strain ranging from 0.001 to 100 % at a constant frequency of 1 Hz. All analyses were performed on the same sample during heating from 20 to 80 °C using a Peltier heating element fitted with an Active Hood Peltier Plate cartridge to prevent heat loss to the sample environment, thereby minimizing the sample thermal gradient. Three experiments were carried out for each cheese formulation. Measurements were carried out at a shear strain of 0.05 % and a constant frequency of 1 Hz. The recorded data included the elastic component G' (storage modulus) and the viscous component G'' (loss modulus).

## 2.10. Data standardization

To facilitate direct comparison and enhance the visualization of differences between the effects of OLE and E-OLE across the various physicochemical, color (L*, a*, b*), and texture (TPA) assays, data normalization was applied. The standardization was performed using the following formula:

$$ \text{Standardised data} = \frac{\text{value measured for each sample}}{\text{value measured for the control}} $$

This approach makes it possible to standardize the results, highlighting the relative variations compared with the control while facilitating a more accurate and consistent interpretation of the data obtained.

## 2.11. Statistical analysis

The results were analyzed for variance (ANOVA) using Minitab Release 16.2.1 (Minitab Inc.). The data were reported as mean ± standard deviation. The effects of the storage period and OLE/E-OLE concentrations were compared by Tukey's test at $p \le 0.05$.

# 3. Results and discussion

## 3.1. Cheese composition

The chemical composition of Cantal-type cheese enriched with E-OLE and OLE is presented in Table 1. A significant increase ($P < 0.05$) in the pH of Cantal cheese was observed with increasing concentration of E-OLE, while no such effect was observed in the OLE-enriched group. A significant effect of storage time was also observed, as the pH of the cheese decreased significantly ($P < 0.05$) after 63 days of storage in all samples (OLE-enriched, E-OLE-enriched, and control samples).

The progressive pH reduction in E-OLE and OLE supplemented samples during storage likely results from increased lactic acid production by LAB, potentially stimulated by phenolic compounds present in the extracts (El-Sayed & Shazly, 2024). According to Piekarska-Radzik and Klewicka (2021), low concentrations of phenolic compounds
<caption>**Table 1**</caption>

Effect of olive leaf extract on the composition and physicochemical characteristics of Cantal-type cheese during refrigerated storage (A) for the control (0 % OLE) and for (B) samples after standardization.

<table><thead><tr><th colspan="3">A</th></tr><tr><th></th><th>Storage period (days)</th><th>Control (0 %)</th></tr></thead><tbody><tr><td rowspan="2">pH</td><td>1</td><td>5.29<sup>B</sup> ± 0.02</td></tr><tr><td>63</td><td>5.35<sup>A</sup> ± 0</td></tr><tr><td rowspan="2">Fat (%)</td><td>1</td><td>33.69<sup>A</sup> ± 0.38</td></tr><tr><td>63</td><td>34<sup>A</sup> ± 0</td></tr><tr><td rowspan="2">Chloride (%)</td><td>1</td><td>1.34<sup>A</sup> ± 0.02</td></tr><tr><td>63</td><td>1.29<sup>A</sup> ± 0.04</td></tr><tr><td rowspan="2">Dry matter (%)</td><td>1</td><td>63.71<sup>Aa</sup> ± 0.56</td></tr><tr><td>63</td><td>63.17<sup>A</sup> ± 0.44</td></tr><tr><td rowspan="2">Protein (%)</td><td>1</td><td>24.70<sup>A</sup> ± 0.18</td></tr><tr><td>63</td><td>24.58<sup>A</sup> ± 0.18</td></tr></tbody></table>

<table><thead><tr><th colspan="8">B</th></tr><tr><th rowspan="3"></th><th rowspan="3">Storage period (days)</th><th colspan="6">Standardized data</th></tr><tr><th colspan="3">E-OLE</th><th colspan="3">OLE</th></tr><tr><th>E-OLE (T<sub>1</sub>)</th><th>E-OLE (T<sub>2</sub>)</th><th>E-OLE (T<sub>3</sub>)</th><th>OLE 1 %</th><th>OLE 2 %</th><th>OLE 3 %</th></tr></thead><tbody><tr><td rowspan="2">pH</td><td>1</td><td>1.01<sup>Ac</sup> ± 0</td><td>1.02<sup>Ab</sup> ± 0</td><td>1.04<sup>Aa</sup> ± 0</td><td>1.00<sup>Ad</sup> ± 0</td><td>1.00<sup>Ad</sup> ± 0</td><td>1.00<sup>Ad</sup> ± 0</td></tr><tr><td>63</td><td>0.95<sup>Bb</sup> ± 0</td><td>0.95<sup>Bb</sup> ± 0</td><td>0.96<sup>Bb</sup> ± 0.01</td><td>0.98<sup>Ba</sup> ± 0.01</td><td>0.98<sup>Ba</sup> ± 0.01</td><td>0.98<sup>Ba</sup> ± 0</td></tr><tr><td rowspan="2">Fat</td><td>1</td><td>0.93<sup>Ac</sup> ± 0.01</td><td>0.87<sup>Bd</sup> ± 0.01</td><td>0.84<sup>Ac</sup> ± 0</td><td>0.98<sup>Aa</sup> ± 0.01</td><td>0.96<sup>Ab</sup> ± 0.01</td><td>0.96<sup>Ab</sup> ± 0</td></tr><tr><td>63</td><td>0.96<sup>Aa</sup> ± 0.02</td><td>0.91<sup>Ab</sup> ± 0.01</td><td>0.84<sup>Ac</sup> ± 0.01</td><td>0.97<sup>Aa</sup> ± 0.03</td><td>0.97<sup>Aa</sup> ± 0.01</td><td>0.95<sup>Aa</sup> ± 0</td></tr><tr><td rowspan="2">Chloride</td><td>1</td><td>0.94<sup>Ab</sup> ± 0</td><td>0.90<sup>Ac</sup> ± 0</td><td>0.82<sup>Bd</sup> ± 0</td><td>0.97<sup>Aa</sup> ± 0.02</td><td>0.96<sup>Aa</sup> ± 0.01</td><td>0.96<sup>Aab</sup> ± 0.01</td></tr><tr><td>63</td><td>0.95<sup>Aab</sup> ± 0.02</td><td>0.91<sup>Ab</sup> ± 0.02</td><td>0.84<sup>Ac</sup> ± 0</td><td>1.00<sup>Aa</sup> ± 0.02</td><td>0.98<sup>Aa</sup> ± 0.04</td><td>0.97<sup>Aa</sup> ± 0.04</td></tr><tr><td rowspan="2">Dry matter</td><td>1</td><td>1.03<sup>Ac</sup> ± 0</td><td>1.05<sup>Ab</sup> ± 0</td><td>1.08<sup>Ba</sup> ± 0</td><td>0.99<sup>Ad</sup> ± 0</td><td>0.99<sup>Bd</sup> ± 0</td><td>0.99<sup>Bd</sup> ± 0</td></tr><tr><td>63</td><td>1.02<sup>Bc</sup> ± 0.05</td><td>1.05<sup>Bb</sup> ± 0</td><td>1.08<sup>Aa</sup> ± 0</td><td>1.00<sup>Be</sup> ± 0.01</td><td>1.00<sup>Ade</sup> ± 0</td><td>1.01<sup>Ad</sup> ± 0</td></tr><tr><td rowspan="2">Protein</td><td>1</td><td>0.99<sup>Ac</sup> ± 0.01</td><td>1.08<sup>Ab</sup> ± 0</td><td>1.09<sup>Aa</sup> ± 0.01</td><td>1.00<sup>Ad</sup> ± 0.01</td><td>0.99<sup>Ad</sup> ± 0</td><td>0.99<sup>Ad</sup> ± 0</td></tr><tr><td>63</td><td>1.05<sup>Ab</sup> ± 0.01</td><td>1.07<sup>Ba</sup> ± 0</td><td>1.08<sup>Aa</sup> ± 0.01</td><td>0.99<sup>Acd</sup> ± 0.01</td><td>1.00<sup>Ac</sup> ± 0.01</td><td>0.98<sup>Ad</sup> ± 0.01</td></tr></tbody></table>

Different uppercase superscripts (A-B) in columns and lowercase superscripts (a-e) in rows indicate significant differences (P < 0.05).

like gallic acid can promote lactic acid bacteria growth. However, concentrations exceeding 3 mM may damage microbial cell walls, interfere with pH homeostasis, and impair carbohydrate metabolism. The acidic nature of phenolic compounds could also explain the decrease in pH observed in the medium following the addition of bioactive compounds (Soliman et al., 2022). A decrease in the pH of cheese enriched with phenolic compounds was also observed by Lee et al. (2016) when fortifying Cheddar-type cheese with Inula britannica extract, and by Azarashkan et al. (2022) when adding free and encapsulated broccoli sprout extract into ricotta cheese during storage.

Further, the increase in the pH of Cantal-type cheese with increasing concentration of E-OLE may be associated with the properties of the materials used for encapsulation. While sodium caseinate solutions generally have a neutral to slightly alkaline pH (Bian & Plank, 2012), adding maltodextrin can reduce acidity and increase pH in various food matrices (Ekpong et al., 2016).

The addition of E-OLE caused a reduction in the fat content of cheese compared to the control and showed a significant (P < 0.05) decreasing trend with increasing concentration of E-OLE. The addition of OLE caused a reduction in the fat content of cheese compared to the control and an effect of concentration (P < 0.05) was observed on day 1, the same trend was observed on day 63 of storage. Our findings align with those of Lee et al. (2016), who observed similar effects when incorporating Inula britannica extract into Cheddar-type cheese. In contrast to our findings, Farrag et al. (2020) reported no significant change in the fat content of soft cheese samples enriched with E-OLE or OLE. Similarly, Balabanova et al. (2020) found that incorporating encapsulated pepper extracts into Labneh cheese did not result in significant changes to the fat, moisture, or protein contents. The observed reduction in fat content with higher levels of OLE may be attributed to interactions between OLE and lipids within the cheese matrix. The plant polyphenols can penetrate lipid bilayers and alter their structure and properties (Karonen, 2022). The polyphenols in OLE could form complexes with lipids, thereby decreasing the detectable free fat in the cheese. These interactions may restrict fat mobility and availability, leading to a lower

measured fat content during analysis. Additionally, this reduction could be influenced by interactions involving the wall material or wall material polyphenols and the fat present in the cheese.

The addition of E-OLE led to a significant (P < 0.05) reduction in the chloride content of cheese compared to the control, with a more pronounced decrease at higher concentrations on both days 1 and 63 of storage. While OLE also lowered chloride content, its effect remained independent of concentration on both days. This reduction in chloride content can be attributed to the encapsulation materials, particularly sodium caseinate, which has a strong ion-binding capacity (Liu et al., 2022). As a protein with functional groups, sodium caseinate binds to ions present in the cheese (Li et al., 2023). When used for encapsulation, it attracts and retains chloride ions, effectively isolating them from the cheese matrix and limiting their mobility, thereby reducing their availability in free form. Furthermore, maltodextrin, known for its moisture-absorbing properties (Letona et al., 2023), helps trap water molecules along with certain ions, including chloride, within the encapsulation structure. By creating moist micro-environments inside the capsule, maltodextrin reduces the outward diffusion of chloride ions. As a result, this encapsulation mechanism contributes to a more pronounced reduction in chloride content in cheese enriched with E-OLE compared to non-encapsulated extracts. The combination of these encapsulation materials—sodium caseinate and maltodextrin—seems to create a controlled environment that limits ion mobility and enhances the retention of chloride ions within the encapsulating matrix. Additionally, OLE contains phenolic compounds like oleuropein and hydroxytyrosol, which are known for their ability to bind ions (Ivanov et al., 2018). These compounds may influence chloride ion balance through redox reactions, particularly in the presence of hypochlorite, reducing it to chloride and altering the equilibrium, thus lowering detectable free chloride (Krych-Madej et al., 2016).

The addition of E-OLE resulted in an increase in the dry matter and protein content of the cheese compared with the control and showed a significant increase (P < 0.05). In contrast, the changes in dry matter and protein content in OLE-enriched samples were less pronounced and
remained relatively close to control values. The increase in E-OLE samples may be due to sodium caseinate and maltodextrin, which are low in moisture, with sodium caseinate being protein-rich. These results are consistent with those of Farrag et al. (2020) who documented an increase in the protein content of soft cheese enriched with OLE encapsulated using a mixture of whey protein isolate, maltodextrin and skim milk. These findings confirm the impact of encapsulation materials on the final nutritional composition of the product. Lee et al. (2016) observed an increase in protein content when Inula britannica extract was added to Cheddar-type cheese. This increase in protein was likely attributed to interactions between the polyphenolic compounds and milk proteins, leading to cross-linking or precipitation.

## 3.2. Total phenolic compound and antioxidant activity of cheese

Table 2 presents the total phenolic content (TPC) and antioxidant activity (ABTS radical scavenging assay) of Cantal-type cheese enriched with E-OLE and OLE. Both E-OLE and OLE significantly increased TPC, with higher concentrations resulting in higher values on both days 1 and 63 of storage (Boli et al., 2022). Our results agree with those of Zandona et al. (2024), who also observed a comparable increase in TPC in rennet-curd cheese fortified with OLE. A comparable trend was also reported by Azarashkan et al. (2022) with free and nano-encapsulated broccoli sprout extract in ricotta cheese. Similarly, the addition of OLE to yogurt showed the same trend (Cho et al., 2020b; Peker & Arslan, 2017; Tarchi et al., 2025; Zoidou et al., 2017). TPC increases are positively correlated with antioxidant activity due to the higher presence of antioxidants capable of scavenging ABTS radicals.

However, while E-OLE samples showed a continuous increase in TPC with storage, OLE samples showed a decrease by day 63. This difference is likely due to the controlled release of polyphenols from the encapsulated E-OLE, which may be more stable during storage. This aligns with Farrag et al. (2020) and Azarashkan et al. (2022). Encapsulation appears to protect polyphenols from degradation as previously noted in cheeses added with Inula britannica (Lee et al., 2016).

As expected, a positive effect of the addition of E-OLE and OLE was found on the antioxidant potential (ABTS radical scavenging) of the Cantal-type cheese. The samples containing E-OLE or OLE showed higher ABTS radical scavenging activity compared to the control cheese and a significant increasing pattern was observed with increasing concentration of E-OLE or OLE. This pattern was found on both days 1 and

63 of storage. Regarding the storage time, a significant decrease was observed in the radical scavenging activity of the cheese samples on day 63 compared to day 1. While Farrag et al. (2020) reported higher antioxidant activity for the cheese samples containing E-OLE compared to the samples containing OLE, we observed higher antioxidant activity for samples containing 1.0 and 2.0% E-OLE on day 1 of the storage. Similar to our results, Farrag et al. (2020) found a decrease in the antioxidant activity of cheese samples containing OLE with storage time. The decline in antioxidant activity of cheese during storage may result from the progressive scavenging of antioxidant compounds caused by the ongoing oxidation of lipids and proteins. In addition, changes in the interactions between proteins and phytochemicals during storage could influence their bioavailability. Proteins can bind to plant phenolics via covalent or non-covalent bonds (Jiang et al. 2018; Sui et al., 2018; Utrera & Estevez, 2013), and the resulting complexes may exhibit distinct physicochemical and functional properties depending on the nature of their binding (Sun et al., 2021). For example, covalent complexes tend to exhibit greater oxidation stability, polyphenol preservation and heat resistance compared to non-covalent interactions (Liu et al., 2021).

## 3.3. Microbiological analysis

The data related to the microbiological quality of Cantal-type cheese enriched with OLE and E-OLE is presented in Table 3. The highest total aerobic mesophilic counts (TAMF) were observed for the control cheese samples and the addition of E-OLE or OLE reduced the microbial counts. An effect of the concentration was noticed and the samples containing the highest levels of E-OLE or OLE showed the lowest TAMF counts on both days 0 and 63 of the storage. A maximum inhibition of 91% was achieved for the cheese samples containing 3% OLE. Aerobic mesophilic bacteria growth was significantly inhibited by E-OLE and OLE, possibly because of the synergistic antimicrobial properties of OLE's bioactive components (Boli et al., 2022; Ruzzolini et al., 2020). It is noteworthy that the encapsulation seems to slightly mask the antimicrobial effect of OLE.

The incorporation of E-OLE initially stimulated yeast and mould proliferation, with a significant concentration-dependent increase (P < 0.05) observed on day 1 of storage. However, after 63 days, higher E-OLE concentrations significantly suppressed (P < 0.05) fungal growth. A similar inhibitory effect was noted in OLE-treated samples, where

<table><caption>**Table 2**
Effect of OLE and E-OLE on the antioxidant potential of Cantal-type cheese for the (A) control samples (0% OLE) and (B) samples after standardization.</caption><thead><tr><th colspan="2">(A)</th><th rowspan="2">Storage period (days)</th><th rowspan="2">Control (0%)</th></tr><tr><th></th><th></th></tr></thead><tbody><tr><td rowspan="2">Total phenolic content (mg GAE/100 g)</td><td>1</td><td>210.48<sup>B</sup> ± 3.68</td></tr><tr><td>63</td><td>281.25<sup>A</sup> ± 8.56</td></tr><tr><td rowspan="2">ABTS radical scavenging activity (µmol TE/ 100 g)</td><td>1</td><td>1038.78<sup>A</sup> ± 70.27</td></tr><tr><td>63</td><td>667.97<sup>B</sup> ± 19.97</td></tr></tbody></table><table><thead><tr><th colspan="2">(B)</th><th rowspan="2">Storage period (days)</th><th colspan="6">Standardized data</th></tr><tr><th></th><th></th><th colspan="3">E-OLE</th><th colspan="3">OLE</th></tr><tr><th></th><th></th><th></th><th>E-OLE (T<sub>1</sub>)</th><th>E-OLE (T<sub>2</sub>)</th><th>E-OLE (T<sub>3</sub>)</th><th>OLE 1 %</th><th>OLE 2 %</th><th>OLE 3 %</th></tr></thead><tbody><tr><td rowspan="2">Total phenolic content</td><td>1</td><td>2.20<sup>Bc</sup> ± 0.05</td><td>2.50<sup>Bb</sup> ± 0.04</td><td>2.80<sup>Ba</sup> ± 0.05</td><td>1.95<sup>Af</sup> ± 0.04</td><td>2.84<sup>Ae</sup> ± 0.03</td><td>4.08<sup>Ad</sup> ± 0.03</td></tr><tr><td>63</td><td>4.42<sup>Ac</sup> ± 0.12</td><td>5.12<sup>Ab</sup> ± 0.07</td><td>6.57<sup>Aa</sup> ± 0.02</td><td>1.50<sup>Bc</sup> ± 0.05</td><td>2.07<sup>Bb</sup> ± 0.03</td><td>2.94<sup>Ba</sup> ± 0.05</td></tr><tr><td rowspan="2">ABTS radical scavenging activity</td><td>1</td><td>3.03<sup>Ad</sup> ± 0.13</td><td>4.45<sup>Ab</sup> ± 0.14</td><td>4.49<sup>Ab</sup> ± 0.10</td><td>2.53<sup>Be</sup> ± 0.05</td><td>3.50<sup>Bc</sup> ± 0.09</td><td>7.46<sup>Ba</sup> ± 0.16</td></tr><tr><td>63</td><td>2.23<sup>Be</sup> ± 0.09</td><td>3.56<sup>Bd</sup> ± 0.04</td><td>4.65<sup>Ab</sup> ± 0.08</td><td>3.94<sup>Ac</sup> ± 0.12</td><td>4.77<sup>Ab</sup> ± 0.13</td><td>9.41<sup>Aa</sup> ± 0.22</td></tr></tbody></table>

Different uppercase superscripts (A-B) in columns and lowercase superscripts (a-e) in rows indicate significant differences (P < 0.05).
<table><caption>**Table 3**
Effect of OLE and E-OLE on the microbiological quality (UFC/g) of Cantal-type cheese for the (A) control samples (0 % OLE) and (B) samples after standardization.</caption><thead><tr><th colspan="3">(A)</th></tr><tr><th>Microbial counts</th><th>Storage period (days)</th><th>Control (0 %)</th></tr></thead><tbody><tr><td rowspan="2">Total aerobic mesophilic flora (TAMF)</td><td>1</td><td>2.3E+08</td></tr><tr><td>63</td><td>3.2E+08</td></tr><tr><td rowspan="2">Yeasts and molds</td><td>1</td><td>4.4E+03</td></tr><tr><td>63</td><td>1.9E+05</td></tr><tr><td rowspan="2">Lactococcus</td><td>1</td><td>2.2E+05</td></tr><tr><td>63</td><td>4.2E+08</td></tr><tr><td rowspan="2">Lactobacillus</td><td>1</td><td>2.5E+07</td></tr><tr><td>63</td><td>1.2E+07</td></tr><tr><td rowspan="2">Enterococcus</td><td>1</td><td>4.0E+05</td></tr><tr><td>63</td><td>5.8E+06</td></tr><tr><td rowspan="2">Staphylococcus spp</td><td>1</td><td>3.3E+03</td></tr><tr><td>63</td><td>3.0E+02</td></tr><tr><td rowspan="2">Leuconostocs</td><td>1</td><td>&lt;100</td></tr><tr><td>63</td><td>&lt;100</td></tr><tr><td rowspan="2">E. coli</td><td>1</td><td>&lt;100</td></tr><tr><td>63</td><td>&lt;100</td></tr><tr><td rowspan="2">Sporulated bacteria</td><td>1</td><td>&lt;100</td></tr><tr><td>63</td><td>&lt;100</td></tr><tr><td rowspan="2">Listeria monocytogenes</td><td>1</td><td>Absence</td></tr><tr><td>63</td><td>Absence</td></tr><tr><td rowspan="2">Salmonella</td><td>1</td><td>Absence</td></tr><tr><td>63</td><td>Absence</td></tr></tbody></table>

<table><thead><tr><th colspan="3">(B)</th></tr><tr><th rowspan="3">Microorganism</th><th rowspan="3">Storage period (days)</th><th colspan="6">Standardized data</th></tr><tr><th colspan="3">E-OLE</th><th colspan="3">OLE</th></tr><tr><th>E-OLE (T<sub>1</sub>)</th><th>E-OLE (T<sub>2</sub>)</th><th>E-OLE (T<sub>3</sub>)</th><th>OLE 1 %</th><th>OLE 2 %</th><th>OLE 3 %</th></tr></thead><tbody><tr><td rowspan="2">Total aerobic mesophilic flora (TAMF)</td><td>1</td><td>0.94</td><td>0.92</td><td>0.77</td><td>0.74</td><td>0.33</td><td>0.09</td></tr><tr><td>63</td><td>1.03</td><td>1.05</td><td>0.92</td><td>0.84</td><td>0.34</td><td>0.26</td></tr><tr><td rowspan="2">Yeasts and molds</td><td>1</td><td>1.43</td><td>1.79</td><td>3.57</td><td>0.59</td><td>0.70</td><td>0.52</td></tr><tr><td>63</td><td>1.42</td><td>0.81</td><td>1.08</td><td>0.63</td><td>0.89</td><td>0.33</td></tr><tr><td rowspan="2">Lactococcus</td><td>1</td><td>0.92</td><td>0.83</td><td>0.73</td><td>0.91</td><td>90.91</td><td>277.27</td></tr><tr><td>63</td><td>0.22</td><td>0.12</td><td>0.01</td><td>0.57</td><td>0.21</td><td>0.15</td></tr><tr><td rowspan="2">Lactobacillus</td><td>1</td><td>0.95</td><td>0.79</td><td>0.67</td><td>0.84</td><td>1.20</td><td>0.02</td></tr><tr><td>63</td><td>0.79</td><td>0.58</td><td>0.67</td><td>6.58</td><td>5.17</td><td>4.50</td></tr><tr><td rowspan="2">Enterococcus</td><td>1</td><td>0.08</td><td>0.36</td><td>0.14</td><td>0.55</td><td>0.45</td><td>0.40</td></tr><tr><td>63</td><td>0.55</td><td>0.27</td><td>0.27</td><td>0.78</td><td>0.38</td><td>0.17</td></tr><tr><td rowspan="2">Staphylococcus spp</td><td>1</td><td>0.79</td><td>0.48</td><td>0.53</td><td>0.67</td><td>0.15</td><td>0.03</td></tr><tr><td>63</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td></tr><tr><td rowspan="2">Leuconostocs</td><td>1</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td></tr><tr><td>63</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td></tr><tr><td rowspan="2">E. coli</td><td>1</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td></tr><tr><td>63</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td></tr><tr><td rowspan="2">Sporulated bacteria</td><td>1</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td></tr><tr><td>63</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td><td>&lt;100</td></tr><tr><td rowspan="2">Listeria monocytogenes</td><td>1</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td></tr><tr><td>63</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td></tr><tr><td rowspan="2">Salmonella</td><td>1</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td></tr><tr><td>63</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td><td>Absence</td></tr></tbody></table>

elevated doses consistently reduced yeast and mould counts on both day 1 and day 63, indicating that prolonged exposure to higher concentrations of either extract restricts microbial viability. Notably, the most substantial inhibition (48 %) occurred in cheese samples fortified with 3 % OLE.

The addition of E-OLE decreased the *Lactococcus* and *Lactobacillus* counts compared to the control samples and a significant decreasing trend was observed with increasing concentration of E-OLE on days 1 and 63 of the storage. The highest concentration of E-OLE almost inhibited all the *Lactococci* present in the cheese samples, indicating a prolonged inhibitory effect of E-OLE on the growth of this bacterium over time. In contrast, the samples containing OLE showed different results and the samples containing 1 % OLE showed lower *Lactococcus* counts than the control on day 1. However, the counts increased significantly with concentration. On day 63, a significant decreasing trend was observed with increasing concentration of OLE and a maximum reduction of 85 % was achieved in the samples containing 3 % OLE. The stronger and long-lasting effect of E-OLE could be attributed to

encapsulation, which enables controlled and prolonged release of polyphenols in the product. Overall, the OLE seemed to inhibit the growth of *Lactobacillus* on day 1 and enhanced the growth during storage, indicating that the inhibitory effect of OLE was not maintained over a longer period. Korukluoglu et al. (2010) reported antibacterial effects of OLE on lactic acid bacteria, linking this activity to specific phenolic compounds (oleuropein, caffeic acid, tyrosol) in the extract.

Overall, a strong inhibitory effect of E-OLE and OLE was observed on the growth of *enterococcus* and *Staphylococcus spp* on day 1 and during the storage. A maximum inhibition of 86 % and 47 % was achieved against *enterococcus* and *Staphylococcus spp* in T<sub>3</sub> samples (E-OLE 3%) on day 1. Similarly, a maximum inhibition of 60 % and 97 % was achieved against *enterococcus* and *Staphylococcus spp* in the samples containing 3 % OLE on day 1. A marked inhibitory effect of OLE against *enterococcus* has been reported by Farrag et al. (2020). After 63 days of storage, *Staphylococci* were almost absent (<100 CFU/g) in all E-OLE and OLE-enriched samples, indicating strong antimicrobial activity of the extract with and without encapsulation. Borjan et al. (2020) observed an
antimicrobial effect of OLE against *Staphylococcus* and attributed it to the presence of bioactive phytochemicals such as caffeic acid, oleuropein, rutin, and verbascoside.

Microbiological analysis revealed that *Leuconostoc* spp., *Escherichia coli*, *spore-forming bacteria*, *Listeria monocytogenes*, and *Salmonella* were either undetectable or present at levels below the detection threshold (<100 CFU/g) throughout storage, confirming the high microbiological quality and absence of contamination. Both encapsulated (E-OLE) and non-encapsulated (OLE) olive leaf extracts exhibited potent antimicrobial activity against spoilage microorganisms and potential pathogens, particularly at higher concentrations (2–3 %), with sustained efficacy during storage.

While the extracts showed some inhibitory effects on beneficial bacteria (*Lactococcus* and *Lactobacillus* spp.), the suppression was notably less pronounced compared to their action against undesirable microbiota. These findings demonstrate that supplementing Cantal-type cheese with 2–3 % OLE or E-OLE significantly improves microbiological stability. However, the antimicrobial activity of non-encapsulated OLE diminished over time, whereas E-OLE maintained stronger and more persistent inhibition, highlighting the protective effect of encapsulation in preserving bioactive compounds and their antimicrobial properties.

### 3.4. Texture profile analysis

The effect of the addition of E-OLE and OLE on the hardness of Cantal-type cheese during storage is presented in Fig. 1. The cheese samples containing E-OLE showed significantly higher hardness compared to the samples containing OLE on both days 1 and 63 of the storage. A significant increasing pattern was observed with increasing concentrations of E-OLE on both days 1 and 63. Incorporating OLE into Cantal-type cheese had no statistically significant impact (*P* < 0.05) on its hardness. Throughout the storage period, including both day 1 and day 63, the hardness remained consistent across samples with varying concentrations of OLE. Azarashkan et al. (2022) found no effect on the hardness of ricotta cheese when free broccoli sprout extract was added. A similar increase in the hardness and firmness of white soft cheese has been reported by Farrag et al. (2020) upon the addition of E-OLE. A significant effect of storage time was also recorded and the hardness of

all cheese samples increased significantly on day 63 except T₁ samples (E-OLE 1%). Similar trends were documented by Piñeiro-Lago et al. (2019), with their study showing increased stiffness in Afuega'l Pitu cheese as storage time progressed. Microstructural analysis in their study indicated the fusion of fat globules and the development of thicker protein strands, both of which played a role in enhancing the rigidity. Furthermore, prolonged storage contributed to a denser, less porous structure and an elevated complex modulus, which are indicative of increased cheese hardness. The increase in cheese hardness with higher concentrations of E-OLE can also be attributed to moisture loss caused by the presence of wall materials. This results in a reduction in water content and a higher concentration of dry matter, thereby making the cheese firmer. A direct correlation between hardness and moisture has been established by Tejada et al. (2007). The reduction in water content promotes a higher concentration of casein and a greater number of casein bonds which increases the hardness (Álvarez & Fresno, 2021; Solowiej et al., 2014). While higher water and fat contents tend to reduce the hardness of cheese, higher dry matter and protein contents increase the hardness (Sekhavatizadeh et al., 2023).

The effect of the addition of E-OLE and OLE on the cohesiveness of Cantal-type cheese during storage is presented in Fig. 2. The cheese samples containing E-OLE showed significantly higher cohesiveness compared to the control and the samples containing OLE on both days 1 and 63 of the storage. A significant increasing pattern was observed with increasing concentrations of E-OLE on both days 1 and 63. The addition of OLE did not affect (*P* < 0.05) the cohesiveness of Cantal-type cheese. Almost no significant difference was observed between the cohesiveness of the samples containing different levels of OLE on days 1 and 63 of the storage. In contrast to our results, Farrag et al. (2020) observed no change in the cohesiveness of white soft cheese upon the addition of E-OLE or OLE. Research by Akazawa et al. (2021) and Konno et al. (1999) indicates that oleuropeindial, the aglycone derivative of oleuropein, as well as its oxidized quinone form, demonstrate significant protein cross-linking capabilities. These compounds, derived from oleuropein, contribute to protein binding within food systems. When E-OLE and OLE are incorporated, they enhance the protein matrix by promoting interactions between polyphenols and proteins, leading to improved structural cohesion. Such findings underscore the diverse

<figure><img src="image_4.png" /></figure>

Different uppercase letters (A-B) for the same concentration and extract at different storage times indicate significant differences (*P*<0.05), different lowercase letters (a-c) at the same time and for the same extract at different concentrations indicate significant differences (*P*<0.05).

**Fig. 1.** Normalized hardness of cheese enriched with E-OLE and OLE during storage. The concentrations are colour-coded (1, 2 and 3%), with the orange representing the E-OLE extract and the blue representing the OLE extract, where lighter shades correspond to lower concentrations and darker shades indicate higher concentrations.

Different uppercase letters (A-B) for the same concentration and extract at different storage times indicate significant differences (*P* < 0.05), different lowercase letters (a-c) at the same time and for the same extract at different concentrations indicate significant differences (*P* < 0.05).
<figure><img src="image_5.png" /></figure>

Different uppercase letters (A-B) for the same concentration and extract at different storage times indicate significant differences (P<0.05);
different lowercase letters (a-c) at the same time and for the same extract at different concentrations indicate significant differences (P<0.05).

<caption>**Fig. 2.** Normalized cohesiveness of cheese enriched with E-OLE and OLE during storage. The concentrations are colour-coded (1, 2 and 3%), with the orange representing the E-OLE extract and the blue representing the OLE extract, where lighter shades correspond to lower concentrations and darker shades indicate higher concentrations.</caption>

Different uppercase letters (A-B) for the same concentration and extract at different storage times indicate significant differences (P < 0.05); different lowercase letters (a-c) at the same time and for the same extract at different concentrations indicate significant differences (P < 0.05).

functionality of these extracts in altering cheese texture. Soliman et al. (2022) reported that the incorporation of encapsulated plant extracts could be one of the factors contributing to the increased hardness and cohesiveness of the cheese.

A significant effect of storage time was also recorded in our study and the cohesiveness of all cheese samples increased significantly on day 63 except T₁ samples (E-OLE 1%). This increase in cohesiveness of the

cheese sample during storage could be due to the decrease in moisture content, leading to a less porous microstructure and enhanced rigidity and structural stability, thereby improving cheese network cohesion (Soliman et al., 2022).

The effect of the addition of E-OLE and OLE on the elasticity of Cantal-type cheese during storage is presented in Fig. 3. By comparing the two samples, it is clear that the cheese enriched with OLE presented

<figure><img src="image_6.png" /></figure>

Different uppercase letters (A-B) for the same concentration and extract at different storage times indicate significant differences (P<0.05);
different lowercase letters (a-c) at the same time and for the same extract at different concentrations indicate significant differences (P<0.05).

<caption>**Fig. 3.** Normalized elasticity of cheese enriched with E-OLE and OLE during storage. The concentrations are colour-coded (1, 2 and 3%), with the orange representing the E-OLE extract and the blue representing the OLE extract, where lighter shades correspond to lower concentrations and darker shades indicate higher concentrations.</caption>

Different uppercase letters (A-B) for the same concentration and extract at different storage times indicate significant differences (P < 0.05); different lowercase letters (a-c) at the same time and for the same extract at different concentrations indicate significant differences (P < 0.05).
higher elasticity compared to the E-OLE-enriched samples. This difference was reduced after storage time. The polyphenols in OLE interact directly with the cheese protein matrix, in particular with the caseins, forming hydrogen or covalent bonds (Han et al., 2019). These interactions strengthen the cohesion of the protein network, which improves elasticity. However, in samples containing E-OLE, the polyphenols are trapped in the encapsulation material, limiting their availability to interact with the proteins (Ali Redha et al., 2024) or may interact differently. Encapsulation materials can interact with the protein matrix and alter its structure, an essential element for preserving the desired texture of the cheese. For example, droplets stabilized by sodium caseinate strengthen the protein network (Luo et al., 2018) which can explain why, after 63 days of storage, the elasticity of the cheese remained relatively stable in all samples.

### 3.5. Colour measurement

The colour is one of the most important parameters for consumers. Table 4 represents the change in the L\* (lightness), a\* (red/green), and b\* (yellow/blue) values of Cantal-type cheese during storage. The values indicate the effect of the addition of E-OLE and OLE and the influence of the wall materials (sodium caseinate and maltodextrin) on the colour of the cheese. The L\* values significantly decreased (P < 0.05) as E-OLE and OLE concentrations increased. Similarly, Siyar et al. (2021) reported a decrease in lightness in ricotta cheese samples containing nanoliposomal saffron extract.

Concerning storage, the clarity of the OLE-enriched samples decreased slightly over time and increased for the E-OLE-enriched samples. OLE contains natural pigments, such as chlorophylls and polyphenols, which are dark in hue (Ekpong et al., 2016). These compounds can gradually diffuse into the cheese matrix, reducing its overall brightness and causing a decrease in the L\* values. Furthermore, during storage, enzymatic reactions such as proteolysis and lipolysis modify the structure of cheese, altering its light reflection properties (Karaca & Güven, 2018). The combination of the structural modifications and the dark pigments of olive leaf amplifies this decrease in L\* values. However, an increase in the L\* values can be observed in the presence of encapsulating wall materials. Encapsulation forms a protective barrier around the bioactive compounds of OLE, limiting their release and interaction with the cheese matrix (Akpo et al., 2024). This preserves and even improves the natural clarity of the cheese over time. In addition, maltodextrin and sodium caseinate, as light-tinted wall materials,

contribute to this effect (Ilyasoglu & El, 2014; Xiao et al., 2022). When the encapsulated extract remains stable and does not diffuse notable pigments, the overall appearance of the cheese appears brighter, with an increase in the L\* values due to the optical properties of these materials.

Cheese samples enriched with OLE or E-OLE showed higher and positive a\* values within the redness range which might be ascribed to the presence of olive leaf pigments and phenolic compounds (Yinbin et al., 2018). A clear increase in a\* values was observed as the concentrations of E-OLE and OLE increased, both on day 1 and day 63 of storage. For the E-OLE-enriched samples, redness decreased significantly over time, while the OLE-enriched samples showed a significant increase in redness during storage. The decrease in a\* values over storage time is primarily driven by the protective nature of the encapsulation process, which stabilizes the bioactive compounds, limits their oxidation, and minimizes their interaction with the cheese matrix. Additionally, the dilution and optical effects of the encapsulating materials contribute to the observed trend. Further, chlorophyll, a natural pigment in olive leaves, can degrade over time into pheophytins and other derivatives. These degradation products can impart yellowish or reddish tones, causing an increase in a\* values.

The b\* values of all the samples were positive and overall, the addition of E-OLE and OLE increased the b\* values compared to the control, giving the Cantal-type cheese a more yellowish appearance. However, no concentration-dependent pattern was noticed. The yellowness of the cheese became more prominent with storage time. El-Messery et al. (2021) found the same effect of OLE on the yellowness of yoghurt. Siyar et al. (2021) reported an increase in yellowness in ricotta cheese samples containing nanoliposomal saffron extract compared to the control sample. Similarly, Hosseini et al. (2023) observed that the addition of chia seed extract enhanced the yellowness of cheese compared to the control. In contrast, Flamminii et al. (2020) observed no effect on the yellowness of mayonnaise incorporated with OLE encapsulated in alginate and pectin microparticles.

### 3.6. Dynamic oscillatory experiment

The changes in the elastic component (G': storage modulus) and viscous component (G": loss modulus) during heating (from 20 to 80 °C) are shown in Fig. 4. The results indicate that the extracts (E-OLE and OLE) had opposite effects on the storage modulus (G'). For example, increasing the concentration of E-OLE led to an increase in G', which suggests that its addition makes the cheese firmer and more elastic. This

<table><caption>**Table 4**

Effect of the addition of E-OLE and OLE on the colour of Cantal-type cheese for the (A) control samples (0 % OLE) and (B) samples after standardization.</caption><thead><tr><th colspan="2">(A)</th><th rowspan="2">Control (0 %)</th></tr><tr><th>Storage period (days)</th><th>Control (0 %)</th></tr></thead><tbody><tr><td>L*</td><td>1</td><td>84.89<sup>B</sup> ± 0.42</td></tr><tr><td></td><td>63</td><td>85.58<sup>A</sup> ± 0.39</td></tr><tr><td>a*</td><td>1</td><td>3.55<sup>A</sup> ± 0.13</td></tr><tr><td></td><td>63</td><td>3.54<sup>A</sup> ± 0.13</td></tr><tr><td>b*</td><td>1</td><td>32.71<sup>A</sup> ± 0.47</td></tr><tr><td></td><td>63</td><td>31.16<sup>B</sup> ± 0.60</td></tr></tbody></table><table><thead><tr><th colspan="2">(B)</th><th rowspan="2">Standardized data</th></tr><tr><th>Storage period (days)</th><th>Control (0 %)</th></tr></thead><tbody><tr><td>L*</td><td>1</td><td>0.84<sup>Bb</sup> ± 0.01</td></tr><tr><td></td><td>63</td><td>0.87<sup>Ab</sup> ± 0.01</td></tr><tr><td>a*</td><td>1</td><td>3.33<sup>Ac</sup> ± 0.18</td></tr><tr><td></td><td>63</td><td>2.66<sup>Bc</sup> ± 0.09</td></tr><tr><td>b*</td><td>1</td><td>1.06<sup>Ba</sup> ± 0.02</td></tr><tr><td></td><td>63</td><td>1.12<sup>Aab</sup> ± 0.03</td></tr></tbody></table><table><thead><tr><th rowspan="2"></th><th rowspan="2">Storage period (days)</th><th colspan="3">E-OLE</th><th colspan="3">OLE</th></tr><tr><th>E-OLE (T<sub>1</sub>)</th><th>E-OLE (T<sub>2</sub>)</th><th>E-OLE (T<sub>3</sub>)</th><th>OLE1 %</th><th>OLE 2 %</th><th>OLE 3 %</th></tr></thead><tbody><tr><td>L*</td><td>1</td><td>0.84<sup>Bb</sup> ± 0.01</td><td>0.75<sup>Bd</sup> ± 0</td><td>0.68<sup>Be</sup> ± 0.01</td><td>0.90<sup>Aa</sup> ± 0.01</td><td>0.81<sup>Ac</sup> ± 0.01</td><td>0.76<sup>Ad</sup> ± 0.01</td></tr><tr><td></td><td>63</td><td>0.87<sup>Ab</sup> ± 0.01</td><td>0.79<sup>Ac</sup> ± 0.01</td><td>0.75<sup>Ad</sup> ± 0.01</td><td>0.89<sup>Aa</sup> ± 0.01</td><td>0.81<sup>Ac</sup> ± 0.01</td><td>0.73<sup>Be</sup> ± 0.02</td></tr><tr><td>a*</td><td>1</td><td>3.33<sup>Ac</sup> ± 0.18</td><td>4.18<sup>Bb</sup> ± 0.14</td><td>4.79<sup>Aa</sup> ± 0.16</td><td>1.60<sup>Be</sup> ± 0.07</td><td>2.00<sup>Bd</sup> ± 0.10</td><td>2.12<sup>Bd</sup> ± 0.09</td></tr><tr><td></td><td>63</td><td>2.66<sup>Bc</sup> ± 0.09</td><td>3.07<sup>Ab</sup> ± 0.09</td><td>3.75<sup>Ba</sup> ± 0.09</td><td>1.70<sup>Ac</sup> ± 0.10</td><td>2.36<sup>Ad</sup> ± 0.12</td><td>2.63<sup>Ac</sup> ± 0.23</td></tr><tr><td>b*</td><td>1</td><td>1.06<sup>Ba</sup> ± 0.02</td><td>1.06<sup>Aab</sup> ± 0.02</td><td>1.03<sup>Bbc</sup> ± 0.02</td><td>1.03<sup>Bbcd</sup> ± 0.02</td><td>1.02<sup>Bcd</sup> ± 0.02</td><td>1.00<sup>Bd</sup> ± 0.02</td></tr><tr><td></td><td>63</td><td>1.12<sup>Aab</sup> ± 0.03</td><td>1.09<sup>Abc</sup> ± 0.04</td><td>1.15<sup>Aa</sup> ± 0.04</td><td>1.06<sup>Acd</sup> ± 0.01</td><td>1.09<sup>Abc</sup> ± 0.04</td><td>1.03<sup>Ad</sup> ± 0.01</td></tr></tbody></table>

Different uppercase exponents (A-B) for the same concentration in different storage times indicate significant differences (P < 0.05); different lowercase exponents (a-c) at the same time for different concentrations indicate significant differences (P < 0.05).
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Elastic component (G') and viscous component G'' (loss modulus) of cheese enriched with E-OLE and OLE during storage (D1: day 1, D63: day 63). The concentrations are colour-coded (1, 2, and 3%), with the orange representing the E-OLE extract and the blue representing the OLE extract, where lighter shades correspond to lower concentrations and darker shades indicate higher concentrations.</figcaption></figure>

may be due to the structuring effect of ingredients like maltodextrin, which reduce free water content and strengthen the protein network. In contrast, the addition of OLE led to a reduction in G', suggesting a softening effect on the texture. This phenomenon may be attributed to polyphenol-protein interactions, which could promote the unfolding of proteins and relaxation of the network structure. As a result, hydrophobic regions that were initially buried may become more exposed to

the surrounding hydrophilic environment (Quan et al., 2019; Yan et al., 2023). These changes weaken the protein network and reduce elasticity, as seen in the lower G' values. Generally, there is an inverse relationship between moisture content and the G' and G'' of cheeses, so that the reduction of moisture in cheese samples causes their texture to be firmer and more solid, and therefore increases the G' and G'' (Azarashkan et al., 2022).
During storage, a gradual reduction in the storage modulus (G') was observed, reflecting a decline in the structural integrity of the cheese matrix. For the T3 formulation, G' measured at 20 °C showed a modest decrease, moving from 32.41 × 10³ Pa on the initial day to about 30.07 × 10³ Pa by day 63. In samples containing 3% OLE, this effect was more pronounced, as the G' dropped significantly from 87.58 × 10³ Pa to 42.74 × 10³ Pa over the same storage period, further confirming the destabilization of the gel network with time."

As shown in Fig. 4 both G' and G'' declined progressively with increasing temperature, ranging from 20 to 80 °C. A significant reduction occurred between 20 and 40 °C, while the decrease was more gradual between 40 and 80 °C. These results align with previous studies on cheese (Boubellouta & Dufour, 2012; Sharma et al., 2016; Udayarajan et al., 2005; Udyarajan et al., 2007). The sharp decrease in G' between 20 and 40 °C is likely due to the melting of the fat phase (Sharma et al., 2016; Yang et al., 2011), while the more moderate decrease between 40 and 80 °C may be linked to the mechanical strength of the cheese network and the thermal stability of the proteins (Sharma et al., 2016).

## 4. Conclusions

This study aimed to evaluate the impact of E-OLE on the quality of Cantal-type cheese during storage. The results showed that the addition of E-OLE, particularly at higher concentrations, improved the antioxidant properties and microbial stability of the cheese without negatively affecting its other quality attributes. E-OLE increased protein, dry matter, and antioxidant potential, while decreasing fat, chloride content, and total aerobic mesophilic counts in a concentration-dependent manner. Additionally, it influenced textural properties, enhancing hardness and cohesiveness while reducing elasticity.

## Ethical statement - studies in humans and animal

This study required no animal or human experiment.

## CRediT authorship contribution statement

**Ines Tarchi:** Writing – original draft, Software, Formal analysis, Data curation.
**Mohamed Bouaziz:** Resources, Funding acquisition, Conceptualization.
**ZuhaibF. Bhat:** Writing – review & editing, Visualization.
**Abderrahmane Aït-Kaddour:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

## Declaration of competing interest

No conflict of interest.

## Data availability

Data will be made available on request.

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