Application of Freeze-Dried Olive Leaf Powder in Cracker Formulation: Effects on Phenolics, Antioxidant Activity, Volatile Profile, and Sensory Quality

Abstract

Crackers and cookies have become the most widely consumed snacks due to their low production costs, long shelf life, and ability to deliver essential nutrients. Increasing consumer health consciousness has shifted preferences toward foods perceived as natural and beneficial. This shift elevates demand for cracker formulations with novel, health-promoting natural ingredients. This study examined the effects of incorporating freeze-dried olive leaf powder (FDOLP) into crackers on their physicochemical properties, phenolic and volatile compound profiles, antioxidant capacity, and sensory acceptability. Total polyphenol content of crackers was determined using the Folin–Ciocalteu method, while antioxidant capacity was evaluated by FRAP and DPPH assays. The UHPLC-ESI-HRMS analysis evaluated olive-derived compounds, including tyrosol, oleuropein derivatives, and pinoresinol 4-O-glucoside, present in olive leaf-enriched crackers. The characterisation of volatile compounds in crackers was performed using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME/GC-MS). A darker colour was observed in the enriched crackers compared to the control samples. Results demonstrated that increasing the proportion of FDOLP led to enhanced phenolic composition and antioxidant activity, as well as changes in the volatile profile of the crackers. Sensory analyses indicated that crackers enriched with moderate levels of FDOLP maintained acceptable overall sensory scores, suggesting a potential for the development of functional snacks. These findings demonstrate that olive leaves can be effectively utilised as a natural functional ingredient in cracker formulations to enhance their nutritional value and bioactive properties.

1. Introduction

In Mediterranean countries, olive trees (Olea europaea L.) have long been cultivated, primarily for olive oil production. In recent years, the global significance of the olive oil sector has substantially grown, driven by rising consumer demand linked to the oil’s nutritional value and numerous health benefits. However, both the pruning of olive trees and the processing of olive fruits generate large quantities of agricultural by-products that often remain underutilised [1]. The primary uses of olive oil production’s by-products are animal feed, organic fertilisers, and energy production in bioreactors [2]. Olive leaves represent one of the major by-products of olive cultivation, accounting for roughly 10% of the total olive harvest weight, with about 25 kg per tree generated during pruning [3]. There is growing awareness that this underused biomass is a potentially valuable, health-enhancing resource with considerable market prospects in the food and dietary industries if exploited efficiently [4]. They also represent one of the most abundant by-products of the olive oil industry and have the potential to provide a cost-effective source of valuable phenolic compounds [5]. In addition, olive leaf extracts are a rich natural source of bioactive compounds that exhibit a variety of health-promoting effects, including phenolics, terpenoids, polysaccharides, proteins and bioactive peptides, which can contribute to their biological activity and antioxidant potential, as well as antihypertensive, hypocholesterolemic, hypoglycemic, cardioprotective, and anti-inflammatory actions [6,7,8,9,10]. Recent studies have further highlighted the growing interest in plant-derived bioactive compounds, including antioxidant peptides, as functional ingredients with significant antioxidant potential with possible applications in food systems [11].

The phenolic content of olive leaves is significantly influenced by numerous factors, such as the olive cultivar, the harvest season, and the used leaf-drying method [12]. The major groups of phenolic compounds found in olive leaf extract include phenolic acids, phenolic alcohols, flavonoids, and secoiridoids. Key constituents are vanillic acid, caffeic acid, hydroxytyrosol, tyrosol, rutin, verbascoside, luteolin, quercetin, oleuropein, demethyloleuropein, and ligstroside [13]. Interest in these compounds is largely due to numerous studies demonstrating their health-promoting properties. In fact, olive leaves have a long history of use in traditional Mediterranean folk medicine [5,13].

While olive leaves and their extracts are commonly sold as dietary supplements, their application as food ingredients is still largely confined to research. They have been suggested as natural additives in food formulations because of their health-enhancing and functional properties, aiming to create functional foods or to extend the shelf life of products [5]. Also, recent studies have explored the use of olive leaf extract for food enrichment. Noori et al. [14] reported improved Lactobacillus casei survival in cheese without sensory deterioration, while Guglielmotti et al. [15] showed increased polyphenol content and antioxidant activity in beer, although high levels affected sensory properties [16].

According to Mustapha et al. [17], crackers are often regarded as a healthier alternative to more calorie-dense biscuits, prompting interest in incorporating novel ingredients to improve their nutritional profile and functional properties. Crackers are low-moisture bakery products formulated primarily from wheat flour and are among the most widely consumed ready-to-eat snack foods worldwide, owing to their long shelf life, affordability, and wide variety of flavours. However, their typical formulation results in high energy density but low nutritional value, which can be attributed to the high content of rapidly digestible carbohydrates and fats and the limited levels of dietary fibre [18]. Consequently, the growing demand for functional food products has encouraged the food industry to develop new bakery snacks enriched with bioactive compounds that exhibit antioxidant activity, aiming to improve nutritional quality and provide potential health benefits. Cedola et al. [16] showed that olive leaf extract, rich in health-promoting bioactive compounds, improved the nutritional quality of Southern Italy’s traditional cereal-based baked product, “taralli,” by increasing total polyphenol and flavonoid contents and antioxidant activity.

According to Medina et al. [19], olive leaves are being valued and marketed as premium herbal products or extracts due to their bioactive potential. In this context, olive leaf powder, owing to its high polyphenol content, represents a promising ingredient for producing functional, health-promoting crackers while simultaneously valorising this valuable agricultural by-product. The aim of this study was to evaluate the effects of adding olive leaf powder on the phenolic composition, antioxidant activity, volatile profile, and sensory acceptability of cracker formulations.

2. Materials and Methods

2.1. Preparation of Freeze-Dried Olive Leaf Powder

Olive leaves of the Oblica variety were picked from the local organic olive orchard in Šestanovac, Dalmatia, Croatia (43°27′31.6″ N 16°54′53.5″). Olive leaves were freeze-dried in LIO-20 FP (freeze-dry, FreezeDry GmbH, Bad Reichenhall, Germany) at a temperature of around −48 °C and pressure below 1 mbar. The leaves were ground into powder by the IKA MultiDrive control (IKA-Werke GmbH & Co. KG, Staufen, Germany) and sieved through a 500 µm RETSCH AS sieve (Retsch GmbH, Haan-Gruiten, Germany).

Freeze-dried olive leaf powder was characterised before incorporation into cracker formulations by determining moisture content, total phenolic content (TPC), FRAP, and DPPH activity.

2.2. Preparation of Crackers

For the purpose of this study, four cracker formulations were prepared: a control, and three formulations with varying amounts of freeze-dried olive leaf powder (FDOLP). All cracker samples were prepared according to the recipe listed in

Table 1. Formulations of cracker samples.

Ingredients	Cracker Composition (g)
	C-C	C-OL1	C-OL2	C-OL3
Oat flour	30	30	30	30
Almond flour	15	13.5	12	10.5
Rice flour	5	5	5	5
Spelt flour	10	10	10	10
Freeze-dried olive leaf powder	-	1.5	3	4.5
Sourdough	15	15	15	15
Butter	15	15	15	15
Extra-virgin olive oil	2	2	2	2
Salt	1	1	1	1
Sugar	1	1	1	1
Water	10	10	10	10

Composition of control (C-C) and freeze-dried olive leaf powder (FDOLP)-enriched cracker formulations (C-OL1, C-OL2, and C-OL3). C-C: control sample without FDOLP; C-OL1, C-OL2, and C-OL3: cracker samples enriched with 2.5%, 5.0%, and 7.5% FDOLP, respectively.

. Enriched crackers were prepared by partially replacing almond flour with FDOLP. The ingredients were weighed and mixed by hand, left at room temperature for 1 h, then stored in the refrigerator overnight for cold fermentation. The next day, the mixtures were left at room temperature for 20 min, then mixed again and rolled out to a thickness of approximately 3–4 mm. The crackers were cut into squares (15 pieces/dough) and baked in a ventilated electrical oven (Bosch HBA573EB0, Robert Bosch GmbH, Gerlingen, Germany) at 170 °C for 20 min (Figure 1). The crackers were cooled at room temperature, packed in zip plastic bags, and stored in a cool, dark place until further analysis.

2.3. Chemicals

For the determination of total phenolic content, the following reagents were used: Na₂CO₃, Folin–Ciocalteu reagent, and gallic acid (100% p.a., VWR BDH Chemicals, Rosny-sous-Bois, France). To determine antioxidant activity by the FRAP method, the following reagents were used: acetate buffer (300 mmol/L); hydrochloric acid solution (40 mM); TPTZ (2,4,6-tris(2-piridil)-s-triazinom) (Merck KGaA, Darmstadt, Germany); iron (III) chloride hexahydrate solution (20 mmol/L) (GRAM-MOL d.o.o., Zagreb, Croatia). For the antioxidant activity determined by the DPPH method, DPPH (2,2-diphenyl-1-picrylhydrazyl) solution (0.04 mg/mL) (Sigma-Aldrich, Steinheim, Germany) reagent was used. All chemicals and solvents used were of analytical or LC-MS grade (Merck KGaA, Darmstadt, Germany).

2.4. Physicochemical Evaluation of Crackers

Length (cm), width (cm), height (cm), spread ratio, and spread factor were determined for physical evaluation. Dimensions of crackers (cm) were measured using a vernier calliper, according to Kamal et al. [20].

Moisture content, water activity, pH, sodium chloride, and acidity were determined by chemical analysis. All measurements were conducted in triplicate, and all instrumental analyses (moisture content, water activity, and pH) were performed according to the manufacturers’ instruction. Moisture content was quantified using a KERN DAB 100-3 moisture analyser (KERN & SOHN GmbH, Balingen, Germany) by the thermogravimetric method. The sample was placed in the moisture analyser and heated using a halogen lamp. As the moisture evaporates, the difference between the initial and final mass is used to calculate the moisture content (%). An AquaLab Series 3 TE water activity metre (Decagon Devices, Pullman, WA, USA) was used to analyse the water activity of the cracker samples. The pH value was determined using a pH metre model FiveEasy F20 (Mettler Toledo AG, Langacher, Switzerland). At the same time, sodium chloride (salt) content was measured by the Mohr titration method, according to the procedure described by Delaš-Aždajić et al. [21].

2.5. Colour Analysis of Crackers

Colour is a particularly important indicator of product quality, and the product’s visual acceptability depends on it. Colour analysis of crackers was performed with a Konica Minolta Chroma Metre CR-5 (Konica Minolta, Inc., Tokyo, Japan) according to the manufacturer’s operating instructions. Measurements were performed using reflectance with an opening diameter of 30 mm. The results are presented in the CIELAB colour system, where L represents lightness (0 = black, 100 = white), a* shows us how similar the colour is to green or red (negative values = green, positive values = red), and b* describes how much the colour leans towards blue or yellow (negative values = blue, positive values = yellow). Measurements were conducted at room temperature in three repetitions. Cracker samples, as well as freeze-dried olive leaves, were ground before measurement.

2.6. Extraction of Crackers

Two types of extraction were performed on cracker samples, namely ultrasonic-assisted extraction (UAE) and microwave-assisted extraction (MAE), using a slightly modified method adapted from Veršić-Bratinčević et al. [22]. Briefly, 5 g of powdered samples and 50 mL of solvent (distilled water) were used for both extractions. The samples were subjected to ultrasonic extraction for an hour at 60 °C and 40 kHz, while microwave-assisted extraction was performed at 65 W for 15 min. Temperature during the microwave-assisted extraction reached a maximum of 50 °C.

2.7. Assessment of Total Phenolic Content

Total phenolic content in all extracts was determined by the Folin–Ciocalteu method as described by Singleton, with slight modification [23]. A total of 25 µL of sample, 1975 mL of distilled water, and 125 µL of Folin–Ciocalteu reagent were added to a cuvette. After 5 min, 375 µL of Na₂CO₃ was added, and the mixture was left in the dark for 2 h, after which the absorbance was measured at 765 nm. A spectrophotometer, SPECORD 200 Plus, Edition 2010 (Analytik Jena AG, Jena, Germany), was used to measure absorbance (UV/VIS). The results are expressed in mg GAE/100 g sample. Three measurements were performed for each sample.

2.8. Assessment of Antioxidant Capacity

The antioxidant capacity was measured using the DPPH (2,2-diphenyl-1-picryl-hydrazyl) method, as described by Brand-Williams et al. [24], and the FRAP (Ferric reducing antioxidant power) method, according to Benzie and Stain [25], both with slight modifications. For each sample and method, three measurements were performed.

For the DPPH method, 2 mL of DPPH solution is added to a cuvette, and its absorbance is measured at 517 nm (A₀). Then, 50 µL of the sample is added, and, after an hour, the absorbance (A_t) is measured using the spectrophotometer SPECORD 200 Plus, Edition 2010 (Analytik Jena AG, Jena, Germany). DPPH radical scavenging activity is calculated according to the formula:

$$\%inhibition={\displaystyle \frac{A_0-A_t}{A_0}} \times 100$$

where

A₀—is the absorbance of the blank.

A_t—is the absorbance of the samples.

For the FRAP method, it is necessary to prepare the FRAP reagent (acetate puffer: TPTZ: FeCl₃ = 10:1:1), then add it to the cuvette (3 mL) and measure its absorbance at 593 nm (A₀). A total of 100 µL of the sample was added to the same cuvette along with the previously measured FRAP reagent, and, after 4 min, absorbance was measured again. For the calculation of the results, the following formula is used:

A_k = A₄ − A₀

where

A_k—is the difference between the two measured absorbances.

A₄—is the absorbance of the sample and FRAP reagent after four minutes.

A₀—is the absorbance of the FRAP reagent.

A spectrophotometer, SPECORD 200 Plus, Edition 2010 (Analytik Jena AG, Jena, Germany), was used to measure absorbance (UV/VIS). The results are expressed in µM Trolox per L of extract, while using the extract for the calibration curve.

2.9. UHPLC-ESI-HRMS Analysis of Phenols

UHPLC-ESI-HRMS analysis was performed according to a previously published method with slight modifications [26,27]. The chemical composition of the extracted samples by MAE was analysed using an ExionLC UHPLC system (AB Sciex, Concord, ON, Canada) coupled with a Sciex TripleTOF 6600+ high-resolution quadrupole time-of-flight (QTOF) mass spectrometer (AB Sciex, Concord, ON, Canada). Chromatographic separation was carried out on a reversed-phase ACQUITY UPLC CSH Phenyl-Hexyl column (130 Å, 1.7 µm, 2.1 × 100 mm; Waters, Milford, MA, USA) maintained at 40 °C, using a flow rate of 0.4 mL/min. The mobile phases consisted of ultrapure water containing 0.1% formic acid (A), and acetonitrile containing 0.1% formic acid (B). The gradient elution programme applied for compound separation is presented in

Table 2. Gradient elution programme. A is aqueous and B is organic mobile phase.

Time (min)	A (%)	B (%)
0	100	0
5	100	0
20	0	100
26	0	100
26.1	100	0
27	100	0

.

Analyses were performed in positive electrospray ionisation mode (ESI+) using information-dependent acquisition (IDA) with collision-induced dissociation (CID) for MS/MS acquisition. Mass spectra were recorded in the m/z range 100–1000, while MS/MS spectra were acquired in the range 50–1000. Source parameters included a temperature of 300 °C, an ion spray voltage of 5.5 kV, and nitrogen as the curtain and collision gas. Instrument calibration was performed automatically prior to each run using an ESI Positive Calibration Solution 5600 (AB Sciex, Concord, ON, Canada). Data processing was carried out using ACD/MS Workbook Suite 2024.2.1 (ACD/Labs, Toronto, ON, Canada), and compound identification was based on accurate mass measurements, isotopic patterns, MS/MS fragmentation, and comparison with the ChEBI, PubChem, and ChemSpider databases.

2.10. Headspace Solid-Phase Microextraction

Volatile compounds were extracted by headspace solid-phase microextraction (HS-SPME) using a DVB/CAR/PDMS fibre (50/30 μm; Supelco Inc., Bellefonte, PA, USA). Prior to use, the fibre was conditioned according to the manufacturer’s instructions. For each extraction, 2 g of the ground sample was placed in a 15 mL glass vial containing 2 mL of deionised water and 1 g of sodium chloride, which was then sealed with a PTFE/silicone septum. The samples were equilibrated at 60 °C for 15 min under continuous stirring, followed by fibre exposure to the headspace for 45 min. Subsequently, the extracted analytes were thermally desorbed in the GC injector at 250 °C for 7 min. The extraction conditions were based on the method described by Wei et al. and were consistent with those employed in our previous studies [28].

2.11. Gas Chromatography–Mass Spectrometry

Volatile compounds were analysed using a gas chromatography–mass spectrometry (GC–MS) system consisting of an Agilent Technologies 8890 gas chromatograph coupled with a 5977E mass selective detector (Agilent Technologies, Inc., Santa Clara, CA, USA). Chromatographic separation was performed on an HP-5MS capillary column (5% phenyl-methylpolysiloxane; Agilent Technologies, Inc., Santa Clara, CA, USA). The oven temperature programme was initiated at 70 °C and maintained for 2 min, followed by a temperature increase to 200 °C at a rate of 3 °C/min and a final holding period of 15 min. Helium served as the carrier gas at a constant flow rate of 1.0 mL/min. Mass spectrometric detection was carried out in electron ionisation (EI) mode at 70 eV, scanning over a mass range of 30–350 m/z. The GC operating conditions were developed within the Department of Organic Chemistry, Faculty of Chemistry and Technology, University of Split, and have been reported previously [29]. Chromatographic data were processed using Enhanced Data Analysis software integrated into Agilent MSD ChemStation (version F.01.03.2357). Compound identification was achieved through comparison of the obtained mass spectra with those contained in the NIST 17 and Wiley 9 spectral libraries using the ChemStation library search algorithm. Additional confirmation was obtained by comparing experimentally determined retention indices (RI), calculated relative to a homologous series of n-alkanes (C8–C25; Fluka Chemie GmbH, Buchs, Switzerland), with the literature values [30]. Relative abundances of the identified compounds were estimated from GC peak areas using the normalisation method. The reported values represent the mean percentages obtained from triplicate HS-SPME/GC–MS analyses.

2.12. Sensory Analysis

A sensory evaluation was performed to assess consumer acceptability of the cracker samples. A total of 20 semi-trained panellists, mainly students and staff from the Faculty of Chemistry and Technology, participated in the evaluation of the sensory quality of crackers. Informed consent was obtained from all panellists before participation. The analysis was carried out according to the Ethics Committee of the Faculty of Chemistry and Technology University of Split, Croatia’s approval (Approval Number 20 May 2026, 2181-234-01-01-26-01). The panellists evaluated appearance, colour, aroma, taste, texture, and overall acceptability using a 5-point hedonic scale (1 = “dislike extremely” to 5 = “like extremely”). The samples were coded with 3-digit numbers and served to the panellists on white plates. To minimise sensory fatigue between tastings, panellists were provided with potable water for oral rinsing. The collected data were subsequently analysed to determine the acceptability of each sample among potential consumers.

2.13. Statistical Analysis

Statistical analysis was carried out using one-way analysis of variance (ANOVA), followed by Tukey’s HSD test, where differences at p < 0.05 were considered statistically significant. Statistical analyses were performed using JASP v.0.97.0 (JASP team, Amsterdam, The Netherlands, 2026) statistical software. All measurements were performed in triplicate and expressed as mean ± standard deviation.

3. Results and Discussion

3.1. Physicochemical Properties of Crackers

The results show that cracker dimensions (length, width, and height) were quite similar across all samples, although the spread ratio and spread factor tended to decrease slightly with higher levels of added FDOLP (

Table 3. Results of the crackers’ dimensions.

Sample	Length (cm)	Width (cm)	Height (cm)	Spread Ratio	Spread Factor
C-C	4.73 ± 0.12 a	3.70 ± 0.10 a	0.43 ± 0.06 a	11.03 ± 1.27 a	100 ± 0.00 a
C-OL1	4.70 ± 0.20 a	3.57 ± 0.15 a	0.40 ± 0.00 a	11.75 ± 0.50 a	107.44 ± 13.13 a
C-OL2	4.60 ± 0.17 a	3.57 ± 0.12 a	0.40 ± 0.00 a	11.50 ± 0.43 a	104.89 ± 8.66 a
C-OL3	4.50 ± 0.10 a	3.67 ± 0.15 a	0.40 ± 0.00 a	11.25 ± 0.25 a	102.89 ± 12.38 a

Data are expressed as mean ± standard deviation (n = 3). Different superscripts in each column indicate significant differences between formulations (p < 0.05). C-C—control cracker sample; C-OL1—crackers enriched with 2.5% of FDOLP; C-OL2—crackers enriched with 5.0% of FDOLP; C-OL3—crackers enriched with 7.5% of FDOLP.

). Nonetheless, these variations indicate that the inclusion of olive leaf powder does not markedly affect dough expansion or shape development during baking. Some of the differences could be attributed to the hand-cutting of dough before baking the crackers.

The mean chemical characteristics of the analysed crackers are summarised in

Table 4. Chemical parameters of crackers enriched with freeze-dried olive leaf powder.

Sample	pH	Water Activity (aw)	Moisture (%)	NaCl (%)
C-C	5.48 ± 0.01 a	0.231 ± 0.00 c	4.61 ± 0.07 c	1.94 ± 0.02 a
C-OL1	5.46 ± 0.01 b	0.282 ± 0.00 b	5.31 ± 0.06 a	1.86 ± 0.03 a
C-OL2	5.46 ± 0.01 b	0.283 ± 0.00 b	5.19 ± 0.03 a	1.50 ± 0.02 b
C-OL3	5.36 ± 0.00 c	0.320 ± 0.00 a	4.92 ± 0.04 b	1.39 ± 0.05 c

Data are expressed as mean ± standard deviation (n = 3). Different superscripts in each column indicate significant differences between formulations (p < 0.05), as determined by one-way ANOVA and Tukey’s HSD test. C-C—control cracker sample; C-OL1—crackers enriched with 2.5% of FDOLP; C-OL2—crackers enriched with 5.0% of FDOLP; C-OL3—crackers enriched with 7.5% of FDOLP. The moisture content of FDOLP was 6.4%.

. The results show that the pH decreased with increasing levels of freeze-dried olive leaf powder (FDOLP). In contrast, water activity increased, with the control sample (C-C) exhibiting the lowest a_w (0.231 ± 0.00) and C-OL3 the highest a_w value (0.320 ± 0.00). In line with the increase in water activity, moisture content was higher in the fortified crackers (4.92–5.31%) compared to the control (4.61%). Our results are consistent with those reported in other studies [31,32,33]. Salihu et al. [34] reported an increase in moisture content in samples enriched with fruit powder, which was attributed to the higher water-holding capacity of powdered cranberry and blueberry extracts compared with wheat flour. The addition of 7.5% olive leaf powder decreased the pH. This effect can be attributed to the presence of acidic bioactive compounds, particularly phenolic constituents, in olive leaves, which increase the overall acidity of cracker products. Additionally, salt content declined as the proportion of FDOLP increased, with the control cracker showing the highest NaCl level (1.94 ± 0.02%) and the C-OL3 sample the lowest (1.39 ± 0.05%). Incorporating FDOLP into cracker formulations can serve as a partial substitute for ingredients like flour, salt, and olive oil, while also improving the product’s nutritional profile [31].

3.2. Colour

Colourimetric analysis was conducted to determine the effect of adding freeze-dried olive leaf powder on the colour of each cracker formulation using a Konica Minolta CR-5 colourimeter (Konica Minolta, Inc., Tokyo, Japan), with results expressed in the CIELAB three-dimensional colour space (

Table 5. Results of colour analyses of crackers.

Sample	L*	a*	b*
C-C	68.12 ± 0.27 a	7.76 ± 0.04 a	29.37 ± 0.36 a
C-OL1	64.71 ± 0.26 b	6.58 ± 0.07 b	29.48 ± 0.13 a
C-OL2	63.86 ± 0.40 c	5.26 ± 0.03 d	27.83 ± 0.06 b
C-OL3	61.63 ± 0.18 d	5.71 ± 0.05 c	27.75 ± 0.22 b

Data are expressed as mean ± standard deviation (n = 3). Different superscripts in each column indicate significant differences between formulations (p < 0.05), as determined by one-way ANOVA and Tukey’s HSD test. C-C—control cracker sample; C-OL1—crackers enriched with 2.5% of FDOLP; C-OL2—crackers enriched with 5.0% of FDOLP; C-OL3—crackers enriched with 7.5% of FDOLP.

).

Instrumental colour analysis showed that the addition of freeze-dried olive leaf powder (FDOLP) significantly affected the colour parameters of crackers (p < 0.05). Lightness decreased with increasing freeze-dried olive leaf powder, indicating the development of a darker colour, which may be related to the natural pigments and phenolic compounds present in olive leaves, as well as the formation of brown pigments during baking. Similar results were reported for bakery products enriched with olive leaf powder and other plant-based ingredients [25].

The a* values decreased in all enriched samples compared to the control, indicating a shift towards greener tones due to the presence of chlorophyll pigments naturally occurring in olive leaves. In addition, thermal processing may have promoted Maillard reactions and chlorophyll degradation, contributing to the development of darker brownish-green shades during baking [35]. Cracker sample C-OL1 showed the highest b* value, whereas higher FDOLP concentrations resulted in lower yellowness values, probably due to the increasing dominance of darker pigments and brown reaction products formed during baking [13].

3.3. Total Phenol Content and Antioxidant Activity

Right before incorporation into cracker formulations, freeze-dried olive leaf powder was characterised by determining total phenolic content (TPC), FRAP, and DPPH activity. UAE and MAE extracts were analysed to compare extraction efficiency and antioxidant activity (

Table 6. Total phenols and antioxidant activity of freeze-dried olive leaf powder extracts.

Sample	Extraction Method	TPC (mg GAE/L)	FRAP (µM TE/L Extract)	DPPH (% Inhibition)
FDOLP	UAE	6619.33	2486.46	92.11
	MAE	3486.67	2760.72	89.64

).

The total phenolic content of the cracker extracts is illustrated in Figure 2. The results clearly demonstrate a positive relationship between the proportion of FDOLP in the formulation and the total phenolic content of the crackers. Accordingly, the control cracker sample (C-C) exhibited the lowest total phenolic content (154–163 mg GAE/100 g), whereas an increasing amount of the FDOLP in the formulation resulted in a proportional increase in total phenols, with the highest values recorded in sample C-OL3 (412 and 467 mg GAE/100 g). Similarly, adding FDOLP resulted in increased antioxidant capacity in the crackers (417–1818 µmol TE/100 g). These findings are consistent with previous studies evaluating the effects of incorporating powders derived from various parts of olives into bakery products [31,36,37].

According to Sahin et al. [38], the biological and antioxidant activities of polyphenols greatly differ according to their composition. The variability in phenolic concentrations may arise from differences in the raw material (e.g., cultivar, harvest time, and growing conditions) and the extraction process (e.g., method and parameters), which influence the extract’s chemical profile. Recent studies have reported that ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) are efficient green techniques for obtaining phenolic compounds from plant matrices, due to improved mass transfer and cell disruption mechanisms [39,40].

A comparison of the extraction methods showed that both techniques (UAE and MAE) were effective for extracting phenolic compounds. However, microwave-assisted extraction (MAE) generally yielded slightly higher total phenolic values than ultrasonic extraction (UE), indicating greater extraction efficiency of microwave energy in releasing phenolics from the plant matrix [7,41,42]. Furthermore, Li et al. [43] showed that different extraction methods significantly affect both the recovery of bioactive compounds and the measured antioxidant activity, despite differences in matrices and compound classes.

These results confirm that the inclusion of natural sources of bioactive compounds can be an effective strategy for enhancing the functional properties of bakery products by increasing their content of health-promoting constituents such as phenolic compounds and antioxidant activity (Figure 3 and Figure 4). These findings are consistent with previous studies investigating the incorporation of olive leaf extracts into bakery products [2,16], as well as the fortification of cookies with various herbal materials [44].

3.4. UHPLC-ESI-HRMS Analysis of Phenols

The UHPLC-ESI-HRMS analysis of samples extracted by MAE confirmed that the addition of olive leaf influenced the phenolic profile of the crackers. Compared with the control sample, which was prepared only with olive oil, the olive leaf-enriched crackers showed a broader and more diverse phenolic composition. The control cracker contained only tyrosol glucuronide, whereas the enriched samples contained several additional olive-derived compounds, including tyrosol, oleuropein derivatives, and pinoresinol 4-O-glucoside (Figure 5).

Tyrosol was detected in all crackers containing olive leaf, with an increase from C-OL1 to C-OL3. A similar trend was observed for tyrosol glucuronide, which was present in the control sample but increased with the addition of olive leaf. These results clearly indicate that olive leaf proportion increment contributed to the enrichment of the cracker with phenolic compounds.

Oleuropein, its aldehyde form, and pinoresinol 4-O-glucoside, were detected only in C-OL2 and C-OL3 samples. Among these compounds, oleuropein showed the clearest increase, particularly in C-OL3. This suggests that higher levels of olive leaf addition resulted in more pronounced enrichment of secoiridoids and lignans characteristic of olive leaf. Oleuropein aglycone was detected in all enriched crackers and showed a moderate increase with higher olive leaf content.

Overall, C-OL3 showed the richest phenolic profile, while C-OL1 exhibited enrichment with limited phenolic compounds. The results indicate that increasing the proportion of olive leaf in the cracker formulation enhanced the phenolic composition of the final product, with the strongest effect observed in the C-OL3 sample.

The phenolic profile obtained in the present study confirms that olive leaf can be considered an effective source of olive-derived phenolic compounds, which is relevant from both nutritional and technological perspectives [5,16,45,46,47,48,49]. The results are in agreement with previous studies reporting olive leaves as a particularly rich source of phenolic compounds, especially secoiridoids, simple phenols, flavonoids, and lignans, which are widely recognised for their antioxidant properties and potential health-promoting effects [4,6,45,50]. Quirantes-Piné et al. reported oleuropein as one of the major compounds in olive leaf extracts, together with other secoiridoid derivatives [51]. Various studies also described the presence of ligstroside, oleuroside, verbascoside, and several flavonoids in olive leaves [52,53]. Mir-Cerdà et al. reported more than 60 tentatively annotated phenolic compounds in olive leaf extracts obtained using natural deep eutectic solvents, with tyrosol derivatives followed by flavonoids described among the most abundant phenolic groups, with oleuropein, oleuropein aglycone and glucoside forming one of the most abundant [54]. Therefore, the presence of oleuropein, oleuropein aldehyde form, and oleuropein aglycone in the enriched crackers reflects their transfer from olive leaves into the cracker matrix and demonstrates that these characteristic olive phenolics can be retained, at least partially, after baking.

The differences between C-OL1, C-OL2, and C-OL3 indicate that the final phenolic profile depended on both the amount of olive leaf added and the behaviour of individual compounds during baking. Oleuropein and related secoiridoids are known to be affected by processing conditions, including drying, storage, and elevated temperature. Feng et al. showed that drying and storage conditions can significantly influence the phenolic composition of olive leaves [55], while Klisović et al. reported decreases in oleuropein and related secoiridoids during the heating of olive oil at 180–220 °C [56]. In the present study, the detection of oleuropein-related compounds, especially in C-OL2 and C-OL3, indicates that these compounds remained detectable after baking despite thermal processing. From a technological perspective, phenolic compounds are known to possess antioxidant activity and may contribute to limiting oxidative processes in food systems, thereby helping to maintain product quality during storage [4,5,16,49,57]. Therefore, the increased abundance of these compounds in crackers enriched with higher levels of FDOLP suggests an enhancement of the functional value of the final product by increasing its content of olive-derived bioactive compounds.

3.5. Volatile Compounds’ Composition

The volatile compounds of FDOLP as well as cracker samples are listed in

Table 7. Volatile compounds (expressed as percentage of the total area) identified in freeze-dried olive leaf powder (FDOLP) and cracker samples.

No	RI	Volatile Compound	FDOLP (AV ± SD)	C-C (AV ± SD)	C-OL1 (AV ± SD)	C-OL2 (AV ± SD)	C-OL3 (AV ± SD)	Odour Description *
		Aldehydes
1	698	pentanal	nd	5.91 ± 0.21	nd	nd	nd	fermented, bready, fruity, nutty
2	754	(E)-2-pentenal	3.26 ± 0.12	2.51 ± 0.13	nd	nd	nd	apple, fruity, pungent
3	800	hexanal	4.14 ± 0.06	20.67 ± 0.32	6.40 ± 0.05	5.40 ± 0.14	6.10 ± 0.11	fresh, green, fatty, grass, fruity
4	854	(E)-2-hexenal	0.68 ± 0.05	nd	nd	nd	nd	green, leaf
5	909	heptanal	0.84 ± 0.05	0.98 ± 0.07	0.64 ± 0.07	0.46 ± 0.09	0.56 ± 0.04	fat, citrus, rancid
6	956	(E)-2-heptenal	0.52 ± 0.03	0.75 ± 0.05	2.18 ± 0.14	4.96 ± 0.17	6.82 ± 0.30	green, fatty
7	961	benzaldehyde	0.65 ± 0.06	2.05 ± 0.07	2.62 ± 0.14	4.34 ± 0.24	3.89 ± 0.16	almond, burnt sugar
8	1006	octanal	nd	1.85 ± 0.03	3.49 ± 0.15	2.69 ± 0.14	2.51 ± 0.31	fat, soap, lemon, green
9	1007	(E,E)-2,4-heptadienal	29.33 ± 0.36	nd	nd	nd	nd	nutty, fatty, hay
10	1049	phenylacetaldehyde	nd	1.27 ± 0.03	1.64 ± 0.08	4.47 ± 0.24	2.91 ± 0.02	honey, floral rose, sweet
11	1056	(E)-2-octenal	nd	1.22 ± 0.04	0.46 ± 0.06	1.91 ± 0.15	1.71 ± 0.03	fresh, leafy, fatty
12	1467	2-dodecenal	2.49 ± 0.55	nd	nd	nd	nd	herbal
13	1102	nonanal	nd	3.52 ± 0.12	9.87 ± 0.15	7.72 ± 0.25	6.51 ± 0.25	fat, citrus, green
14	1162	(E)-2-nonenal	nd	nd	0.93 ± 0.11	0.29 ± 0.06	0.59 ± 0.06	fatty
15	1200	decanal	0.44 ± 0.04	2.49 ± 0.13	5.37 ± 0.11	2.12 ± 0.04	2.58 ± 0.08	soap, orange peel, tallow
16	1265	(E)-2-decenal	0.29 ± 0.07	nd	1.54 ± 0.12	1.04 ± 0.09	1.10 ± 0.06	tallow
17	1317	(E,E)-2, 4-decadienal	nd	nd	nd	1.75 ± 0.09	0.92 ± 0.07	fatty, fried, citrus
		Alcohols
18	967	1-heptanol	nd	0.66 ± 0.09	nd	nd	nd	chemical, green
19	986	1-octen-3-ol	nd	0.83 ± 0.08	1.13 ± 0.03	2.61 ± 0.14	4.24 ± 0.15	earthy, green
20	1015	2-ethyl-1-hexanol	nd	0.80 ± 0.09	0.90 ± 0.03	0.81 ± 0.10	1.09 ± 0.10	green, vegetable
21	1033	benzyl alcohol	0.35 ± 0.01	3.04 ± 0.07	3.78 ± 0.03	3.53 ± 0.21	2.74 ± 0.10	sweet, floral
22	1066	(E)-2-octen-1-ol	nd	nd	0.56 ± 0.09	1.08 ± 0.05	1.11 ± 0.05	green, citrus
23	1073	1-octanol	nd	1.80 ± 0.04	3.60 ± 0.02	2.86 ± 0.07	2.66 ± 0.12	herbal, fatty, green
24	1139	phenylethyl alcohol	2.09 ± 0.03	1.70 ± 0.09	4.08 ± 0.08	4.32 ± 0.04	3.07 ± 0.06	floral
		Ketones
25	678	1-penten-3-one	5.77 ± 0.24	nd	nd	nd	nd	pungent, peppery, mustard
26	987	6-methyl-5-hepten-2-one	4.43 ± 0.19	nd	nd	nd	nd	earthy, fruity
27	1081	3,5-octadien-2-one	7.58 ± 0.20	nd	nd	nd	nd	fruity, fatty, mushroom
28	1090	2-nonanone	nd	nd	0.52 ± 0.04	0.69 ± 0.07	0.15 ± 0.03	floral, fatty
29	1107	6-methyl-3,5-heptadien-2-one	2.62 ± 0.08	nd	nd	nd	nd	cinnamon, spicy, woody, sweet
30	1294	2-undecanone	nd	0.72 ± 0.03	1.18 ± 4.04	1.89 ± 0.16	1.90 ± 0.19	fruity, fresh
31	1494	2-tridecanone	nd	nd	0.71 ± 90.01	1.83 ± 0.09	1.02 ± 0.08	fatty, waxy, herbal, earthy
		Acids
32	602	acetic acid	2.34 ± 0.11	8.76 ± 0.94	13.98 ± 0.68	11.68 ± 0.34	14.88 ± 0.24	alcoholic
33	981	hexanoic acid	nd	2.21 ± 0.10	2.66 ± 0.11	3.16 ± 0.13	2.18 ± 0.06	rancid, sweaty
34	1191	octanoic acid	nd	0.90 ± 0.07	3.98 ± 0.04	1.02 ± 0.04	0.14 ± 0.04	sweat, cheese
35	1276	nonanoic acid	nd	1.54 ± 0.07	1.01 ± 0.03	1.55 ± 0.22	1.06 ± 0.07	green, fat
36	1964	hexadecanoic acid	nd	5.04 ± 0.08	nd	nd	nd	waxy
		Furans
37	996	2-pentylfuran	nd	0.85 ± 0.07	nd	nd	0.72 ± 0.07	green, earthy, beany
		Esters
38	1197	methyl salicylate	0.56 ± 0.01	nd	nd	nd	nd	mint
		Terpens
39	1021	p-cymene	0.53 ± 0.09	nd	nd	nd	nd	citrus, solvent
40	1030	limonene	1.65 ± 0.05	0.76 ± 0.03	0.71 ± 0.03	0.72 ± 0.07	0.80 ± 0.02	citrus
41	1214	β-cyclocitral	0.85 ± 0.09	nd	nd	nd	nd	tropical, saffron, herbal
42	1273	α-citral	0.88 ± 0.06	nd	nd	nd	nd	citrus
43	1434	neryl acetone	4.36 ± 0.18	nd	nd	nd	nd	fatty
44	1469	β-ionone	1.55 ± 0.09	nd	nd	nd	nd	violet, flower, raspberry
		Others
45	898	oxime-, methoxy-phenyl	nd	1.74 ± 0.07	1.32 ± 0.04	1.55 ± 0.07	2.35 ± 0.14	fresh shrimp and crabs
46	968	3-ethenylpyridine	0.41 ± 0.03	nd	nd	nd	3.92 ± 0.26	tobacco, caramelic

RI = retention indices relative to C₉–C₂₅ alkanes; Av = average area percentage composition for three replicates; SD = standard deviation of the area percentages for three replicates; * = odour description taken from The Good Scents Company Information System and/or Flavornet online databases [58,59]; nd = not detected in sample; C-C—control cracker sample; C-OL1—crackers enriched with 2.5% of FDOLP; C-OL2—crackers enriched with 5.0% of FDOLP; C-OL3—crackers enriched with 7.5% of FDOLP.

. A total of 46 headspace volatile compounds were identified in FDOLP as well as in the cracker samples. Identified volatiles belong to the following chemical classes: aldehydes (17), alcohols (7), ketones (7), acids (5), furans (1), esters (1), terpenes (6), and others (2). Among the identified volatiles, aldehydes were numerous and most abundant.

Among identified volatiles in FDOLP, aldehydes were the major compounds, with (E,E)-2,4-heptadienal as the most dominant (29.33 ± 0.36), with sensory perception as nutty, fatty, and hay. Other identified aldehydes characterised by herbal, green, leaf, and grass descriptions were hexanal (4.14 ± 0.06), (E)-2-pentenal (3.26 ± 0.12), (E)-2-hexenal (0.68 ± 0.05), and 2-dodecenal (2.49 ± 0.55). Most of them have been identified in previous studies in several olive cultivars [60]. Among alcohols, benzyl alcohol and phenylethyl alcohol were identified in FDOLP, with sensory descriptions as sweet and floral. Ester, methyl salicylate was found only in the volatile fraction of FDOLP with a typical mint flavour and was previously identified in the olive leaf volatile fraction [60]. Wang et al. [61] found methyl salicylate in the volatile fraction of teas with different degrees of fermentation. The major volatile compounds for distinguishing volatiles between the FDOLP and analysed crackers could be attributed to terpenes, considering that terpene compounds are secondary plant metabolites and are typical of plant species. As can be seen from Table 7, neryl acetone (4.36 ± 0.18), limonene (1.65 ± 0.05), and β-ionone (1.55 ± 0.09) are the main terpenes found in FDOLP. Among them, only limonene was identified in the control cracker, as well as in crackers enriched with different amounts of FDOLP. Considering that all crackers formulations consisted of extra-virgin olive oil, a possible source of limonene, besides FDOLP, could be attributed to it.

In addition, some ketones identified in the FDOLP, such as 1-penten-3-one, have been detected in studies of olives and olive oil. According to Gomes da Silva et al. [62], 1-penten-3-one is positively correlated with bitter and pungent taste. 3-ethenylpyridine, characterised by a tobacco and caramel description, was found in both FDOLP and cracker, with the highest proportion in FDOLP (C-OL3). This compound was found in the study performed by Campeol et al. [60] in volatile fractions from three cultivars of Olea europaea L. in Italy.

Considering the volatile compounds in cracker samples, aldehydes were the most abundant group, followed by alcohols and acids. In all cracker samples, the main volatile compound from the aldehyde group was hexanal, ranging from 5.40% to 20.67%. A similar finding was reported in a study by Man et al. [63] on the volatiles of biscuits containing roasted flaxseed flour. In the control crackers, hexanal was identified as the predominant volatile compound contributing to the overall volatile profile. However, its relative abundance decreased in FDOLP-enriched samples, suggesting that the incorporation of freeze-dried olive leaf powder may inhibit hexanal formation. Since hexanal and other aldehydes such as pentanal, (E)-2-heptanal, and nonanal are widely recognised as indicators of the oxidative process, especially in baked products such as biscuits and crackers, lipid oxidation is a major contributor to quality deterioration. This reduction suggests a potentially positive effect of FDOLP on the oxidative stability of the product, as shown in Table 7. This effect may be attributed to the higher content of phenolic compounds, which exhibit strong antioxidant activity. Phenolics derived from olive leaves are known for their ability to neutralise free radicals, thereby slowing the oxidation process [26]. Similar findings were reported in study performed by Starowicz et al. [44], investigating the volatiles in cookies enriched by Lamiaceae herbs. Furthermore, de Gennaro et al. [33] reported that the incorporation of olive cake powder into breadstick formulations increased the total phenolic content and antioxidant activity, which may contribute to reducing the concentration of certain aldehydes associated with lipid oxidation. In addition, similar findings were reported in a study performed by Difonzo et al. [64], investigating the influence of incorporation of olive leaf extract on reducing the lipid oxidation into baked snacks. According to Papageorgiou et al. [65], most of the identified carbonyl compounds have already been documented in bakery products. Their occurrence is mainly linked to the lipid fraction of the raw ingredients and to Maillard and Strecker degradation processes. Some of these compounds arise from the autoxidation of unsaturated fatty acids. Specifically, (E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal, (E,E)-2,4-heptadienal, and (E,E)-2,4-decadienal are generated through the oxidation of linoleic and linolenic acids, whereas octanal and nonanal are formed via the oxidation of oleic acid. In baked products such as biscuits and crackers, lipid oxidation is a major contributor to quality deterioration. Aldehydes such as pentanal, hexanal, (E)-2-heptanal, and nonanal are widely recognised as indicators of this oxidative process [33].

Furthermore, alcohols were the most common volatiles in all cracker samples and were associated with green, earthy, sweet, floral, and fatty sensory notes. Among acids, only acetic acid was identified in both FDOLP and in all cracker samples. Other volatile acids identified in the cracker samples listed in Table 7 contribute to the aroma profile of crackers, described as having fatty, sweaty, and cheesy flavours.

3.6. Sensory Analysis of Crackers

Sensory properties, nutritional value, and health-related benefits mainly drive consumer acceptance of food products. According to Salihu et al. [34], the addition of plant extracts to bakery products represents a potential strategy for improving their nutritional and functional quality through phytochemical bioactivity and sensory acceptability. Since volatile compounds are key contributors to aroma and flavour perception through ortho- and retronasal olfaction, their influence on sensory properties is highly complex. Volatile compounds alone cannot fully explain flavour, which emerges from interactions among multiple volatile compounds as well as non-volatile taste components. This perception is further shaped by the food matrix and by multisensory integration involving texture, temperature, trigeminal sensations, and cognitive expectations, making relationships between chemical composition and sensory data often complex and not directly predictive [66,67].

Figure 6 presents the hedonic sensory evaluation results for the control cracker sample (C-C) and crackers enriched with freeze-dried olive leaf powder (C-OL1, C-OL2, C-OL3). Panellists evaluated appearance, colour, odour, taste, texture, and overall acceptability using a five-point hedonic scale. The sensory data indicate distinct differences between the control sample (C-C) and fortified crackers.

Appearance and colour were similarly liked for the C-C sample, C-OL1, and C-OL2, with C-OL3 scoring lower. Aroma scores declined slightly as OLP increased, suggesting lower acceptability at higher levels. The most notable differences were in taste, with the C-C and C-OL2 samples rated highest and the C-OL3 sample lowest due to its increased bitterness. Earlier studies have shown that incorporating high levels of phenolic compounds, such as those derived from olive products, can lead to a bitter taste in the final product [31].

Texture scores were similar across all samples. Overall acceptability slightly decreased with higher OLP content, indicating that lower additions maintained sensory quality, while higher levels negatively affected consumer liking. These results confirm that lower levels of olive leaf powder in the formulation do not compromise sensory quality, whereas higher additions negatively affect product acceptability. Bitterness from phenolic compounds such as oleuropein is known to limit the use of olive leaf extracts in foods, as the intense bitter taste becomes perceptible at higher concentrations [68]. Similar results were reported in the study by Cedola et al. [16], which evaluated the sensory acceptability of “taralli” enriched with olive leaf extract. In contrast, other studies have shown that increasing the concentration of berry powder increases consumer acceptability across all evaluated attributes, particularly taste and mouthfeel [34,69].

4. Conclusions

This study proposes a potential valorisation strategy for olive oil by-products. Specifically, it evaluates the impact of incorporating freeze-dried olive leaf powder into cracker formulations by comparing the resulting products with a control for sensory attributes, volatile profile, and bioactive compounds. The addition of freeze-dried olive leaf powder to cracker formulations slightly affects their physicochemical properties. Total phenolic content and antioxidant activity of cracker extracts increased proportionally with higher olive leaf powder levels. Both microwave and ultrasonic extraction methods were effective, with microwave-assisted extracts showing slightly higher phenolic content and antioxidant activity. Lower addition levels (2.5–5%) were sensory acceptable, while higher levels reduced sensory scores, particularly taste, due to pronounced bitterness. The addition of FDOLP to the cracker formulation resulted in a reduction in aldehydic compounds, particularly hexanal, which are indicative markers of lipid oxidation, as well as a decrease in Maillard reaction-derived products, suggesting a potential contribution to enhanced oxidative stability and extended shelf life of the final product. The results showed that the addition of freeze-dried olive leaf powder increased the total polyphenolic profile and the antioxidant activity of the crackers. Tyrosol, oleuropein derivatives, and pinoresinol 4-O-glucoside were detected, thereby demonstrating that olive leaf powder has significant potential for the development of functional bakery products due to its high bioactive phenolic content, though further studies are needed to determine optimal use. Future research should focus on evaluating the product’s nutritional properties, monitoring lipid oxidation, and conducting microbial analyses throughout storage to provide valuable insights into its stability and shelf life.