Aromatic Herbs of the Lamiaceae Family as Functional Ingredients in Wheat Tortilla

Abstract

The rationale for this research is the investigation of the potential health benefits as well as the antibacterial and antifungal properties of selected aromatic plants from the Lamiaceae family, which may lead to the development of improved functional foods. The present study investigated the effects of incorporating dried aromatic plants Thymus vulgaris, Thymus serpyllum, Thymus × citriodorus, Origanum vulgare and Rosmarinus officinalis at a concentration of 1% in refined wheat flour and wholemeal flour on the production of functional tortillas. Sensory analysis was employed to identify the optimal 1% addition, with the objective of achieving a favorable flavor and aroma profile. It was hypothesized that this addition would affect water activity, moisture, texture, color, antioxidant content and phenolic content, thereby enhancing the tortillas as a source of bioactive compounds. The results indicated that the type of flour used had a significant impact on the water activity of the tortillas, with wholemeal flour resulting in higher water activity than refined flour. The water activity ranged between 0.735 and 0.821, while the water content remained relatively stable. The water activity in whole-grain tortillas was significantly higher than that of refined flour tortillas, with a value exceeding 0.8, which makes them susceptible to mold growth and the production of mycotoxins. The sensory evaluations indicated that the enriched refined flour tortillas with common thyme (Thymus vulgaris), lemon thyme (Thymus × citriodorus) and rosemary (Rosmarinus officinalis) were rated highly; a similar result was observed for the whole-grain tortillas enriched with wild thyme (Thymus serpyllum) and lemon thyme. The whole-grain tortillas with rosemary were rated the highest of all the tortillas. The addition of aromatic plants increased the phenolic content and the antioxidant potential, depending on the flour type and the plant used. The addition of wild thyme and rosemary resulted in a significant increase in the phenolic content of wheat tortillas, while all enriched whole-grain tortillas exhibited a higher phenolic content than the control samples. The highest phenolic content in whole-grain tortillas was found in those fortified with rosemary, oregano and wild thyme. The highest antioxidant content was recorded in tortillas prepared with rosemary, irrespective of whether the flour used was refined or wholemeal. Fourteen phenolic compounds were tentatively identified in aromatic plants tested. The main phenolic compounds in Origanum vulgare were flavonoids. Rosmarinic acid was the dominant phenolic compound in rosemary and all thyme species, reaching the highest level in rosemary. Such high levels of rosmarinic acid may be responsible for the high antioxidant and total phenolic contents observed in rosemary extracts and also in tortillas when this plant is included in the recipe. The results of this study indicate that selected aromatic plants, particularly rosemary, have the potential to be utilized as functional ingredients in bakery products. By incorporating dried aromatic plants from the Lamiaceae family into wheat flour tortillas, food manufacturers can create products that not only taste better but also provide added health benefits. The use of selected herbs can improve the nutritional profile of tortillas by increasing antioxidant properties and, due to the properties of herbs, extend the shelf life of the product.

1. Introduction

Aromatic herbs from the mint family (Lamiaceae) are widely used for culinary and medicinal purposes. Most of them naturally occur in southern Europe, but due to their valuable properties, they are cultivated in other parts of the world. They are commonly used as culinary herbs, especially in meat dishes, soups and sauces. They perfectly complement the taste of vegetables, especially potatoes, tomatoes, eggplants and carrots. They are used in both fresh and dried forms [1,2].

The Lamiaceae family, known for its phytochemical richness, offers a wide range of bioactive compounds with significant health benefits. The biological activity of plants from Lamiaceae family is mainly related to phenolic compounds and essential oil constituents [3]. Studies have highlighted the antimicrobial and antioxidant activities of essential oils from the Lamiaceae plants, making them valuable in food preservation. Key compounds such as rosmarinic acid contribute to the antioxidant and anti-inflammatory properties [4] that are essential for food fortification. In addition to rosmarinic acid and other phenolic compounds, they are often rich in fragrant, volatile terpenes [5]. Such diverse phytochemicals provide sensory and nutritional benefits, making them ideal for functional food applications.

Herbs of the genus Thymus are a rich source of bioactive phenolic compounds and terpenoids, including phenolic monoterpenes, namely thymol and its isomer carvacrol, with strong antimicrobial properties [6,7]. Among the thyme species, Thymus vulgaris L. is the most widely cultivated and used. Vázquez-Sánchez et al. [8] compared the effectiveness of 19 essential oils against Staphylococcus aureus and demonstrated that thyme oil was the most effective, followed by lemongrass oil. Thyme essential oil also demonstrated a significant inhibitory effect on biofilm formation, although it was unable to completely remove the biofilm. The most commonly used essential oil obtained from Thymus vulgaris has been shown to exhibit strong antifungal activity against the species Aspergillus, Penicillium, Cladosporium, Rhizopus and Mucor [9]. Thyme essential oil has also been demonstrated to possess antiviral properties. Due to its antiviral properties, Thymus vulgaris has been used for centuries to treat a range of minor infections, including throat infections, coughs and other respiratory ailments [2]. Some studies have indicated that certain compounds in Thymus vulgaris may possess anti-parasitic properties [10]. Furthermore, reports indicate that thyme extract may possess hepatoprotective properties, which has been confirmed in animal studies [11]. Taking into account the discussed properties of Thymus vulgaris, especially antimicrobial and antioxidant properties, its use as a food additive seems advisable.

Although other Thymus species have many benefits, including antioxidant and antimicrobial properties, they are not widely used and have not received much attention in medicine or food science. Different Thymus species have unique aromatic properties that can diversify the flavor profile of food products. This can lead to the creation of novel and appealing flavors, potentially increasing consumer interest in functional foods. Thymus serpyllum L., also known as wild thyme, is native to the Mediterranean region of Europe and North Africa. It is a naturally occurring species in the mountains [12]. Research conducted by Shahar et al. [13] has demonstrated that T. serpyllum is rich in proteins, vitamins (A, C, E) and minerals. Like other Thymus species, it is rich in thymol, which has strong antimicrobial properties.

Thymus × citriodorus is not a botanical species. It is probably a hybrid of Thymus pulegioides and Thymus vulgaris [14]. It is cultivated in varieties with differently colored leaves. The plant is valued for its pleasant lemon scent and characteristic flavor [14]. Oliveira et al. [15] demonstrated that the essential oil extracted from Thymus × citrodorus effectively inhibits biofilm formation. In contrast to other species of thyme, where thymol and carvacrol are the dominant components in essential oils, geraniol has been reported as the major constituent [15,16]. Other compounds found in the essential oils of Thymus × citrodorus include nerol, the cis-isomer of geraniol and the aldehydes geranial and neral, which are two isomers of citral. These compounds give the plant its characteristic taste and intense lemon-like aroma. The water and alcohol extracts obtained from Thymus × citrodorus are rich in phenolic acids, flavonoids and triterpenic acids, which confer antioxidant, anti-inflammatory and cytoprotective effects [15].

Oregano (Origanum vulgare L.) is a culinary herb that, like thyme, is widely used in Mediterranean cuisine. In addition to its culinary uses, oregano is appreciated for its medicinal properties. The aerial parts of the plant are rich in phenolic compounds, including flavonoids and terpenoids, including phenolic monoterpenes—carvacrol and thymol—as predominant constituents in essential oil [17,18]. The composition of bioactive compounds in oregano renders it a valuable source of antioxidants and antimicrobial agents. Oregano essential oil is employed in a multitude of industries, including pharmaceuticals, food and aromatherapy. It is renowned for its multifaceted properties, which include antifungal, antioxidant, anti-inflammatory and anti-diabetic activity. A significant body of research has demonstrated the efficacy of oregano essential oil against food-borne pathogenic bacteria and bacteria responsible for food spoilage [19,20]. With regard to its antifungal activity, oregano essential oil has been demonstrated to be effective against a diverse range of micro-organisms, including Aspergillus [21], Fusarium [22], Botrytis [23] and Candida [24,25]. This potency is attributed to specific constituents within the essential oil, particularly carvacrol and thymol, which serve as their primary active compounds [17,18]. In the context of combating Fusarium, oregano oil has demonstrated strong fungicidal efficacy. Notably, studies have explored the combined antifungal activity of oregano oil with other essential oils, such as thyme oil. The combined application of these oils demonstrated enhanced inhibitory effects [22].

Rosmarinus officinalis L., commonly known as rosemary, is a perennial herb native to the Mediterranean region. The herb is known for its aromatic, evergreen leaves and distinctive fragrance, which is due to its high essential oil content. Rosemary extracts are known to have a strong antioxidant potential, mainly due to the presence of rosmarinic acid. After rosmarinic acid, carnosol and carnosic acid have been identified as the strongest antioxidants in rosemary [26]. In herbal medicine, rosemary is known for its antimicrobial, antidiabetic and anti-inflammatory properties [27]. Its potential is also being studied in terms of hepatoprotective and anti-obesity properties [28] and therapeutic potential in neurodegenerative diseases, including Alzheimer’s disease [29]. The antimicrobial activity of rosemary has been the subject of numerous studies, which have highlighted its effectiveness against a variety of pathogens, including bacteria, fungi and viruses. Studies have demonstrated the efficacy of this plant against common bacteria, including Salmonella sp., Shigella sp., Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli [30] and Listeria monocytogenes [31]. The essential oil of rosemary has been demonstrated to exhibit antifungal and antiaflatoxigenic activity against Aspergillus flavus [32], as well as antifungal activity against Alternaria alternata, Botrytis cinerea and Fusarium sp. [33] as well as Candida sp. and Penicillium sp. [34].

The growing consumer concern about the potential negative effects of synthetic chemicals in food has led to an increasing interest in the use of natural alternatives to synthetic additives. The high antioxidant and antimicrobial properties of selected herbs make them strong candidates as natural food antioxidants and preservatives. The use of herbs or extracts from thyme, oregano and rosemary in the preservation of bread offers a flavorful alternative to synthetic additives. Research from recent years, although scarce, seems to confirm the potential of spice plants from the Lamiaceae family in the preservation of bread. A series of studies conducted by Skendi et al. [35] have demonstrated that the addition of dried herbs such as thyme and oregano to bread recipes has the potential to inhibit the growth of both Penicillium sp. and Aspergillus sp. fungi. The inhibitory effect of the aromatic plants was most pronounced against Penicillium sp., with thyme identified as the most effective antifungal agent among the plants tested against both fungi. While herbs may not fully replace traditional preservatives, they can certainly enrich the preservation process, providing a more wholesome option for consumers who are wary of synthetic ingredients. Moreover, herbs are a source of antioxidants and may infuse bread with distinctive tastes and scents, thereby enhancing its flavor profile. Nevertheless, it is crucial to recognize that while herbs and herbal extracts can contribute to the prolongation of bread freshness, they may not achieve the same level of preservation efficacy as synthetic additives. The chemical composition of a specific plant can vary considerably due to the presence of different chemotypes within the same species, environmental factors and the time of harvest [36]. Furthermore, the storage conditions and the composition of the bread itself also exert a significant influence on its shelf life.

This research is motivated by the potential health benefits, as well as the antibacterial and antifungal properties of selected herbs, which could lead to the development of improved functional foods. The present study investigated the effect of adding dried aromatic plants from the Lamiaceae family at 1% in wheat flour on the preparation of functional tortillas. The amount of aromatic plants added was determined based on the results of sensory tests. From a sensory point of view, the 1% level on a flour basis was identified as the optimal addition, as it contributed to a favorable flavor and aroma, according to the evaluators. It is hypothesized that the addition of 1% dried aromatic herbs to the wheat tortilla recipe will affect its water activity, moisture content and texture. It may also affect color, antioxidant content and total phenolic content, making tortillas a source of bioactive compounds.

2. Materials and Methods

2.1. Preparation of Tortillas

The tortillas in our experiments were typically thin, round and made with wheat flour without the use of leavening agents. The recipe for wheat tortilla production (flatbread) consisted of wheat flour type 480 (40 g), hot water (18 g), canola oil (4 g), salt (0.6 g) and dried aromatic plants (0.4 g). The recipe for whole-grain tortilla production consisted of wheat flour type 480 (20 g) and wheat whole-grain flour type 1850 (20 g). The proportions of the remaining ingredients remained unchanged. The control tortillas did not contain added herbs. The ingredients were combined for five minutes and then allowed to rest for 20 min at room temperature, covered with a towel. Each piece of dough was rolled and then fried (for one to two minutes) in a medium-hot, dry pan. The herbs were cultivated under optimal growth conditions, harvested and then dried at a temperature of 35 ± 1 °C for 6 h. The dried herbs were traditionally crushed with a mortar and pestle, packed and stored in sterilized plastic bags until use. Wheat flour, canola oil and salt were purchased from a local market.

2.2. Water Activity Determination

Water activity (aw) in obtained tortillas was measured using Hygropalm AW-1 (Rotronic AG, Bassersdorf, Switzerland). The device was calibrated using three Rotronic-certified humidity standards in the form of aqueous salt solutions. Calibration and measurements were performed at a room temperature of 23 ± 0.25 °C. Each freshly prepared tortilla was cut into small pieces (circa 2 g), which were placed in a vial and left in the closed vial for around 30 min at room temperature. Then, the opened vial was placed on the plate of the apparatus and closed with AW-DIO probe. The measurements were conducted in six independent repetitions.

2.3. Water Content Determination

Water content was determined using an MA 50/1 Moisture Analyzer (Radwag, Radom, Poland) and expressed in %. The measurements were conducted in six independent repetitions.

2.4. Texture Analysis

Hardness analysis was conducted with the use of TA.XTplus texture analyzer (Stable Macro System, Godalming, UK) equipped with an HDP/BS probe (Warner-Bratzler blade). The hardness of the sample was expressed as the force required to completely break the continuity of the tested material, which was a fragment of a tortilla, 60 mm wide and 150 mm long. The material was cut several times at intervals of approximately 2–3 cm. The initial force applied was 5 g (0.049 N), the distance was 20 mm and the blade speed during cutting was 1 mm/s. The measurements were conducted in six independent repetitions.

2.5. Organoleptic Evaluation of Tortillas

The organoleptic evaluation of the tortillas was carried out in the sensory analysis laboratory with good lighting and ventilation by a panel of 10 participants. The participants were a group of 10 random consumers aged between 22 and 50. Each was given a set of tortilla samples, with a random number assigned to each sample to avoid bias. Each participant was given a form with a list of attributes to be evaluated. The tortillas were evaluated on the attributes of color, taste, aroma, shape and flexibility using a 5-point scale with corresponding significance coefficients, where 5 points corresponded to the best quality and 1 point to the worst. The final scores were presented on a 5-point scale, with a maximum score of 5 indicating a highly desirable quality and 1 indicating an unacceptable product.

2.6. Color Determination

The color of each tortilla was determined in the CIE L*a*b* system using a Konica Minolta CR-400 trichromatic reflectance colorimeter with Spectra Magic NX, Color Data Softwere CM-S100w (Konica Minolta Sensing INC., Osaka, Japan). The colorimeter was calibrated using a white tile. The measurements were conducted in six independent repetitions. The color of each tortilla was measured at several points. The L* parameter determines the brightness of the product and ranges from black (0) to white (100). The a* coefficient with values from −50 to +50 corresponds to the color from green (−50) to red (50). The b* coefficient with values from −50 to +50 corresponds to a color from blue (−50) to yellow (50). The total difference or distance between two colors on the CIELAB diagram is known as the Delta E (ΔE*). It is a good tool for assessing significant color changes of a given product in relation to the product considered as the standard. ΔE* value was calculated by using formula ΔE* = √(ΔL)² + (Δa)² + (Δb)². The calculated ΔE* value represents the total color difference between the tested tortilla and the control. This value indicates whether or not the color difference is perceptible to the human eye [37].

• ΔE* < 1: color differences are not obvious to the human eye.
• 1 < ΔE* < 3: the human eye can detect subtle color differences depending on the specific hue.
• ΔE* > 3: color differences are obvious to the human eye.

2.7. Lyophilization of Tortillas

The tortillas were cut into smaller pieces and frozen at −80 °C for 24 h. They were then freeze-dried in a DELTA 1-24 LSC Christ freeze dryer Martin Christ (Osterode am Harz, Germany) for 24 h. The condenser temperature was −50 °C, while the pressure was 0.7 millibar. The obtained freeze-dried tortillas were then ground to a fine powder in a knife mill XB-9103 MPM PRODUCT (Milanówek, Poland). Extracts were immediately prepared from the obtained powder for further analyses.

2.8. Preparation of Extracts from Freeze-Dried Tortillas

A total of 0.5 of each dried tortilla powder was added to 10 mL of 60% ethanol solution in a plastic tube, sealed and mixed for 2 h using Rotator Multi Bio RS-24 (Biosan, Riga, Latvia). The solid/solvent ratio equaled 1/20 (w/v). The mixture was centrifuged at 6000 rpm (4800 G) for 10 min using a Centurion Scientific K2015R centrifuge (Stoughton, UK). The supernatants were transferred to new plastic tubes and stored in a freezer at −80 °C until analysis.

2.9. Aromatic Plant Leaf Extraction

A total of 0.5 g of fresh plant tissue was ground in 10 mL of 60% ethanol solution using a mortar and transferred to a plastic tube. The solid/solvent ratio was 1/20 (w/v). The solution was then centrifuged at 6000 rpm (4800 G) for 10 min using a Centurion Scientific K2015R centrifuge (Stoughton, UK). Subsequently, all the supernatants were transferred to new plastic tubes and stored at a temperature of −80 °C until analysis.

2.10. Determination of Total Phenolic Content

The total phenolic content was quantified using the Folin–Ciocalteu method, as originally described by Singleton and Rossi [38]. To each extract (0.1 mL), 3.8 mL of distilled water and 0.1 mL of Folin–Ciocalteu reagent were added. The mixture was then incubated in the dark for 3 min. Subsequently, 1 mL of a 10% (w/v) Na₂CO₃ solution was added, and the mixture was incubated for 60 min at room temperature in the dark. The absorbance was then read at 765 nm using a UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan). The experiments were conducted in six replicates. A standard curve was generated using gallic acid solutions with concentrations ranging from 0.05 to 0.4 mg/mL. The total phenolic content in extracts obtained from fresh leaves was expressed as milligrams of gallic acid equivalent per gram of fresh weight (mg GAE/g FW). In the case of tortillas, the total phenolic content was expressed as milligrams of gallic acid equivalent per 100 g of dry matter (mg GAE/100 g DW).

2.11. Determination of Total Antioxidant Capacity

The total antioxidant capacity was quantified using the FRAP (Ferric-Reducing Antioxidant Power) method, as described by Vignoli et al. [39], with certain modifications. For each extract, 0.1 mL (extracts from fresh leaves) or 0.3 mL (extracts from dried tortilla powder) was combined with 4 mL of the FRAP solution. The FRAP solution was prepared by combining 2.5 mL of a 10 mM TPTZ solution (2,4,6-tripyridyl-1,3,5-triazine) in 40 mM HCl, 2.5 mL of a 20 mM FeCl₃ × 6H₂O solution and 25 mL of a 0.3 mM acetate buffer at pH 3.6 in a 100 mL flask. The flask was filled to a volume of 100 mL with distilled water, sealed and thoroughly mixed. The extracts were incubated with the FRAP solution in the dark for a period of 30 min. Subsequently, the absorbance was quantified at 593 nm using a UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan). The experiments were conducted in six replicates. A standard curve was constructed using Trolox solutions with concentrations ranging from 40 to 4000 μM. The total antioxidant capacity of extracts obtained from fresh leaves was expressed as micromoles of Trolox equivalents per gram of fresh weight (μmol TE/g FW). In the case of tortillas, the total antioxidant capacity was expressed as micromoles of Trolox equivalents per 100 g of dry matter (μmol TE/100 g DW).

2.12. UHPLC-DAD Analysis of Phenolic Compounds in Plant Leaf Extracts

Phenolic compounds were analyzed using UHPLC-DAD method according to procedure described in detail by Kulbat-Warycha et al. [40]. UHPLC + Diodex UltiMate 3000 liquid chromatographic system with a UHPLC pump, an autosampler, a column oven and a diode array detector with multiple wavelengths (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used. Separation was performed on an Accucore™ C18 column (2.1 × 150 mm, 2.6 μm particle size; Thermo Scientific, Pittsburgh, PA, USA) with a two-phase gradient system of 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase B. The mobile phase gradient used was as follows: 0–8 min, 1–5% B; 8–15 min, 5–8% B; 15–20 min, 8–10% B; 20–25 min, 10–15% B; 25–35 min, 15–20% B; 35–40 min, 20–25% B; 40–50 min, 25–90% B; 50–53 min, 90% B; 53–58 min, 90–1% B. Finally, the initial conditions were held for 7 min to allow re-equilibration of the column. The mobile phase flow rate was 0.35 mL/min, and the column temperature was 30 °C. The injection volume was 10 μL. The chromatograms were recorded at three different wavelengths (280, 320 and 365 nm). The identification of the compounds was confirmed by comparison of their UHPLC–DAD retention time and UV–vis profile to those of the phenolic standards. The concentrations of the identified compounds were calculated using calibration curves for analytical standards: procyanidin B1, procyanidin B2, caffeic acid, epicatechin, quercetin-3,7-di-O-glucoside, epicatechin gallate, quercetin 3-O-galactoside, quercetin 3-O-glucoside, luteolin 7-O-glucoside (cynaroside), quercetin, naringenin and apigenin. The content of rosmarinic acid and carnosic acid has been calculated on the basis of the calibration curve for chlorogenic acid.

2.13. Statistical Analysis

Results are presented as the mean of 6 independent replicates ± standard deviations (±SD). Statistical analysis of variance (ANOVA) was carried out. Significant differences between means were estimated by Duncan’s post hoc test for total phenolic content and antioxidant activity, while Tukey’s post hoc test was performed for water activity, water content, hardness and identified phenolic compounds. For all statistical analyses, p < 0.05 was considered as statistical significance. All statistical evaluations were performed using Statistica 13.0 software (StatSoft Inc., Palo Alto, CA, USA).

3. Results and Discussion

3.1. Physicochemical Characteristic and Organoleptic Assessment of Tortillas

Water activity determines the course of biological processes, particularly in the development of micro-organisms. There is a significant correlation between water activity and the shelf life of food products, influenced by microbial activity, enzyme activity and the rate of lipid and dye oxidation [41]. Microbiologists and food scientists have identified that water activity, rather than moisture content, exerts control over microbial responses, including sporulation and toxin production. Water activity is often considered a critical control point in risk analysis, as defined by the Hazard Analysis and Critical Control Points (HACCP) concept. The growth of micro-organisms is dependent on maintaining water activity at a specific level, which is optimal for a given micro-organism. Most bacteria require a water activity above 0.9, while yeast require a water activity of 0.8. A value of 0.7 is a limit for most molds, with a value of 0.8 being the limit for mycotoxin production [42]. The most dangerous molds producing mycotoxins include fungi of the genera Fusarium, Aspergillus and Penicillium. The final two are relatively resistant to low water activity in the environment [43].

Table 1. Physicochemical characteristics and organoleptic assessment of the tested tortillas enriched with various aromatic plants.

Type of Tortilla	Samples	Water Activity	Water Content (%)	Hardness (kg)	Organoleptic Assessment (Points)
wheat tortilla	Control	0.757 ± 0.027 b	21.22 ± 1.58 ab	8.34 ± 1.75 b	3.3 ± 0.4
	Origanum vulgare	0.794 ± 0.009 d	20.14 ± 1.26 a	11.12 ± 2.47 c	3.5 ± 0.3
	Thymus serpyllum	0.746 ± 0.004 a	21.78 ± 1.12 b	11.99 ± 2.19 c	3.6 ± 0.3
	Thymus vulgaris	0.767 ± 0.023 c	20.00 ± 1.36 a	7.54 ± 1.31 b	4.2 ± 0.2
	Thymus × citriodorus	0.735 ± 0.063 a	21.61 ± 2.06 b	11.17 ± 4.87 c	4.3 ± 0,2
	Rosmarinus officinalis	0.774 ± 0.010 c	21.42 ± 1.19 b	8.53 ± 2.57 b	4.3 ± 0.3
whole-grain tortilla	Control	0.808 ± 0.015 e	21.62 ± 0.98 b	7.12 ± 1.20 b	4.1 ± 0.2
	Origanum vulgare	0.809 ± 0.008 e	21.56 ± 1.58 b	7.31 ± 2.65 b	3.7 ± 0.2
	Thymus serpyllum	0.818 ± 0.009 f	22.01 ± 1.12 b	7.81 ± 1.06 b	4.1 ± 0.1
	Thymus vulgaris	0.806 ± 0.002 e	21.04 ± 1.13 ab	5.58 ± 0.60 a	3.8 ± 0.2
	Thymus × citriodorus	0.821 ± 0.023 f	22.36 ± 2.15 b	5.24 ± 0.24 a	4.3 ± 0.1
	Rosmarinus officinalis	0.817 ± 0.004 ef	21.54 ± 0.63 b	7.27 ± 3.07 b	4.6 ± 0.2

The values represent the means + SD from n = 6. Statistical analysis of variance (ANOVA, p < 0.05, post hoc Tukey’s test) was performed. Different letters, a, b, c, d, e and f, indicate that samples are significantly different. The letter “a” marks the lowest value. The values followed by the same lowercase letter (a–f) within the same column do not differ significantly according to Tukey’s HSD test at p < 0.05.

presents the water activity (aw) and water content [%], as well as the hardness [kg] and organoleptic assessment of all the tested tortillas.

Refined wheat flour tortillas typically exhibit a lower water activity compared to whole-grain tortillas. The inclusion of bran and germ in whole-grain flour increases the water-binding capacity, resulting in a higher water activity and water content. The results of the water activity measurements in tested tortillas confirm the initial assumptions indicating that the type of flour, rather than functional enrichment with aromatic herbs, significantly affected the water activity of tortillas. The water activity ranged from 0.735 to 0.821. The water activity in whole-grain tortillas was significantly higher than that of refined flour tortillas, with a value exceeding 0.8. Exceeding 0.8 in whole-grain tortillas not only makes them susceptible to mold growth but also to mycotoxin production. Refined wheat tortillas typically have a lower water content, which is confirmed by the results of our analyses. The absence of bran and germ, which have a high water retention capacity, results in these tortillas losing moisture more rapidly, leading to a drier product over time. Whole-grain tortillas have a higher water content due to the hygroscopic nature of the fiber present in the bran and germ. This higher water content helps maintain moisture, but it can also contribute to a shorter shelf life due to increased microbial activity [44,45,46]. Our observations are consistent with the literature data. A study conducted by Skendi et al. [47] examined the impact of adding aromatic herbs from the Lamiaceae family in a dried form into wheat flour during the bread-making process. All breads with added aromatic plants in dried form retained a higher moisture content after one day of storage compared to the control bread. As expected, the moisture content of all breads decreased after eight days of storage. However, the decrease in moisture content was less pronounced in the breads enriched with aromatic herbs. In addition to their antioxidant and antimicrobial effects, these herbs also improved the sensory quality of the bread. This includes improved flavor, aroma and overall acceptability, which can make the product more appealing to consumers and potentially reduce waste.

The average hardness value (8.34 N) of the control wheat tortillas was found to be comparable to that reported by Mashau et al. [48] for traditional tortillas made with maize flour (8.99 N). The average hardness of whole-grain tortillas in our experiments was slightly lower at 7.12 N. Due to the large standard deviations, no relationship was found between tortilla hardness and the addition of a given herb species. It is likely that the addition of selected herbs did not significantly affect the hardness of the tortillas.

The organoleptic evaluation of the tested tortillas ranged from 3.3 points for the control wheat tortillas to 4.6 points for the whole-grain tortillas enriched with Rosmarinus officinalis. The whole grain control tortillas scored higher in the organoleptic evaluation than the wheat control tortillas. High scores of more than 4 points were obtained for refined flour tortillas enriched with Thymus vulgaris, Thymus × citriodorus and Rosmarinus officinalis, which means that they are highly desirable. Similar values were obtained for control whole-grain tortillas and whole-grain tortillas enriched with Thymus serpyllum and Thymus × citriodorus. Tortillas enriched with Rosmarinus officinalis were rated as highly desirable, with the highest score of 4.6 points.

3.2. Color Determination

Color, along with flavor and aroma, is one of the fundamental attributes that influence the quality of food. The color of the tortillas in our experiments depended on the type of flour and herbs used. The color of each tortilla was determined using the CIE L*a*b* system. The results are presented in

Table 2. CIE L*a*b* color parameters of the tested tortillas enriched with various aromatic plants.

Type of Tortilla	Herbs	L*	a*	b*	ΔE*
wheat tortilla	Control	74.61 ± 1.62	−0.27 ± 0.11	18.68 ± 0.95	0.00
	Origanum vulgare	74.22 ± 1,09	−0.27 ± 0.10	15.05 ± 0.83	3.65
	Thymus serpyllum	76.57 ± 2.23	−0.53 ± 0.21	13.94 ± 1.21	5.14
	Thymus vulgaris	72.62 ± 4.95	−0.41 ± 0.33	18.29 ± 0.92	2.03
	Thymus × citriodorus	66.80 ± 1.14	−0.62 ± 0.18	17.65 ± 0.19	7.89
	Rosmarinus officinalis	78.10 ± 1.61	−0.94 ± 0.12	16.45 ± 0.97	4.20
whole-grain tortilla	Control	64.60 ± 1.26	5.31 ± 0.29	15.03 ± 0.64	0.00
	Origanum vulgare	61.64 ± 1.12	5.31 ± 0.25	15.39 ± 0.46	2.98
	Thymus serpyllum	65.64 ± 0.75	5.13 ± 0.15	15.85 ± 0.48	1.34
	Thymus vulgaris	57.07 ± 2.54	5.28 ± 1.36	15.39 ± 2.02	7.54
	Thymus × citriodorus	58.59 ± 1.98	5.80 ± 0.87	15.66 ± 0.71	6.06
	Rosmarinus officinalis	54.82 ± 0.77	7.26 ± 7.26	19.06 ± 0.16	10.76

The values represent the means + SD from n = 6. Color parameters are reported as L*, a* and b* values, indicating brightness, red-green and yellow-blue components, respectively. The ΔE* represents the total color difference between the tested tortilla and the control. ΔE* < 1: color differences are not obvious to the human eye; 1 < ΔE* < 3: the human eye can detect subtle color differences; ΔE* > 3: color differences are obvious to the human eye.

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The brightness (L*) of the surface of tested tortillas ranged from 54.82 to 78.10, whereas the redness (a*) and the yellowness (b*) ranged from −0.27 to 7.26 and 13.49 to 19.06, respectively. The results indicated that the whole-grain tortillas were darker and more red than the refined flour tortillas. The b* parameter showed less variability, especially for whole-grain tortillas. The calculated ΔE* value was higher than 1 for each tortilla enriched with aromatic plants, which means that the human eye can detect subtle color differences. The value of 3, which means that the color difference is obvious to the human eye, was exceeded in the wheat tortillas enriched with Origanum vulgare, Thymus serpyllum, Thymus × citriodorus and Rosmarinus officinalis and in the whole-grain tortillas enriched with Thymus vulgaris, Thymus × citriodorus and Rosmarinus officinalis. The highest ΔE* value of 10.76 was calculated for whole-grain tortilla with Rosmarinus officinalis, which meant that this variant differed the most from the control. Photos of tortillas enriched with aromatic plants are presented in Figure S1.

3.3. Phenolic Content and Antioxidant Properties of Aromatic Plants and Tortillas

Figure 1 and Figure 2 present the total content of phenolic compounds in selected aromatic plants and their antioxidant potential, respectively.

The results demonstrated that rosemary exhibited the highest phenolic content and the highest antioxidant capacity, with an average of 6.29 mg GAE/g in fresh leaf biomass, followed by Thymus × citriodorus (3.07 mg GAE/g) and Thymus vulgaris (2.88 mg GAE/g). Origanum vulgare had the lowest polyphenol content, with an average of 1.38 mg GAE/g. A comparable pattern was identified in the context of antioxidant potential. Fresh rosemary leaves contained an average of 140.70 μmol TE/g in fresh biomass, while leaves of Thymus × citriodorus and Thymus vulgaris contained 62.40 and 68.80 μmol TE/g, respectively. The lowest antioxidant potential was recorded in Origanum vulgare, with an average of 16.70 μmol TE/g. These findings suggest that rosemary may possess superior antioxidant properties compared to the other herbs tested. Recent studies have demonstrated the considerable variation in phenolic content and antioxidant activity among different culinary herbs, particularly within the Lamiaceae family. When compared to thyme and oregano, rosemary consistently demonstrated the highest total phenolic content, which correlates with high antioxidant activity, as confirmed in a comprehensive study by Vallverdú-Queralt et al. [49]. The very high antioxidant potential of rosemary and the slightly lower but still high potential of other plants from the Lamiaceae family make them valuable not only for culinary use but also for their potential applications in food preservation and nutraceutical formulations.

Taking into account the above, in our research, we added selected aromatic plants to the tortilla recipe in order to increase the content of phenolic compounds and the antioxidant potential of the product. Figure 3 and Figure 4 present the total content of phenolic compounds in wheat and whole-grain tortillas. Figure 5 and Figure 6 present the total antioxidant potential of wheat and whole-grain tortillas, respectively.

The results indicated that the enrichment of tortillas with various aromatic plants can improve the total phenolic content and antioxidant potential of the tortillas, depending on the flour type and plants used as a functional additive. In the case of wheat tortillas, the addition of wild thyme (Thymus serpyllum) and rosemary (Rosmarinus officinalis) resulted in a significant increase in the phenolic content. The average content of total phenolic compounds in tortillas with rosemary was the highest of all variants, with an average of 126.78 mg/100 g DW. The value was approximately twofold that of the control (71.08 mg/100 g DW). Tortillas enriched with Thymus serpyllum also exhibited a significantly higher phenolic content (96.85 mg/100 g DW) in comparison to the control, whereas tortillas enriched with oregano (Origanum vulgare), common thyme (Thymus vulgaris) and lemon thyme (Thymus × citriodorus) exhibited total phenolic contents that were not significantly different from the control. This suggests that although these herbs contribute polyphenols, their levels are comparable to those naturally present in wheat tortillas. In the case of whole-grain tortillas, all variants enriched with aromatic plants exhibited significantly higher content of phenolic compounds compared to the control. Interestingly, of all the wholemeal tortillas studied, the highest values were observed for those fortified with rosemary (131.77 mg/100 g DW), oregano (126.49 mg/100 g DW) and common thyme (106.61 mg/100 g DW).

The addition of the selected aromatic plants to different types of tortillas also resulted in a significant increase in antioxidant activity. It was found that a 1% addition of the tested plants in their dried form increased the total antioxidant activity of all the tortillas compared to the respective controls. The antioxidant activity of the tortillas ranged from an average of 56.27 μmol TE/100 g DW in the control wheat tortillas to 679.17 TE/100 g DW in the whole-grain tortillas with rosemary and 690.67 μmol TE/100 g DW in the wheat tortillas with rosemary. It is also noteworthy that the control tortillas made with wholemeal flour showed a twofold higher antioxidant activity (135.50 μmol TE/100 g DW) than the control tortillas made with refined wheat flour. This result is consistent with the assumption that wholemeal flour and products made with it are nutritionally superior to refined wheat flour, particularly in terms of antioxidants. The removal of the bran and germ during the milling process in the production of refined flour results in a significant loss of dietary fiber, vitamins, minerals and antioxidants, including not only phenolic compounds but also vitamin E and selenium [50]. It is important to remember that changes in the levels of antioxidants and individual polyphenols can be caused not only by the type of flour used but also by thermal processing. Different phenolic compounds in flour may respond differently to food processing. Thermal treatment leads to significant chemical changes in the phenolic profile. High temperatures can alter the biological activity of phenolics, sometimes reducing their antioxidant properties while potentially generating new bioactive compounds. In addition, thermal treatment can affect the bioavailability and functionality of these compounds, thus affecting the overall nutritional quality of the food [51]. Further evaluation is needed to understand how phenolic and flavonoid compounds present in flour and selected herbs are transformed during the production of final bakery products.

All of the above observations are in line with the literature data. Starowicz et al. [52] demonstrated that the incorporation of aromatic plants from the Lamiaceae family (sage, mint, oregano, thyme and rosemary) into oat- and buckwheat-based cookies significantly enhanced the formation of aroma compounds, enriched sensory properties and boosted antioxidant activity. This study demonstrated that selected herbs not only enhance the organoleptic qualities of the cookies but also increase their nutritional value by providing potent antioxidants. The antioxidant content of cookies with the addition of herbs from the Lamiaceae family was found to be significantly higher than that of control cookies. Similarly to our research, rosemary was the most promising in terms of increasing the antioxidant potential and forming a positive aroma profile. These findings indicate that herbs from the Lamiaceae family can be effectively employed as a functional ingredient in the development of healthier and more flavorful baked goods. Research conducted by Skendi et al. [47] investigated the impact of incorporating aromatic herbs from the Lamiaceae family on the quality, antioxidant activity and shelf life of traditional bread. The study found that the addition of dried herbs in an amount of 1% significantly increased the phenolic content in the bread, enhancing its overall antioxidant capacity. Furthermore, the authors claimed that adding essential oils instead of dried aromatic plants did not increase the total phenolic content of the breads. This suggests that phenolics in essential oils are more susceptible to thermal degradation. It seems that the plant matrix protects the phenolics from various reactions during the baking process.

It is well established that the antioxidant capacity of food products is closely linked to their free phenolic content. Studies in which bakery products have been fortified with plants from other botanical families confirm the assumption that fortification increases the content of phenolic compounds, resulting in an increase in the total antioxidant content of the product. Studies conducted by Świeca et al. [53] confirmed that fortification of wheat bread with quinoa leaves (Chenopodium quinoa Willd.) is an effective strategy to improve the antioxidant potential of bread. The final antioxidant potential of a product is not only the result of higher phenolic content but is also influenced by thermal processing and the interactions between phenolics, proteins and starch within the product. Results from other researchers confirm that fortifying bread with plant-based additives rich in phenolics effectively increases the antioxidant capacity of the product. The authors used a 1% addition of green tea powder to a pan bread recipe, which significantly increased the antioxidant potential of the final product, inhibited peroxidation during storage and maintained the baking quality of the bread [54].

The results and literature review indicate that the fortification of bakery products with plant-based additives rich in phenolic compounds is an effective method for enhancing their antioxidant capacity. Nevertheless, future research should investigate a wider variety of polyphenol-rich plants and their impact on both the nutritional and sensory properties of bakery products. Additionally, studies should examine the optimal concentrations for maintaining product quality while maximizing health benefits. Furthermore, research could explore the interactions of polyphenols with other food components during digestion. These efforts would provide a more comprehensive understanding of the potential benefits and challenges associated with fortifying bakery products, particularly with aromatic plants.

3.4. Profile and Concentrations of Phenolic Bioactive Compounds in Herbs

The HPLC results showed a medium variation in phenolic composition between the analyzed species, but the most pronounced differences were observed between the analyzed genus Oregano, Thymus and Rosmarinus. A total of 14 phenolic compounds were tentatively identified in the tested extracts. The results are presented in

Table 3. The content of individual phenolic compounds tentatively identified in aromatic plants (mg/100 g FW).

Phenolic Compound (mg/100 g FW)	RT (min)	Origanum vulgare	Thymus serpyllum	Thymus vulgaris	Thymus × citriodorus	Rosmarinus officinalis
Procyanidin B1	8.47	nd	nd	11.67 ± 0.51 b	9.79 ± 0.96 a	9.60 ± 0.52 a
Caffeic acid	12.04	0.91 ± 0.02 a	3.31 ± 0.14 b	5.36 ± 0.04 c	3.78 ± 0.31 b	3.50 ± 0.34 b
Procyanidin B2	17.20	10.16 ± 1.13 b	9.01 ± 0.58 a	29.99 ± 1.67 d	18.32 ± 0.21 c	nd
Epicatechin	21.46	nd	6.24 ± 0.62 a	8.12 ± 0.86 b	7.92 ± 0.67 b	nd
Quercetin-3,7-di-O-glucoside	26.00	4.10 ± 0.25 c	nd	3.84 ± 0.91 b	3.38 ± 0.03 a	nd
Epicatechin gallate	27.72	22.35 ± 0.98 a	nd	21.59 ± 1.37 a	nd	nd
Quercetin 3-O-galactoside	28.41	nd	18.89 ± 1.12 b	8.25 ± 0.47 a	21.83 ± 2.23 c	18.59 ± 1.25 b
Quercetin 3-O-glucoside	29.50	4.02 ± 0.11 b	5.34 ± 0.07 c	5.80 ± 0.61 c	1.93 ± 0.09 a	nd
Luteolin 7-O-glucoside (Cynaroside)	31.69	nd	32.04 ± 1.08 b	115.64 ± 8.25 d	92.30 ± 6.21 c	6.10 ± 0.17 a
Rosmarinic acid	37.14	nd	42.70 ± 5.78 a	125.30 ± 7.21 b	186.38 ± 6.32 c	416.07 ± 11.26 d
Quercetin	37.83	nd	1.59 ± 0.03 a	11.84 ± 1.01 c	6.07 ± 0.72 b	16.77 ± 1.01 d
Carnosic acid	43.10	nd	nd	nd	nd	8.86 ± 0.49
Naringenin	45.65	7.40 ± 0.26 c	5.25 ± 0.98 b	2.99 ± 0.76 a	5.35 ± 0.08 b	nd
Apigenin	46.38	13.05 ± 1.05 c	nd	4.97 ± 0.84 a	7.00 ± 0.94 b	nd

nd—not detected. Data are presented as means ± SD from n = 3. Statistical analysis of variance (ANOVA, p < 0.05, post hoc Tukey’s test) was performed. Different letters indicate that samples are significantly different within a given compound. The letter “a” marks the lowest value.

. The main phenolic compounds of Origanum vulgare were flavonoids, namely epicatechin gallate and apigenin. Rosmarinic acid, luteolin 7-O-glucoside (cynaroside) and quercetin 3-O-galactoside were detected as major phenolic compounds of all the Thymus species, whereas rosmarinic acid, quercetin and quercetin 3-O-galactoside were identified as major phenolic compounds of Rosmarinus officinalis. Rosmarinic acid was the dominant phenolic compound in rosemary and all thyme species, reaching the highest value with an average of 416.07 mg/100 g in fresh biomass of rosemary. Such a high content of rosmarinic acid may be responsible for the high content of antioxidants and total phenolic content in rosemary extracts as well as in tortillas with its addition. Our previous studies also confirm a relationship between the content of rosmarinic acid and the antioxidant activity of herbal extracts obtained from plants of the Lamiaceae family [40]. Carnosic acid, belonging to the diterpenoids, was the second acid identified after rosmarinic acid, but only present in rosemary, while caffeic acid was present in all the plants studied. According to Mena et al. [55], the diterpenoids carnosic acid and carnosol, together with rosmarinic acid and the flavonoids present in rosemary, are responsible for the bioactivity of rosemary extracts. Among flavonoids, epicatechin gallate was the most abundant in Origanum vulgare and Thymus vulgaris, while luteolin and quercetin derivatives were characteristic for all the Thymus species and Rosmarinus officinalis. Apigenin was detected in Origanum vulgare, Thymus vulgaris and Thymus × citriodorus, while naringenin was present in Origanum vulgare and all the Thymus species. Among anthocyanins, the most abundant was procyanidin B2, present in Origanum vulgare and all the Thymus species.

The results of our study are in agreement with the literature data. Rosmarinic acid is considered to be the major phenolic acid in plants of the Lamiaceae family and has been reported as the major phenolic compound in Thymus vulgaris [49], Thymus x citrodorus [56], Thymus serpyllum and many other species of thyme [57], as well as in Origanum vulgare [49,58] and Rosmarinus officinalis [59]. In the case of flavonoids, which are considered to be the group of phenolic compounds with strong health benefits, apigenin has been reported to be dominant in sage (Salvia officinalis), marjoram (Origanum majorana) and common thyme (Thymus vulgaris) [60], as well as in other species of thyme [57]. On the other hand, Jafari Khorsand et al. [58] reported luteolin as the most abundant flavonoid in Origanum vulgare. Similar to our results, naringenin has been reported but at lower concentrations in Origanum vulgare [58] and many thyme species [57,60] but not in rosemary. Among other flavonoids, quercetin and its derivatives, as well as luteolin derivatives, were reported by many authors in the studied species [49,56,57,60].

For future studies, it is important to carry out a deeper analysis of the phenolic compounds as well as an analysis of the volatile compounds composition of all the species studied. For this reason, it is necessary to extend our investigation for ultra-performance liquid chromatography with mass spectrometry (UHPLC-MS) to confirm all the tentatively identified compounds and gas chromatography (GC) as a good analytical technique for the quantitative determination of volatile compounds belonging to terpenoids.

4. Conclusions

In conclusion, the study demonstrated that the incorporation of aromatic herbs from the Lamiaceae family, including oregano, wild thyme, common thyme, lemon thyme and rosemary, can significantly enhance the antioxidant activity and phenolic content of wheat tortillas, thereby potentially extending their shelf life. This may be achieved through increased phenolic content, reduced lipid oxidation and antimicrobial effects. The results of the texture analysis and organoleptic assessments demonstrated that whole-grain tortillas exhibited superior sensory attributes, including an improved texture and a greater overall desirability among consumers. Furthermore, it can be assumed that whole-grain tortillas offer significant nutritional benefits due to their higher fiber and micronutrient content. The health benefits of whole-grain tortillas make them a valuable alternative, particularly in efforts to improve dietary fiber intake. In addition, the fortification of whole-grain tortillas with selected aromatic plants makes them an additional source of antioxidants. The addition of 1% dried rosemary, a rich source of rosmarinic acid, resulted in the highest antioxidant potential and total phenolic content among the tortilla variants. These findings highlight the potential of using natural herbs as functional ingredients in bakery products to improve their quality and longevity. In particular, the successful incorporation of rosemary highlights the potential for similar applications in a wide range of bakery products, opening up new avenues for innovation in food science. Ultimately, this study emphasizes the importance of harnessing natural resources to enhance food quality and sustainability on a global scale.