Butia odorata was harvested in 2022 in Rio Grande, RS, Brazil (S 32°10.097' W052°24.595'). The beeswax used to prepare the oleogels was purchased from a local market in the city of Pelotas, RS, Brazil. The reagents used were of analytical grade.

Oil was extracted in a Soxhlet extractor using 20 g of Butia odorata seed and 250 mL of hexane p.a. as a solvent. The solvent was evaporated on a rotary evaporator (Büchi®, Rotavapor® RII; Merck, Darmstadt, Germany). The oil extraction yield was calculated gravimetrically based on the initial mass of seeds.

The fatty acid profile was determined using the gas chromatography (GC) method. The equipment is coupled with a GC flame ionization detector (FID) (Clarus 500; PerkinElmer®, Shelton, WA, USA) equipped with a capillary column (30 m×0.25 mm, 0.25 mm Elite-1; PerkinElmer, Waltham, MA, USA). An aliquot of 0.1 mL of Butia seed oil was added to 2 mL of hexane and 2 mL of potassium hydroxide. The sample was in an ultrasonic bath for 5 min and then centrifuged for 2 min at 4000×g (K14-4000; Kasvi, Paraná, Brazil). The supernatant was collected for analysis (16). The operating conditions were as follows: the carrier gas was nitrogen with a constant flow rate of 1.5 mL/min. The column was heated at 120 °C for 1 min, then at a heating rate of 15 °C/min to 160 °C for 3 min, and finally at a heating rate of 3 °C/min to 230 °C and held for 10 min.

Fatty acids were quantified with fatty acid methyl ester (FAME) and a six-point standard curve using mixture C4-C24 (Supelco, Bellefonte, PA, USA) and compounds were identified by comparing elution patterns and retention times with the reference mixture.

For the extraction of polar metabolomics, 0.1 mg of beeswax was placed in a 2-mL bottle, then 1 mL of hexane was added and homogenized using ultrasound for 5 min, and then centrifuged (K14-4000; Kasvi) at 2000×g for 5 min. An aliquot of 0.1 mL was then placed in a vial and derivatizated by adding 0.1 mL of N-methyl-N-(trimethylsilyl) trifluoroacetamide. The vial was then placed in a bath at 60 °C for 40 min. Analytes were quantified and identified using a GC system (QP2010 UltraPlus; Shimadzu Corporation, Kyoto, Japan) equipped with a mass detector (MS) (Shimadzu Corporation) and Rxi-1MS capillary column (30 m×0.32 mm×0.25 μm; Restek, Bellefonte, PA, USA). The ramp temperature was maintained at 70 °C for 2 min, increased to 180 °C at 2.5 °C/min, then to 230 °C at 10 °C/min and maintained under isothermal conditions for 2 min. MS was operated in scan mode (mass range m/z=35–450).

For the extraction of nonpolar metabolomes, 0.1 mg of beeswax was placed in a 2-mL bottle, then 1 mL of hexane was added and homogenized using ultrasound for 5 min, and finally, centrifuged (K14-4000; Kasvi) at 2000×g for 5 min. Analytes were quantified and identified using a GC system equipped with a mass detector and an Rxi-1MS capillary column (30 m×0.32 mm×0.25 μm; Restek). The ramp temperature was maintained at 60 °C for 1 min, increased to 180 °C at 5 °C/min, then to 280 °C at 40 °C/min and maintained under isothermal conditions for 15 min (17).

Butia seed oil and beeswax were prepared using the headspace solid-phase microextraction method with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS: 50 μm DVB and 30 μm CAR/PDMS layer) fiber (Supelco, Sigma-Aldrich, Merck, Bellefonte, PA, USA) preconditioned following the manufacturer's protocol. For the extraction of volatile organic compounds (VOCs), 0.1 mL of sample was placed in a 20-mL bottle, then 1 g of sodium chloride and 10 μL of standard benzophenone solution (2 μg) were added. The sealed flasks containing the extract were submerged in a water bath at 40 °C for 15 min, then the fiber was exposed to the headspace for 15 min under constant agitation. VOCs were quantified and identified by a GC system (QP2010 UltraPlus) equipped with a mass detector (Shimadzu), an Rxi-1MS capillary column (30 m×0.32 mm×0.25 μm; Restek) and LabSolution (GCMS solution v. 4.11 SU2; Shimadzu). MS was operated in full scan mode (m/z=30–450). GC-MS data were analyzed and VOCs were identified by comparing similarity indices and mass spectrum with the NIST11 system database (18). Retention index, the retention index calculated from a homologous series of C8-C40 hydrocarbons, and the quantitative analysis was determined by internal standardization with benzophenone (17).

The oleogels were prepared according to Lim et al. (19), with some modifications. Beeswax was added to the Butia seed oil at mass fractions of 1, 3 and 5 % and Butia seed oil was used as a control. Initially, the Butia seed oil was heated in a thermostatic bath to (90±2) °C to ensure complete melting (Fisatom, São Paulo, Brazil) with stirring using a digital mechanical stirrer at 1107×g (RW20; IKA, Hamburg, Germany). After reaching the desired temperature, the beeswax was added and stirred for 2 min to dissolve completely and then the samples were placed on an acrylic plate of 32.8 mm (Kasvi). After that, the oil-wax constituents formed an oleogel. Finally, the oleogels were cooled to room temperature ((22±2) °C) and the plates were turned off until subsequent analyses.

The oil-binding capacity (OBC) of the oleogels was determined according to the method of Zheng et al. (20), with some modifications. About 1.5 g of sample was centrifuged (5430 R; Eppendorf Hamburg, Germany) at 7870×g for 20 min. The overrun Butia seed oil oleogel was then drained onto a soft paper towel for 2 min and the tube mass was weighed together with the solid oil. The empty tube was weighed previously. The values were calculated according to the following equation:

For the stability of the gel during storage, the oleogels based on Butia seed oil and beeswax (~4 mL) were put in clear glass vials and placed upright (Fig. S1). Then, the oleogels were stored at 5 °C for 24 h. After that, the oleogels were maintained in a temperature-controlled room with an air conditioner at (22±2) °C for 90 days of analysis, including the phase separation and liquid oil exudation on the surface. The classification was based on the scale on which (i) a completely liquid system would be defined as oil-like, with high fluidity and phase separation, (ii) a weak system would be characterized by high viscosity, slow flow and minor liquid oil exudation, (iii) a medium system showed a visual gel property and could easily flow under gravitational force, (iv) a firm system had a visual gel property and could flow slowly under gravitational force, and (v) totally firm system had a visual gel property and could not flow under external force (21).

The thermal properties of Butia seed oil, beeswax and oleogel were analyzed by differential scanning calorimetry (DSC) (Q20; TA Instruments, New Castle, DA, USA). The oleogel (~5 mg) was heated in an aluminum pan at a rate of 10 °C/min. The thermal behavior was evaluated over a temperature range of –20–300 °C. Nitrogen gas with a flow of 25 mL/min was used as the vehicle.

The samples of Butia seed oil, beeswax and oleogels were evaluated by Fourier transform infrared (FTIR) analysis in conjunction with attenuated total reflection (ATR), using a spectrophotometer model SPIRIT (Shimadzu) scanned from 4000 to 400 cm^-1, resolution of 4 cm^-1, and 100 scan readings.

The oxidative and hydrolytic stability of samples were evaluated by peroxide index and acidity index, according to AOCS method Cd 8-53 (22). The Butia seed oil or oleogel (5 g) was dissolved in V(acetic acid):V(chloroform)=3:2. Starch (1 g/100 g) was used as a starch indicator on the dispersion. The oleogel and Butia seed oil were titrated with sodium thiosulfate (0.01 mol/L). The peroxide value was expressed as millimoles of oxygen per kilogram of sample. For acidity index analysis, first, 4 g of the sample were weighed and added to 25 mL of V(ether):V(alcohol)=2:1. Titration was performed with 0.1 mol/L potassium hydroxide solution. The acidity index was expressed as mg of KOH per gram of sample.

The cookies were prepared according to Barragán-Martínez et al. (8), with modifications, using 100 g wheat flour (containing per 100 g: proteins 14 g, moisture 13 g and ash 0.8 %), refined sugar 44 g, hydrogenated vegetable fat Primor (São Paulo, Brazil), 30 g or Butia seed oil 30 g or oleogel 30 g, baking powder (2.2 g; containing: corn starch, chemical leavening agents sodium acid pyrophosphate, sodium bicarbonate and monocalcium phosphate), salt 0.9 g and water 18 g. The oleogel chosen for the cookie formulation was the one with the highest OBC, textural and thermal properties (Butia seed oil oleogel with 5 % beeswax). Cookies with the addition of pure Butia seed oil and cookies with the addition of hydrogenated vegetable fat (control) were also produced. Hydrogenated fats are chemically formed by two saturated fatty acids, stearic acid and palmitic acid (23). The dough was prepared in an electric planetary mixer (Kitchen Aid, Troy, OH, USA). The dry ingredients, part of the flour and hydrogenated vegetable fat were mixed for three minutes at low speed, followed by the addition of water and mixing the dough for one minute at low speed and one minute at medium speed. After adding all the flour, the dough was mixed for two minutes at low speed, rolled to a thickness of 5 mm and cut in a 30-mm diameter. The samples were baked at 180 °C for 9 min in an automatic electric oven (VP 80649; Pratica, Pouso Alegre, Minas Gerais, Brazil). After cooling for 2 h, the physical properties of the cookies were analyzed.

Mass loss of the cookies was measured by weighing before and after baking (in grams) using the following equation:

The expansion factor was calculated by the ratio between the cookie diameter and thickness values. A caliper was used to measure the thickness and diameter of cookies, using a millimeter scale, 1 h after removal from the oven (24). The specific volume of the cookies was calculated as the ratio between the cookie volume (cm³), measured by the millet seed displacement method, and the cookie mass after baking. The analysis was done in triplicate.

The values were calculated in triplicate, and the results, expressed as mean value±standard deviation, were analyzed using Statistica v. 7 software (25). ANOVA based on factorial design and Tukey’s test (p≤0.05) was used for data analysis.

The extraction yield of Butia seed oil was (38.4±3.1) % (m/m). Hoffmann et al. (1) reported a 29 to 56 % extraction of Butia odorata seed oil. In contrast, Pereira et al. (26) reported lower values for the extraction of Butia catarinensis seed oil, with a yield of 17.2 % (m/m). As hypothesized, Butia seed oil consists predominantly of saturated fatty acids, with a combined concentration of 98.84 mg/mL. Among these, short- and medium-chain fatty acids, including caprylic acid at 22.45 mg/mL and lauric acid at 22.87 mg/mL, are predominant (Table 1). Medium-chain triacylglycerols are less likely to accumulate in the human body than long-chain triacylglycerols due to their rapid absorption into the metabolism, which helps prevent mass gain. These fatty acids are beneficial when requiring a rapid energy source or when there are digestion challenges (27).

Among the unsaturated fatty acids, a high concentration of oleic acid (C18:1) at 33.21 mg/mL and linoleic acid (C18:2) at 30.61 mg/mL was observed. Soybean oil, widely used in food, is primarily composed of linoleic acid (53.2 %) (23). In this context, regarding linoleic acid content, Butia seed oil exhibits a considerable concentration (30.61 mg/mL). These fatty acids, commonly found in palm fruit oils, have attracted significant interest due to their fatty acid profile, but their application in the oleogel has not yet been studied (27). Vieira et al. (28), however, obtained similar results with higher percentages of lauric acid of 39.17 % in Butia capitata oil. Unsaturated fatty acids, such as oleic acid (20.73 %), were also found in Butia capitata seed oil (28). Pereira et al. (26) reported that oil extracted from Butia catarinenses seeds had the highest content of lauric acid (39.56 %), followed by oleic (11.34 %), capric (10.42 %) and caprylic acid (10.08 %). The composition and quantification of fatty acids depend on various factors, including edaphoclimatic conditions, raw material, cultivation methods, origin of fatty acids, storage conditions and the methods and solvents used (1).

The presence of saturated fatty acids provides stability to oxidation, as there are no double bonds in the chain (28). Lauric acid, found in significant amounts in Butia seed oil, is a short-chain saturated fatty acid known for its antimicrobial properties (28). Fats with a fatty acid profile characterized by high percentages of unsaturated and non-trans acids remain liquid at room temperature (29). Therefore, oleogel based on Butia seed oil, which provides a balanced proportion of healthy fatty acids such as oleic acid and linoleic acid (Table 1), is a good solution to replace hydrogenated vegetable fats, thereby reducing trans fatty acids in cookie formulations. Moreover, Butia seed oil, with a high percentage of linoleic acid (omega 6) and oleic acid (omega 9), is considered significant for food and health, given their roles in cell membranes and brain function, which affect the central nervous system (1).

The choice of vegetable oil affects the thermal, textural and rheological properties of the oleogel, thereby influencing its properties and concentration. According to Patel (30), oils with different concentrations of saturated and unsaturated fatty acids result in oleogels with different properties. Oils with higher saturated fatty acid content produce oleogels with a firmer texture. Conversely, when the oil used for oleogel preparation has a high content of unsaturated fatty acids, the amount of beeswax must be increased.

Like many other lipids, beeswax is considered a complex mixture as it consists of a mixture of different class of chemical components, over 300 of them. Each class comprises compounds with different chain lengths, including hydrocarbons, free fatty acids, fatty acid and alcohol esters, diesters and exogenous substances (31). Among the chemical compounds in beeswax, fatty acid esters are the main components, formed by combining long-chain fatty acids with long-chain alcohols. Beeswax also contains various hydrocarbons, including medium- and long-chain alkanes, such as triacontane (31.85 %), found in the nonpolar fraction (Table S1), and eicosane, predominantly found in the polar fraction (30.31 %) (Table S2). In addition to esters, beeswax contains free fatty acids, including palmitic acid (24.18 %), linoleic acid (8.26 %) and stearic acid (2.59 %) (Table S2). Long-chain alcohols, such as heptacosanol (1.08 %) and tetracosanol (2.54 %), formed by the reduction of fatty acid esters, are also present in the beeswax. These compounds are part of the nonpolar fraction, with tetratriacontanoic fatty acids comprising 21.92 % and triacontane 31.85 %, making them the major organic compounds.

The use of waxes, particularly beeswax, for structuring edible oils is due to their gel-forming properties at low concentrations and high oil-binding capacity (32). Špaldoňová et al. (33) showed the chromatographic profile of beeswax, which predominantly consists of fatty acid methyl esters with 15-37 carbon atoms, with palmitic acid methyl ester identified as the major compound.

Volatile organic compounds (VOCs) are molecules characterized by high vapor pressure, moderate hydrophilicity and low molecular mass. These compounds play a key role in the sensory properties of foods, affecting aroma and contributing to the acceptance of a product. However, no studies have been conducted on the volatile profile of the oil extracted from Butia seed.

The Butia seed oil had six volatile organic compounds, four ketones (2-pentanone, 2,3-pentadione, 2-hexanone and sulcatone), one acid (hexanoic acid) and one ester (ethyl hexanoate) (Table 2). The ketones identified in this study are natural compounds commonly found in various plant-derived products. Ethyl hexanoate, with a low aroma threshold, was the main volatile compound in the Butia seed oil (23.34 mg/mL). According to Hoffmann et al. (1), this compound was also found in the pulp of Butia odorata and is largely responsible for the aroma associated with this species of Butia. Hexanoic acid, a medium-chain volatile fatty acid found in Butia pulp, is responsible for off-flavor (unpleasant aroma) (1).

The main volatile compound in beeswax was d-limonene (22.50 %), followed by decanal (9.48 %), α-pinene (9.29 %), nonanal (8.30 %) and β-pinene (8.23 %) (Table 2). In their study on sunflower oleogel with beeswax, Sobolev et al. (34) identified alcohols such as 1-hexanol, 1-octanol and 1-nonanol, along with the terpene d-limonene. According to Felicioli et al. (35), the high concentration of decanal aldehyde in beeswax could be associated with antimicrobial activity against Pseudomonas aeruginosa. On the other hand, even in small amounts, limonene in beeswax may contribute to antibacterial activity against Staphylococcus aureus. Similarly, nonanal may be responsible for the antibacterial properties of beeswax against Listeria monocytogenes, even in low concentrations.

The oxidative and hydrolytic stability of the Butia seed oil and oleogels were evaluated using the peroxide and acidity indices, respectively. The peroxide content in the oleogel with 5 % beeswax (0.625 mmol/kg) was higher than in the Butia seed oil (0.2 mmol/kg) (p<0.05). This result depends on factors such as the number of double bonds in the molecule and the quantity of fatty acids present. The evaluation of lipid oxidation is essential for the determination of the extent of oil oxidation and, consequently, its quality and chemical composition. The extraction of Butia seed oil using Soxhlet extraction and the oleogel production at 90 °C are key factors influencing the peroxide and acidity levels in the oil. According to the Codex Alimentarius Commission (36), both Butia seed oil and oleogels are within the acceptable consumption limit, which is a maximum of 10–15 mmol/kg oil. The initial phase of oil oxidation occurs when fatty acids react with oxygen to form odorless compounds, such as peroxides (37).

The acidity index was extremely low for Butia seed oil (0.12 mg/g) and higher for the oleogel with 1 % beeswax (0.18 mg/g) (p<0.05). Increasing the beeswax mass fraction to 5 % (0.07 mg/g) in the oleogels effectively reduced the acidity index compared to other oleogels and to the Butia seed oil. Beeswax has emulsifying properties that can help stabilize the oleogel matrix. This stabilization can reduce the exposure of the oil to potential degradation processes, thus lowering the acidity index by minimizing hydrolysis and oxidation reactions that typically generate free fatty acids. The recommended acidity index for refined oils and fats expressed as KOH should not exceed 0.6 mg/g (36), indicating that both Butia seed oil and oleogels are suitable for food applications.

The oil-binding capacity (OBC) values of the oleogel produced with beeswax and Butia seed oil are shown in Fig. 1. There was an increase in the OBC with the mass fraction of beeswax, with the highest value observed for the oleogel with 5 % beeswax (99.9 %) and the lowest for the oleogel with 1 % beeswax (45.1 %). Hwang et al. (32) investigated the effect of the beeswax mass fraction from 0.5 to 15 % on oleogel production. These authors reported that very high mass fractions of wax resulted in a strong flavor and made the emulsion firm, which hindered its incorporation into products and increased the final price of the product with the oleogel. In another study, Zheng et al. (20) produced an oleogel with oil rich in diacylglycerol. They used ethylcellulose as a gelling agent and added γ-oryzanol as an antioxidant to obtain the results for OBC above 98.5 %. However, increasing the amount of ethylcellulose in the oleogel did not result in a significant increase in the OBC.

Other important factors include the source of vegetable oil, the oleogelator used, and their concentrations, all of which can affect the thermal, textural and rheological properties of the oleogel. Patel (30) analyzed five different oils with different concentrations of saturated and unsaturated fatty acids, which gave different results. This author concluded that oils with higher saturated fatty acid concentration form oleogels with a firmer texture. Butia seed oil has a higher concentration of saturated fatty acids (98.84 mg/mL) than unsaturated fatty acids (63.82 mg/mL). A higher percentage of saturated fatty acids leads to the formation of a stronger oleogel (10).

The visual analysis on the first day showed that the oleogel with 1 % beeswax completely disintegrated, with oil flowing through the tube wall (resulting in 100 % oil release) (Fig. S1). From the first day to the 90th day, the oleogels stabilized with 3 and 5 % beeswax showed no oil loss, remaining stable throughout storage. These results show that the beeswax as a stabilizer in mass fractions above 3 % is effective in absorbing and retaining Butia seed oil during storage at room temperature ((20±3) °C). Thus, oleogels produced with 3 and 5 % beeswax had a stable structure for at least 90 days.

The differential scanning calorimetry (DSC) curves of Butia seed oil, beeswax and oleogel in the temperature range of -20 to 150 °C are shown in Fig. 2. All thermograms show only one endothermic peak. For Butia seed oil, the endothermic peak occurred between 12 and 16.5 °C, with an enthalpy variation of 4.2 J/g. The oleogel showed an endothermic peak starting at 16 to 20 °C, and peaks wider than that of beeswax, with an enthalpy variation of 4.56 J/g. For beeswax, the endothermic peak occurred at around 65.5 °C (melting), with an enthalpy variation of 5.59 J/g^.

The oleogel had a lower melting point and broader peak than beeswax (Fig. 2). This suggests that Butia seed oil contains a variety of compounds, some of which have lower melting points, such as butyric, caprylic and capric acids (Table 1). According to Li et al. (38), oleogelators with low molecular mass, such as monoacylglycerol, sodium stearoyl lactylate, rice bran wax and beeswax, have melting points at around 60 °C. Waxes are considered among the most efficient structuring agents for the production of oleogels, as they crystallize at low mass fractions added, forming a crystalline network with a high oil retention capacity (32, 39). Hwang et al. (32) observed a similar change in the melting peak of an organogel containing natural waxes (sunflower wax, rice bran wax, beeswax and candelilla wax). They attributed this phenomenon to the dilution of the wax upon the addition to the oil, which alters the melting temperature of the oleogel. This results in a decrease in temperature and enhances the band signal.

The thermal stability of oleogels is influenced by the stability of the materials used for their stabilization, with enhanced thermal stability typically observed when polymers are combined. Previous studies have documented that oleogels stabilized with certain lipophilic gelling agents, such as fatty acids and waxes, usually have melting temperatures between 50 and 75 °C. Additionally, the melting temperature varies depending on the specific oleogelator used (39). However, in this study, the endothermic peaks of both Butia seed oil and the oleogel occurred at similar temperatures (16.5 and 20 °C, respectively). This suggests that the crystallization caused by the addition of 5 % beeswax was not significant enough to substantially enhance the thermal stability of the oleogel.

Fig. 3 shows the FTIR spectra of Butia seed oil and beeswax oleogels at mass fractions of 1, 3 and 5 %. When analyzing the pure compounds (beeswax and Butia seed oil), characteristic bands specific to each compound were observed. Bands between 2990 and 2800 cm^-1, at 2885–2780 cm^-1 and at 1750 cm^-1 for Butia seed oil, oleogels and beeswax are related to free fatty acids and the methyl group (-CH₃), methylene group (-CH₂) and ester group of fatty acids, respectively (8). The bands around 1150 cm^-1 correspond to the stretching of bonds of aliphatic esters and bending vibrations of CH₂ (11); for the spectra of the oleogels with different mass fractions of beeswax, not all characteristic bands of beeswax were identified. This may be due to the low mass fractions of beeswax used.

The color parameters of Butia seed oil and oleogels based on Butia seed oil containing different mass fractions of beeswax are shown in Table 3. Lightness was higher for Butia seed oil (72.46) and decreased with the addition of beeswax, resulting in values of 53.9, 42.7 and 44.1 for oleogels with 5, 3 and 1 % beeswax, respectively. The a* (red/green) coordinate showed negative values for Butia seed oil and all oleogels, reflected in greenish tones. Conversely, the b* (yellow/blue) coordinate value increased as the beeswax mass fraction increased. No statistical difference was observed between the Butia seed oil and the oleogel with 1 % beeswax regarding the b* coordinate (p>0.05), nor between the oleogels with 3 and 5 % beeswax, indicating that the yellow color of the oleogels is primarily due to the Butia seed oil and not the beeswax. Butia seed oil contains carotenoids, antioxidant compounds from secondary metabolism, which impart a yellow-orange color (1). The chroma value, which measures color purity, was higher for the oleogel with 1 % beeswax and for Butia seed oil, with no significant difference between them (p>0.05). However, increasing the beeswax mass fraction (to 3 and 5 %) in the oleogels resulted in a reduction in the chroma value.

The texture profile analysis of the oleogels is shown in Table 3. Hardness, defined as the maximum force during the first compression, was the highest in the oleogel with the highest mass fraction of beeswax compared to the other samples (p<0.05). The oleogel with the lowest beeswax mass fraction (1 %) showed greater springiness than the other formulations (p<0.05). Overall, all oleogels produced in this study had low springiness values, indicating a limited ability to return to their original form after deformation, a property inherent to oleogels. Fats are classified by their plasticity, where lower elasticity is associated with greater plasticity (32). The springiness and cohesiveness values of the oleogels containing 3 and 5 % beeswax were quite similar, both lower than that of the oleogel with 1 % beeswax, which had the highest values. In terms of hardness and chewiness, the oleogel with 5 % beeswax showed the highest values, which is in agreement with the high OBC results observed (Fig. 1).

Beeswax has a diverse chemical composition, containing esters, hydrocarbons (eicosane), free fatty acids (palmitic, linoleic, oleic and stearic), and free fatty alcohols (tetracosanol). These compounds, particularly the esters with long chains, crystallize and form networks that trap the liquid vegetable oils. This crystallization changes the physical properties of the oil, affecting the elasticity and hardness of the oleogel (32). The texture profile of oleogels is crucial for their optimal industrial application.

The evaluation of texture, color, mass loss, expansion factor and specific volume of the cookies made with hydrogenated vegetable fat (control), Butia seed oil and oleogel containing Butia seed oil and 5 % beeswax is shown in Table 4 and Fig. S2. Oleogel with 5 % beeswax was selected for cookie production due to its gel stability and high oil-binding capacity, ensuring the maintenance of the desired cookie texture.

The cookies made with the oleogel showed the lowest hardness, followed by the cookies with Butia seed oil, and those with hydrogenated vegetable fat (control). Hardness refers to the ability to resist deformation under applied force. The fracturability (or brittleness) of a cookie is the distance it deforms until it breaks under stress (34, 40). Other authors have obtained lower values for fracturability of cookies containing oil gels modified with olive oil, soybean oil, linseed oil, and wax, than in this study (40).

In this study, cookies made with Butia seed oil had lower hardness than those made with hydrogenated vegetable fat. The reduced hardness of the cookie produced with oleogel and pure Butia seed oil may be attributed to the chemical composition of the Butia seed oil and the beeswax present in the oleogel formulation. The primary components of beeswax are fatty acid esters, which act as emulsifiers, along with monoglycerides and fatty acids (stearic acid and oleic acid) (32). The presence of these emulsifiers can improve the incorporation of air into the dough and thus reduce its hardness.

No significant difference was observed in the fracturability of the cookies (p>0.05). Fracturability is related to crispness and is a key factor for cookies. Fat affects the softness of the cookies by lubricating the dough, while sugar impacts the characteristics of fracturing or breaking. The thermal stability of the materials and their melting temperature used in cookie production also influence the texture properties (22).

The fracturability (or brittleness) of the biscuit is defined as the deformation (in millimeters or centimeters) that the sample undergoes until it breaks under particular conditions (40). Despite the relatively small thickness of the biscuit, a force 10 to 12 times greater is required for it to fracture. The applied force influences the positioning of the probe, causing it to travel a greater distance before rupture occurs. It is important to note that the texture analysis was conducted 1 h after baking. Until that time, the biscuits had already cooled and had a dry structure with no residual moisture. This condition contributed to the increased hardness and the reduced fracturability (40).

Hydrogenation, or the use of saturated fats, is necessary to structure liquid oil into semi-solid plastic pastes for use in spreads, margarine and shortenings (40). However, hydrogenated fats are composed primarily of saturated fatty acids, such as palmitic and stearic acid (23). Bakery products have been the focus of studies that aim to replace hydrogenated fats with structured oils, due to the global rise in overweight and obesity associated with the high consumption of unhealthy, energy-dense diets, including processed foods (8). Zhao et al. (41) used oleogels in an attempt to replace commercial fat in biscuits. He concluded that biscuits with corn oil oleogel had properties comparable to the control (visual appearance, hardness and firmness), resulting in healthier biscuits with a high content of unsaturated fats.

There was no significant difference in the color parameters between the cookies made with oleogel, Butia seed oil, and hydrogenated vegetable fat (p>0.05). Visual analysis showed that all cookies had a spherical shape with some small grooves on the surface and the color (Table 4 and Fig. S2) was predominantly yellow (Fig. S1 and Fig. S2). The cookies made with oleogel had the lowest mass loss during baking, followed by the cookies with Butia seed oil (p<0.05), while the cookies with hydrogenated vegetable fat had the highest mass loss (p<0.05). These results indicate that using oleogel increased mass yield during baking. Goldstein and Seetharaman (42) reported a relationship between cookie height and moisture content in the cookies made with a monoglyceride emulsion, where greater height was associated with increased moisture. Moisture loss affects cookie volume and expansion capacity, which may explain the lower expansion factor observed in the cookie made with Butia seed oil (4.01, p<0.05) (Table 4), as no gel was formed to retain moisture during baking.

The cookies made with oleogel had the highest specific volume, while the cookies with Butia seed oil and the control did not differ from each other (p>0.05). There was an inverse relationship between specific volume and hardness: the cookies with oleogel had the highest specific volume (1.3 g/cm³) and the lowest hardness. In contrast, the cookies made with hydrogenated vegetable fat had the lowest specific volume (0.81 g/cm³) and the highest hardness compared to those made with Butia seed oil and oleogel. These results may be related to the higher mass loss observed in these cookies, 14.1 % (p<0.05) (Table 4). Specific measurements of volume, thickness and width in cookies are influenced by several factors, including ingredient quality, texture (softness or hardness), types and quantities of ingredients, and processing conditions.

Regarding the expansion factor, there was a significant difference between all cookies, with the highest expansion factor observed for the control cookies. Factors such as cookie diameter, thickness and expansion factor are commonly used to assess product quality. According to Jacob and Leelavathi (40), the spreading of the cookie dough is related to the viscosity of the dough since a dough with low viscosity takes longer to stop spreading in the mold and harden, leading to larger sizes. However, cookies with very high or very low expansion factors cause problems in the processing of industrialized foods, resulting in products with low or high mass.

Oleogelation is a promising method, as it does not alter the unsaturated fatty acid profile of liquid oils. The replacement of 100 % hydrogenated fat with Butia seed oil oleogel containing beeswax aims to reduce the content of saturated and hydrogenated fats, as well as to improve the lipid profile of cookies, since Butia seed oil is rich in polyunsaturated fatty acids such as linoleic and oleic acids (Table 1). Furthermore, the oleogel production process does not use chemical reagents to transform liquid oils into thermo-reversible and three-dimensional gel networks with solid-like properties.