Different types of raw materials are used to prepare the granola bar. FOS was sponsored by a local ingredient supplier (DKSH, Selangor, Malaysia) in the form of powder. The other ingredients, such as rolled oats, puffed rice, pumpkin seeds, chia seeds, peanuts, almonds, glucose syrup, raisins, olive oil, and brown sugar, were purchased from the local market.

Absolute alcohol (Sigma-Aldrich, USA), petroleum ether (Merck, Germany), concentrated sulphuric acid (H₂SO₄, 95–98%; Merck, Germany), catalyst tablet (potassium sulphate and selenium; Merck, Germany), 50% sodium hydroxide (NaOH, Sigma-Aldrich, USA), 2% boric acid (Sigma-Aldrich, USA), 0.1 M methyl red (Sigma-Aldrich, USA), 0.255 N H₂SO₄ (Merck, Germany), 0.313 N NaOH (Sigma-Aldrich, USA), 1% hydrochloric acid (HCl, Sigma-Aldrich, USA), Fehling’s A solution and Fehling’s B solution (Merck, Germany), methylene blue indicator solution (Sigma-Aldrich, USA), invert sugar standard solution (Sigma-Aldrich, USA).

About 200 g of FOS powder was placed in 280 mL of water. This solution was warmed slowly over low heat until the mixture became slightly thicker. This solution was stirred continuously to prevent the coagulation of the FOS [16].

The bars were prepared according to the following steps, as shown in Figure 1. The ingredients were selected based on their functional roles and nutritional contribution. Rolled oats and puffed rice provided structure, complex carbohydrates, and desirable texture. Nuts and seeds (almonds, peanuts, chia seeds, pumpkin seeds) contributed protein, healthy fats, and dietary fiber, while raisins added natural sweetness. Glucose syrup acted as a binder and sweetener in the control formulation, and FOS syrup was included in varying amounts to assess its prebiotic effect. Olive oil improved mouthfeel and binding, and vanilla essence and salt enhanced flavour and balance.

Dry ingredients were added separately before mixing and nuts were crushed for uniformity. For agglutination of granolas, the slurry was prepared in a stainless-steel container, where the ingredients (vanilla flavour, salt, glucose syrup, and olive oil) were heated and mixed with dry ingredients until a uniform mixture was obtained. The mixture was deposited in the mold, compacted with a roller, and baked at 108°C for approximately 20 min until golden. After cooling, bars were cut and stored at room temperature for three days to stabilize chemical reactions and ingredient properties. Five formulations were prepared as shown in Figure 2, with the control (F0) prepared according to de Carvalho et al. [17] without FOS syrup (Table 1).

Six analyses were conducted to determine the proximate analysis in the granola bar sample, namely, moisture, fat, protein, ash, crude fiber, and carbohydrate content. The granola bar was evaluated for its physicochemical features in triplicate (n = 3).

Sugar concentration was determined using the Lane and Eynon titration method following the AOAC Official Method 923.09, 2005 guideline [19]. All titrations were performed in triplicate.

Granola bar extraction: Approximately 5 g of granola bar was homogenized and extracted with Milli-Q water to a final volume of 100 mL. The extract was filtered and sugar analysis was conducted on the aqueous phase. Therefore, results are expressed as sugar concentration in the extract solution.

Determination of initial reducing sugar concentration: Reducing sugar concentration was determined by titrating appropriately diluted sample extract against boiling Fehling’s A and B solutions using methylene blue as an indicator. The endpoint was identified by the disappearance of the blue colour, yielding a brick-red precipitate.

Total sugar concentration analysis: An aliquot of the sample extract was hydrolyzed with concentrated HCl at 68°C for 30 min to convert sucrose into reducing sugars. The hydrolyzed solution was neutralized, diluted, and titrated following the same procedure for reducing sugars.

Standardization of sucrose solution: Standardization of sucrose solution was performed by preparing a 10% invert sugar solution adjusted to pH 8.0. It was titrated with 10 mL of boiling Fehling’s A and B solutions using 1% methylene blue as an indicator. The endpoint was defined by the disappearance of the blue colour (brick-red precipitate). Blank titrations were conducted without sample hydrolysis. All titrations were performed in triplicate and the mean value was used for calculations.

Method validation and quality control: Recovery tests were performed by spiking three sample extracts with known amounts of invert sugar and sucrose. Sugar concentrations were calculated using the dextrose factor equation (Equation 8).

(8) S u g a r   c o n c e n t r a t i o n   ( g / 100   m L ) = E + A × B - C × 1 D × d i l u t i o n   f a c t o r × 100 1000

Where the concentration of the standard sucrose = A mg/mL. Required volume of hydrolyzed standard sucrose solution to titrate with Fehling’s A and B = B mL. Calculated dextrose factor = (A × B) mg. Dextrose factor from table = C mg. Correction factor = (A × B) – C mg. Required volume of sample to titrate with Fehling’s A and B = D mL. Dextrose factor of sample from table = E mg.

The colour of the granola bar samples was measured using a Minolta Chromameter CR 400 (Minolta, Osaka, Japan). Colour parameters were recorded as L* (lightness), a* (green to red), and b* (blue to yellow). The instrument was calibrated using a white standard tile before each use. Samples were spread evenly on a plastic microbiological plate, and empty spaces were covered to ensure accurate reading. Measurements were taken at three random locations per sample and the mean values were used for statistical analysis.

The pH value of the sample was determined using a HANNA instrument pH meter calibrated with pH 4 and pH 8 buffer solutions. Ten g of the sample were homogenized with 100 mL of distilled water prior to measurement.

Water activity (a_w) was measured using an AquaLab™ CX-2T water activity meter at 25.0 ± 0.3°C. Samples were homogenized in a porcelain mortar before analysis. Each measurement was repeated three times, and the mean value was used for statistical analysis [20].

The texture of the granola bars was assessed using a texture profile analysis (TPA) with a Texture Analyser TA-XT2i (Stable Micro Systems, UK) equipped with a 3-point bending rig. Sample dimensions were measured using a vernier caliper (width = 3 cm, thickness = 1 cm) before analysis. Each granola formulation was tested in triplicate. Bars were placed right-side up on the rig supports and compressed with a cylindrical probe (diameter = 1.2 cm) at a trigger force of 25 g. Pre- and post-test speeds were 10 mm/s, with a compression distance of 35 mm and a dwell time of 3 s. Instrument calibration was performed according to the manufacturer’s instructions before each session. Force-time curves were generated and textural parameters including hardness, fracturability, chewiness, springiness, and cohesiveness, were calculated using the instrument software.

Sensory evaluation of the granola bars was conducted with 30 untrained and semi-trained panellists (students from UiTM Shah Alam, aged 20–25 years). Samples were cut into 1.5 × 1.5 × 1 cm pieces and coded with a randomized three-digit number to ensure blinding. Panellists evaluated seven attributes, which are taste, odour, colour, texture, crispiness, appearance, and overall acceptability using a 7-point hedonic scale (1 = “dislike extremely”, 7 = “like extremely”). To minimize order and carry-over effects, samples were presented in a randomized order and panellists evaluated them from left to right while rinsing their mouths with mineral water between samples. Prior to testing, panellists received a brief orientation on the evaluation procedure to ensure consistent understanding.

The data were analyzed using Statistical Package Social Sciences (SPSS) version 26. One-way analysis of variance (ANOVA) and Tukey’s HSD post-hoc test were used to compare for any significant differences between samples. The mean and standard deviation were expressed as the values of different parameters. Differences were considered significant at p < 0.05. All analyses were carried out in triplicate.

The total sugar concentration in the extracts decreased significantly with increasing FOS substitution, consistent with the replacement of glucose syrup by FOS which has lower sweetness intensity, limited digestibility, and a low glycemic index [35]. Previous studies have demonstrated that partial replacement of conventional sugars with FOS can reduce sugar concentration without adversely affecting product quality in baked goods, dairy products, and snack foods [33, 36–38]. The present findings confirm that FOS is effective in lowering sugar concentration in granola bars while maintaining overall product acceptability and nutritional enhancement through increased fiber content. These findings also suggest that FOS may act as a valuable ingredient in developing functional foods that cater to health-conscious consumers.

The significant increase in L* values with higher FOS levels indicates the production of lighter-coloured granola bars (Table 3). This effect is primarily attributed to the reduced intensity of Maillard reactions during baking, as FOS provides fewer reducing sugars than glucose syrup. Similar observations were reported by Ramli et al. [39] who observed lighter colour tones in dark chocolate formulated with Nypa fruticans sugar substitute due to suppressed non-enzymatic browning.

The decrease in both a* and b* values further supports the reduced formation of brown pigments such as melanoidins during thermal processing. Comparable trends were also observed by Pallavi et al. [23] and Nadeem et al. [27], who reported that prebiotic syrups yielded lighter colour and improved visual appeal in cereal and fruit bars. These findings suggest that FOS substitution effectively modulates browning reactions, resulting in visually lighter products.

The incorporation of FOS did not have a significant impact on the pH values of the granola bars, as formulations F1, F2, and F3 exhibited values comparable to the control (F0). A slight but significant increase was observed in F4 (100% FOS), with a pH of 6.15. Nevertheless, all formulations remained within the pH range reported for FOS-enriched products (5.98–6.06) by Hartmann et al. [34]. Moreover, FOS is known to be stable across typical food pH ranges (4.0–7.0), indicating that its incorporation does not compromise pH stability [40].

a_w values decreased significantly (p < 0.05) with increasing FOS substitution, ranging from 0.359 in F0 to 0.218 in F4. This reduction suggests that FOS limits the availability of free water within the product matrix. The lowest a_w observed in F4 indicates a reduced potential for microbial growth. Souiy et al. [41] reported a_w values between 0.45 and 0.53 for gluten-free and sugar-free energy bars, while Sánchez-Maldonado et al. [42] classified foods with a_w < 0.85 as low-moisture foods. Accordingly, all granola bar formulations in this study can be categorized as low-moisture products, which do not require refrigeration for microbial stability.

Texture results revealed increased hardness and fracturability with higher FOS incorporation. This trend is consistent with the reduced fat content observed in the proximate analysis, as lower fat levels promote stronger inter-particle bonding and a denser product structure. Hartmann et al. [34] observed similar effects in FOS-fortified low-fat meat products.

The irregular variation in cohesiveness and chewiness may be attributed to the heterogeneous particle distribution and differences in syrup viscosity. FOS syrup is generally less viscous than glucose syrup, which can lead to firmer but less cohesive matrices [26, 33]. Comparable hardness and chewiness values were reported by Austina Ribanar and Hemalatha [43] in snack bars, supporting that the textural properties obtained in this study fall within acceptable ranges for consumer products.

Overall, increasing FOS levels produced granola bars with greater structural rigidity, reduced stickiness, and acceptable texture suitable for consumer acceptance.