SJA_41_4_1837-1845

Introduction

Celiac disease and gluten intolerance are increasingly prevalent autoimmune and gastrointestinal conditions that necessitate the development of nutritionally optimized gluten-free (GF) food products. According to the World Gastroenterology Organization (2021), the global incidence of celiac disease is rising, underscoring the critical demand for safe and health-promoting GF alternatives. Although rice flour is a widely accepted base in GF formulations due to its hypoallergenic properties, it is inherently deficient in essential macronutrients such as protein and dietary fiber. This limitation warrants the incorporation of bioactive-rich functional ingredients to enhance the nutritional profile and technological functionality of GF baked goods.

Flaxseed (Linum usitatissimum) is a well-established functional ingredient, recognized for its high content of α-linolenic acid (omega-3 fatty acids), lignans, and dietary fiber (Goyal et al., 2014). Its regular consumption has been associated with improved cardiovascular health, modulation of inflammatory pathways, and enhanced gastrointestinal function. Technologically, flaxseed’s mucilage a natural hydrocolloid contributes to improved water retention and textural integrity in GF matrices, mitigating the common quality defects in gluten-free baking (Singletary, 2019).

Cinnamon (Cinnamomum verum) is another functional additive with significant antioxidant, antimicrobial, and anti-inflammatory properties (Ranasinghe et al., 2013). It has been shown to inhibit lipid peroxidation and microbial growth, thereby enhancing the oxidative stability and microbiological shelf life of food products (Shan et al., 2004). These attributes position cinnamon as a promising natural preservative in clean-label formulations. Flaxseed is rich in alpha-linolenic acid, dietary fiber, and lignans, which offer antioxidant and anti-inflammatory effects. Cinnamon, particularly Cinnamomum verum, contributes natural antioxidants and exhibits strong antimicrobial properties, which enhance food preservation without synthetic additives.

Contemporary food industry trends increasingly favor clean-label products, driven by consumer preference for minimally processed foods free from synthetic additives (Bigliardi and Galati, 2013). Conventional preservatives such as sodium benzoate and potassium sorbate, though effective, have raised health concerns including allergenicity and gastrointestinal distress (Sharma, 2015). In contrast, natural preservative systems based on flaxseed and cinnamon offer multifunctional benefits improving nutritional quality, extending shelf life, and ensuring consumer safety thereby aligning with both regulatory and market demands.

The objective of this study was to develop gluten-free muffins enriched with flaxseed and cinnamon to improve their nutritional and functional properties while extending shelf life without synthetic preservatives. Additionally, the study evaluated the effectiveness of gamma irradiation as a preservation technique to ensure microbial safety and quality retention over an extended storage period.

Materials and Methods

Procurement of ingredients

All ingredients, including rice flour, all-purpose flour, flaxseed, cinnamon powder, sugar, eggs, vegetable oil, milk, and baking soda, were sourced from local markets in Peshawar. Flaxseed was ground into fine powder before incorporation into the muffin formulations to ensure uniform distribution.

Formulation and preparation of muffins

Five treatments were formulated as T0: Control (100% all-purpose flour), T1: 100% rice flour, T2: 90% rice flour + 10% flaxseed, T3: 98% rice flour + 2% cinnamon, and T4: 90% rice flour + 8% flaxseed + 2% cinnamon. The dry ingredients (flour, flaxseed, cinnamon, and baking soda) were sieved to ensure uniform particle size and then mixed. Separately, eggs, sugar, vegetable oil, and milk were whisked until a homogeneous mixture was achieved. The wet ingredients were gradually incorporated into the dry mixture, and the batter was mixed thoroughly to maintain consistency. The muffin batter was poured into pre-greased molds and baked at 180°C for 25 minutes in a preheated oven. Once baked, muffins were cooled at room temperature before being packed for further analysis.

Packaging and storage

The muffins were packed in air-sealed zip-lock bags and stored at room temperature for 28 days. The packaging was done to minimize moisture loss and oxidative degradation during storage.

Irradiation process

The packed muffins were subjected to gamma irradiation at a dose of 4 kGy for 4 hours using a Cobalt-60 gamma radiation source at the Nuclear Institute of Food and Agriculture (NIFA), Peshawar. Irradiation was performed to assess its impact on shelf-life extension and microbial stability.

Analytical tests

A series of analytical tests were conducted at 0, 14, and 28 days of storage to assess the quality and stability of the muffins. All the samples of Gluten free muffins enriched with flaxseed and cinnamon were subjected to evaluate physico-chemical analysis (proximate analysis, texture analysis, water activity, Free fatty acid, Peroxide Value, total phenolic content, and Total viable count (TVC) was assessed using the standard plate count method on nutrient agar, incubated at 37 °C for 24–48 hours, following AOAC (2012) protocols. and sensory evaluation (color, taste, texture and overall acceptability) by 9 points Hedonic scale (Larmond, 1977).

Sensory analysis

All the samples were studied for 28 days with every 14-day interval. Sensory evaluation of gluten-free muffins was conducted using a 9-point hedonic scale, where 1 = dislike extremely and 9 = like extremely.

Stastical analysis

Data were analyzed using a completely randomized design (CRD) (Steel et al., 1997). Statistical comparisons were performed using analysis of variance (ANOVA) followed by the Lest Significant Difference (LSD) test to determine significant differences (p < 0.05). The software used for statistical analysis was SPSS version 25.0 (IBM Corp., Armonk, NY, USA).

Results and Discussion

Proximate composition and free fatty acids (%)

The proximate analysis of gluten-free muffins showed significant variations across treatments (Figure 1). The protein content was highest in T₄ (6.40± 0.13%), attributed to flaxseed’s high protein content (Goyal et al., 2014). Similarly, fiber content increased significantly in T₄ (3.22± 0.09%), as flaxseed is an excellent source of dietary fiber, contributing to improved gastrointestinal health (Singletary, 2019). Fat content was also highest in T₄ (8.43± 0.16%) due to the presence of flaxseed’s omega-3 fatty acids, essential for cardiovascular health (Kajla et al., 2015).

Moisture content decreased over the storage period, with T₄ retaining more moisture compared to T₀, likely due to flaxseed’s water-binding capacity. Ash content, indicating the mineral content, was highest in T₄ (1.83±0.09%), further supporting the enhanced nutritional benefits of flaxseed and cinnamon (Jayaprakasha et al., 2006). The statistical analysis showed that these compositional changes were significant (p<0.05), aligning with previous studies on flaxseed-fortified baked products (Grasso et al., 2019).

Free fatty acid (FFA) content is an important indicator of lipid hydrolysis and fat degradation in food products. The results (Figure 1) showed a significant (p<0.05) increase in FFA levels over storage in all formulations. However, the increase was lowest in T₄ (1.21 ± 0.06%), suggesting that flaxseed and cinnamon contributed to lipid stability. The lower FFA content in T₄ may be due to the antioxidant properties of cinnamon, which help prevent fat oxidation (Ranasinghe et al., 2013).

The breakdown of fats leads to an increase in FFA content, which can negatively impact the flavor and shelf life of muffins. The results are consistent with previous studies that indicate antioxidants help maintain lipid quality in bakery products (Jayaprakasha et al., 2006).

Texture hardness increased significantly over storage in all treatments (Figure 2), with the smallest increase observed in T₄ (Initial: 1.85 ± 0.07 N, Day 28: 2.65 ± 0.10 N). This suggests flaxseed and cinnamon delayed staling and preserved crumb softness. Flaxseed’s mucilage content has been reported to improve texture retention in gluten-free products (Muhammad and Dewettinck, 2017). Springiness and cohesiveness were also better retained in T₄ (Figure 2), aligning with previous research on flaxseed’s mucilage improving texture in gluten-free products (Grasso et al., 2019).

Water activity (Aw)

Water activity (Aw) significantly (p<0.05) decreased in all formulations, with T₂ and T₄ recording the lowest values (Figure 3). Lower aw values are associated with improved microbial stability and extended shelf life (Suthar and Das, 2004).

The presence of flaxseed and cinnamon contributed to the decreased water activity, as flaxseed’s fiber content helps bind water, reducing its availability for microbial growth (Kajla et al., 2015). Cinnamon’s antimicrobial properties further enhance product stability (Ranasinghe et al., 2013).

Peroxide value and total phenolic content

Peroxide value (POV) is a key indicator of lipid oxidation, which directly affects the shelf life and sensory attributes of bakery products. Higher POV levels indicate oxidative rancidity, leading to undesirable flavors and reduced product quality. The results (Figure 4) showed a significant (p<0.05) increase in POV across all formulations over storage, with T₄ (1.43 ± 0.05 meq/kg) having the lowest increase. This suggests that flaxseed and cinnamon contributed to oxidative stability by reducing lipid peroxidation (Ranasinghe et al., 2013).

The lower POV in T₄ may be attributed to the antioxidant properties of cinnamon, which contains bioactive compounds such as cinnamaldehyde that inhibit oxidation (Jayaprakasha et al., 2006). Flaxseed, rich in omega-3 fatty acids, also plays a role in enhancing lipid stability by forming a protective matrix around fats, preventing excessive oxidation (Kajla et al., 2015).

TPC values were significantly higher (p<0.05) in T₄ (23.4 ± 0.7 mg GAE/g) compared to other treatments, confirming the role of flaxseed and cinnamon as polyphenol-rich ingredients (Figure 4). Jayaprakasha et al. (2006) highlighted the antioxidant benefits of these ingredients, which support oxidative stability over storage. Cinnamon is well-known for its rich polyphenolic content, particularly cinnamaldehyde and proanthocyanidins, which contribute to its strong antioxidant properties (Ranasinghe et al., 2013). Flaxseed also contains lignans and flavonoids that enhance the overall phenolic profile, improving the muffins’ resistance to oxidative degradation (Kajla et al., 2015).

Total viable count (TVC)

TVC was significantly lower in T₂ and T₄ (Day 28: 1.9 ± 0.2 log CFU/g for T₄), indicating enhanced microbial stability (Table 1). Cinnamon’s antimicrobial effects have been widely reported, reducing bacterial and fungal contamination (Ranasinghe et al., 2013). Total viable count (TVC) is a key indicator of microbial load in food products and is used to assess their microbial stability and safety during storage. Higher TVC values indicate increased microbial contamination, which can negatively impact shelf life and food safety (Ranasinghe et al., 2013). The presence of antimicrobial ingredients such as cinnamon, combined with irradiation, plays a crucial role in controlling microbial growth.

Sensory analysis

Sensory evaluation of gluten-free muffins was conducted using a 9-point hedonic scale, where 1 = dislike extremely and 9 = like extremely. Sensory attributes assessed included taste, color, texture, and overall acceptability (Figures 5). The taste scores decreased over storage, but T₄ maintained the highest acceptability (Day 0: 8.5 ± 0.3; Day 28: 7.9 ± 0.2), followed closely by T₀ and T₁. The incorporation of cinnamon contributed aromatic sweetness, while flaxseed added a subtle nuttiness, improving flavor complexity. For color, T₄ and T₀ achieved the highest ratings (8.4 ± 0.2), attributed to the warm brown hue imparted by cinnamon and flaxseed. In contrast, T₂ received the lowest color scores (7.4 ± 0.3), likely due to flaxseed’s darker appearance dominating the visual appeal.

Table 1: Total viable count (CFU/g) of muffins

Treatment	CFU/g
T0	10000
T1	17000
T2	200
T3	20000
T4	700

Texture ratings were best in T₄ and T₀ (8.5 ± 0.2), indicating enhanced softness and chewiness, retained over storage. Flaxseed mucilage likely played a key role in moisture retention and crumb integrity, reducing textural degradation. In terms of overall acceptability, T₄ (8.3 ± 0.2) and T₁ (8.4 ± 0.2) were most favored, combining improved sensory traits with better shelf stability. These findings suggest that flaxseed and cinnamon act synergistically to improve the organoleptic properties of gluten-free muffins, even over extended storage.

Conclusions and Recommendations

This study successfully developed a gluten-free muffin formulation using rice flour enriched with flaxseed and cinnamon, enhanced through gamma irradiation for shelf stability. The T₄ treatment (90% rice flour, 8% flaxseed, 2% cinnamon) demonstrated superior nutritional content higher protein, fiber, and antioxidant levels and better sensory scores than the control and other treatments. Additionally, flaxseed and cinnamon contributed to reduced microbial load and oxidative rancidity, thereby extending product shelf life.

These results support the potential of natural functional ingredients and non-thermal preservation techniques in producing clean-label, gluten-free bakery products with improved consumer acceptability and stability during storage.

Novelty Statement

This study uniquely combines rice flour-based gluten-free muffin formulation with natural enrichment using flaxseed and cinnamon, alongside a non-thermal preservation technique through gamma irradiation. It is the first to comprehensively evaluate the synergistic impact of these functional ingredients and irradiation on both the nutritional enhancement and microbial stability of gluten-free baked products during storage. The findings highlight a novel approach to improving the shelf life, nutritional profile, and sensory acceptability of gluten-free muffins, addressing key challenges in gluten-free product development.

Author’s Contribution

The study was designed, conducted and written by Komal Fatima. Majid S. Hashmi designed, supervised, and assisted in finalizing the manuscript. Muhammad Asim Irshad supervised and assisted in drafting the manuscript. Ayaz Ahmad assisted in data analysis, proof reading, formatting and preparing the final manuscript. Aysha Riaz and Sahib Alam assisted in data analysis and finalization of the manuscript.

This work has been carried out as a requirement for M.Sc. (Hons) degree of first author.

Funding

No funding has been received for this study.

Generative AI or AI assisted technology statement

The authors declare that no Genrative AI was used in the creation of this manuscript.

Conflict of interests

The authors show no conflict of interests.

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