Introduction

Rice (Oryza sativa L.) is one of the most important cereal crops globally and serves as a staple food for more than half of the world’s population. Approximately 95% of global rice production originates from Asia, underscoring its critical role in ensuring global food security [1]. Two primary rice species dominate cultivation: Oryza sativa, widely grown in Asia, and Oryza glaberrima, native to Africa [2]. The grain’s structure, comprising bran, endosperm, and germ, provides essential carbohydrates, proteins, and micronutrients that contribute substantially to dietary energy and nutrition [3]. However, the milling process that converts brown rice to white rice commonly consumed in most countries removes the bran and germ layers, resulting in significant nutrient losses, particularly in vitamins, minerals, and dietary fiber [4].

Rice plays a pivotal role not only as a staple food but also as a symbol of cultural heritage, forming the base of traditional cuisines across diverse ethnic groups [5]. The nation’s dependency on rice as a primary carbohydrate source highlights the importance of enhancing local rice production and exploring innovative uses of indigenous rice varieties to support food security and economic resilience [6]. Ongoing efforts in Malaysia focus on improving rice quality, yield, and diversification through the utilization of nutrient-rich pigmented varieties and by-products as functional food ingredients [7, 8].

The rising prevalence of gluten intolerance and celiac disease has spurred growing interest in gluten-free bakery products [9, 10]. Gluten-free biscuits, in particular, have become a popular alternative due to their versatility, wide consumer acceptance, and potential for incorporating nutrient-dense local ingredients [11–14]. Formulating gluten-free biscuits using composite flours such as red rice and cassava not only provides a safe option for individuals with gluten sensitivity but also enhances product diversity, promotes the use of indigenous crops, and reduces reliance on imported wheat [6].

Among the nutrient-dense alternatives, pigmented rice varieties such as red rice have gained attention for their superior nutritional and functional properties [15, 16]. Red rice contains high levels of bioactive compounds, including anthocyanins, proanthocyanidins, γ-oryzanol, phenolic acids, and γ-aminobutyric acid, all of which contribute to antioxidant, anti-inflammatory, and cardioprotective effects [17]. Its mineral content, particularly iron, magnesium, calcium, and zinc are also significantly higher than that of polished white rice [18]. These attributes make red rice a promising functional ingredient for developing health-promoting food products. Cassava (Manihot esculenta Crantz), another key tropical crop, presents additional advantages for sustainable food production. It is highly tolerant to drought, thrives in marginal soils, and is rich in starch (up to 80% dry weight), making it an excellent energy source and a potential raw material for flour-based applications [19, 20]. In Malaysia and other tropical regions, cassava flour has been explored as a partial substitute for wheat flour in bakery products due to its low cost and local availability [21].

The growing demand for wheat-based products such as biscuits has placed increasing pressure on wheat imports, particularly in countries such as Malaysia, where wheat is not cultivated locally. To reduce dependency on imported wheat and enhance product nutritional quality, composite flours prepared by blending wheat with locally available non-wheat ingredients have gained considerable attention [11, 22, 23]. Previous studies have explored the incorporation of various alternative flours, including rice, cassava, and other plant-based ingredients, into bakery products to improve nutritional value and diversify product formulations [24–27]. However, many of these studies have primarily focused on individual flour substitutions or limited aspects of product quality.

Pigmented rice varieties such as red rice have attracted growing interest due to their superior nutritional and functional properties, including high levels of phenolic compounds, antioxidants, and essential minerals [28, 29]. Likewise, cassava (Manihot esculenta Crantz) is a widely cultivated tropical crop known for its high starch content and adaptability to marginal soils, making it an attractive ingredient for sustainable food production [19, 20]. Although both red rice and cassava flours have been individually investigated in bakery formulations, studies examining their combined application in gluten-free biscuit systems remain limited, particularly with regard to their integrated effects on physicochemical properties, shelf-life stability, and consumer acceptance.

Therefore, the novelty of this study lies in the development and comprehensive evaluation of gluten-free biscuits formulated using composite flours derived from red rice and cassava. Unlike previous studies that have primarily focused on single flour substitutions or limited product attributes, this research provides a holistic assessment encompassing physicochemical characteristics, microbiological stability during storage, and consumer sensory acceptance. The findings contribute new insights into the potential of combining nutrient-rich pigmented rice and locally abundant cassava flour as sustainable ingredients for developing value-added gluten-free bakery products.

Accordingly, this study aims to evaluate the physicochemical properties, shelf-life stability, and consumer acceptability of gluten-free biscuits formulated using red rice and cassava composite flours, providing a potential strategy for enhancing the nutritional value of bakery products while promoting the utilisation of locally available crops.

Materials and methods

Results

A total of 50 sensory panelists participated in the nine-point hedonic test, and the results are expressed as mean ± standard deviation (Table 2). No significant difference (p > 0.05) was observed in color scores between the control biscuit and those produced with composite flours at different substitution levels. Among all formulations, F3 (50% red rice flour:50% cassava flour) recorded the highest mean color score, followed by F2. The control, F1, and F4 showed similar values, while F5 (100% cassava flour) had the lowest score. For aroma, F3 achieved the highest score and was significantly higher than the control (p < 0.05). Taste scores for F3, F4, and F5 were significantly higher than the control (p < 0.05), with F3 recording the highest mean value. Crispiness varied significantly among samples, and F3 exhibited the highest score, followed by F4. Significant differences were observed between F3 and the control, F1, F2, and F5 (p < 0.05). Overall acceptability increased significantly from F1 to F3 as cassava flour substitution increased up to 50%, followed by a non-significant decline at higher substitution levels. Formulation F3 achieved mean scores above 7 for crispiness and overall acceptability. Panelists described F3 biscuits as pleasant, flavourful, and well-balanced in texture. Consequently, F3 was selected for subsequent analyses. Figure 1 shows biscuits produced using the F3 formulation and the wheat-based control.

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Figure 1. Biscuits produced with red rice-cassava composite flour (F3, left) and biscuits made with 100% wheat flour (control, right). The figure is for illustration purposes only and does not depict the actual size ratio of the samples.

The proximate composition, total energy, and dietary fiber contents of the control biscuit and the optimized F3 formulation are presented in Table 3. The moisture content of the control biscuit was significantly higher than that of F3 (p < 0.05), whereas the ash content was significantly higher in F3 (p < 0.05). Crude protein content was significantly higher in the control than in F3 (p < 0.05). No significant differences (p > 0.05) were observed in crude fat and crude fiber contents between the samples. However, the total dietary fiber content increased significantly in F3 compared to the control (p < 0.05). Carbohydrate content in F3 was significantly higher than in the control (p < 0.05), and the total energy content was also slightly but significantly higher in F3 (p < 0.05).

Physical analysis showed that the control biscuit exhibited significantly greater hardness than the F3 formulation (p < 0.05) (Table 4). Color measurements revealed significant differences between samples, with F3 showing lower lightness, higher redness, and lower yellowness values (p < 0.05). F3 demonstrated a significantly higher spread ratio than the control (p < 0.05), and its bulk density was also significantly higher than that of the control (p < 0.05).

Microbiological count, a_w, and hardness of the control and F3 biscuits during the 8-week storage period are presented in Table 5. Shelf-life evaluation indicated that total plate counts for both control and F3 biscuits remained below 10 CFU g⁻¹ throughout eight weeks of storage. a_w increased gradually over time but remained ≤ 0.65 by week 8. At all storage intervals, F3 exhibited significantly lower a_w than the control (p < 0.05). Hardness declined progressively for both formulations during storage; however, F3 remained consistently softer than the control at all time points (p < 0.05).

Figure 2 depicts consumer acceptance of sensory attributes of composite flour biscuits. A total of 100 respondents participated in the consumer acceptance study, and over 70% expressed positive acceptance across all evaluated attributes for the red rice-cassava biscuit, indicating favourable consumer perception of the composite formulation.

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Figure 2. Consumer acceptance of sensory attributes of composite flour biscuits (50% red rice flour + 50% cassava flour) (n = 100).

Discussion

Sensory performance of composite biscuits is strongly influenced by flour characteristics, processing conditions, and consumer expectations. In this study, different ratios of red rice flour and cassava flour (100:0 to 0:100) were systematically evaluated to investigate how variations in starch composition, fiber content, and functional properties influence biscuit quality. Red rice flour contributes higher dietary fiber, minerals, and bioactive compounds, whereas cassava flour provides a high starch content that can influence dough rheology and texture development during baking. Evaluating a gradient of flour ratios, therefore, allowed identification of the optimal balance between nutritional enhancement and desirable sensory properties in gluten-free biscuit formulations. The comparable color perception between composite and wheat-based biscuits suggests that substitution with red rice and cassava flours does not necessarily compromise visual acceptability. Similar observations have been reported when wheat flour was partially replaced with sweet potato flour without significantly altering biscuit color [36]. The minimal visual difference may be explained by the intrinsic properties of the raw materials. Cassava flour is naturally white and visually similar to wheat flour [37], while the washing step during red rice flour preparation removes portions of pigmented bran containing anthocyanins and proanthocyanidins [38]. As a result, the composite flour appears lighter and closer in color to refined wheat flour, which supports acceptable color development in the final baked product.

Aroma and taste remain critical drivers of consumer liking and are often more influential than appearance. The limited differentiation among some formulations may relate to the use of untrained panelists, who are generally more suited for overall acceptability assessment than for detecting subtle aroma nuances [39]. Nevertheless, aroma plays a dominant role in food enjoyment, contributing up to 80% of overall satisfaction and shaping perceptions of freshness and safety [40]. Taste, encompassing sweetness, saltiness, sourness, bitterness, and umami [41], is widely recognised as a primary determinant of consumer preference and market success [42]. The favourable taste perception of the balanced red rice-cassava blend may reflect complementary flavour interactions between the two flours. This interpretation is consistent with reports that gluten-free bakery products exhibit broad taste variability depending on flour type and formulation strategy [43].

Texture, particularly crispiness, is a defining quality attribute in biscuits. Crispiness is closely associated with hardness and moisture distribution within the product matrix [44], and consumers typically favor thin, crisp biscuits [45]. The improved crispiness observed in balanced composite formulations likely relates to optimal starch gelatinisation and structural setting during baking. Composite and gluten-free biscuits frequently encounter issues such as excessive hardness or dryness; therefore, achieving a balanced texture is crucial for acceptance. Overall acceptability reflects the combined perception of color, aroma, taste, and texture [46], and the sensory performance of the composite formulation indicates that these attributes can be successfully optimised even in gluten-free systems.

Sensory benchmarks further contextualise product quality. Products with mean hedonic scores ≥ 5.0 are generally considered acceptable, while scores ≥ 7.0 indicate high acceptability and can serve as sensory targets in product development [47]. The strong sensory performance of the balanced composite formulation suggests that appropriate flour combinations can yield biscuits with high consumer appeal. This supports the broader understanding that gluten-free or composite flour biscuits can achieve desirable sensory quality when formulation and processing are carefully optimised.

The proximate composition results revealed that the composite biscuit formulation exhibited lower moisture content compared to the wheat-based control, which can be attributed to the higher dietary fiber content of red rice and cassava flours. Dietary fiber has a strong water-binding capacity during dough formation and reduces moisture retention after baking. This explanation is consistent with Shareenie et al. [48], who reported reduced moisture in fiber-enriched composite biscuits. Ng et al. [49] similarly observed that increased fiber promotes water redistribution within the dough matrix, resulting in lower final moisture. Reduced moisture is advantageous in biscuit systems because it enhances crispiness and prolongs shelf-life [50].

The elevated ash content in the composite formulation indicates greater mineral contribution from the raw materials. Similar increases in ash content have been documented in multigrain and legume-based composite biscuits [51, 52]. Red rice flour is known to contain higher levels of minerals such as iron and potassium compared to cassava and refined wheat flours [53], which likely explains this observation. From a nutritional standpoint, higher ash values suggest improved mineral density.

The lower protein content in composite biscuits is mainly due to the absence of gluten-forming proteins in red rice and cassava flours, both of which are low in protein and high in starch [54, 55]. Since gluten proteins constitute about 80% of wheat’s total protein fraction [56, 57], replacing wheat flour naturally leads to protein dilution. This phenomenon is commonly reported in gluten-free bakery formulations and reflects ingredient composition rather than processing inefficiency.

Comparable crude fat levels among formulations are expected because red rice, cassava, and wheat flours contain similar lipid contents. In biscuit systems, fat levels are largely influenced by added shortening or margarine, which primarily contribute to tenderness and mouthfeel [58]. The slightly higher crude fiber content in the composite biscuit is related to the inherent fiber present in red rice and cassava flours. Fiber content depends strongly on flour type [59], and both ingredients contain more insoluble fiber than refined wheat flour [60, 61]. More importantly, the increase in total dietary fiber is consistent with previous reports on fiber-enriched composite biscuits [48]. Dietary fiber, composed of indigestible polysaccharides such as cellulose, hemicellulose, lignin, and pectins, contributes positively to nutritional quality.

The higher carbohydrate content in the composite biscuit reflects the starch-rich nature of red rice and cassava flours [62, 63] and shows an inverse relationship with protein content. Because carbohydrates are the main contributors to caloric value in bakery products, this also explains the slightly higher energy content observed. Such compositional patterns are typical in gluten-free and root-crop-based formulations where starch replaces gluten proteins as the structural backbone. Furthermore, these compositional trends indicate that substituting wheat flour with red rice and cassava flours enhances mineral and dietary fiber content while maintaining comparable fat and energy levels. This supports the potential of composite flours as nutritionally improved alternatives, particularly for consumers seeking fiber-rich or gluten-free bakery products.

These compositional modifications are not only nutritionally relevant but also influence the structural and textural behaviour of the resulting biscuits. The softer texture observed in composite biscuits compared to wheat-based counterparts is widely associated with the absence of a gluten network. Gluten proteins normally form a cohesive, elastic matrix that provides structural rigidity in wheat dough. When wheat flour is replaced with gluten-free flours such as red rice and cassava, this structural framework is weakened, leading to a softer and more friable texture. This reduction in structural rigidity also contributes to the greater spread ratio observed in the composite biscuits, as a weaker dough structure allows increased lateral expansion during baking. Similar textural reductions in composite biscuits have been reported [31, 48, 52]. Higher lipid contributions can further tenderize the crumb by interfering with starch-protein interactions [64]. In addition, composite flours often possess coarser particle size distributions (typically < 250 µm) than refined wheat flour (< 130 µm), which can promote a more open structure and reduce resistance to compression [65].

Color differences in composite biscuits are primarily linked to ingredient pigmentation and thermal reactions during baking. Red rice flour contains natural pigments from the bran layer that contribute to darker and more reddish tones, while Maillard browning further intensifies color development during baking [59]. Reduced yellowness can be explained by the thermal degradation of carotenoids naturally present in cassava and rice flours, a phenomenon frequently observed in gluten-free and composite baked products [65]. Such color shifts are expected when pigmented or whole-grain flours are incorporated into bakery formulations.

Greater biscuit spread during baking is commonly related to dough viscosity and water-binding behaviour. Composite flours generally exhibit lower water-binding capacity than wheat flour, which can produce softer doughs that spread more readily during baking [51, 66]. Larger particle size also reduces the specific surface area available for hydration, limiting water absorption and promoting lateral expansion [65]. These factors collectively influence dough flow and final biscuit geometry.

Denser biscuit structures are often reported when wheat flour is replaced with alternative flours, as shown by Zouari et al. [67] and Dada et al. [68]. Bulk density is governed by flour composition, particle packing, and internal air cell structure [69]. It plays a critical role in determining product texture, packaging efficiency, and mouthfeel perception [70]. Variations in starch composition, fiber content, and particle morphology in composite flours can modify packing behaviour and internal structure, contributing to these differences.

Taken together, the physicochemical behaviour of red rice-cassava composite flours explains the softer texture, darker appearance, greater spread, and denser structure typically observed in gluten-free biscuits. These characteristics are consistent with the known functional roles of gluten absence, flour composition, and water-fat interactions in composite bakery systems and help explain the sensory responses reported for such products.

Shelf-life evaluation is essential for guiding formulation and packaging decisions in bakery products, particularly those formulated with gluten-free composite flours. In this study, microbiological stability, a_w, and hardness were selected as key indicators of storage quality because they represent the primary factors influencing the safety, texture, and consumer acceptability of low-moisture baked products such as biscuits. Low-moisture systems and reduced a_w are well known to suppress microbial growth and contribute to the extended stability of products such as cookies and crackers. Maintaining a_w below the critical limits for yeast and mould development is therefore a key factor in ensuring microbiological safety and shelf stability in baked goods.

Differences in a_w between formulations can be partly explained by compositional factors, especially dietary fiber content. Fiber-rich ingredients enhance water binding during dough formation and can facilitate greater moisture loss during baking, resulting in lower a_w in the final product. Over storage, gradual increases in a_w are commonly associated with the hygroscopic nature of biscuits and the permeability of packaging materials such as LDPE, which allow moisture transfer from the environment.

Textural changes during storage are also strongly influenced by moisture redistribution within the biscuit matrix. Softening over time is a typical phenomenon in baked products and is often linked to rising a_w and internal water migration. Fiber incorporation may further influence texture by modulating starch retrogradation, potentially reducing the rate of structural firming or fracture development. Collectively, the relationships among microbiological stability, a_w, and texture highlight the importance of moisture control and formulation design in maintaining the quality and acceptability of composite biscuits during storage. These factors play a critical role in determining shelf-life performance and consumer-perceived freshness in gluten-free and composite bakery products.

Consumer acceptance of composite biscuits is closely linked to familiarity, sensory expectations, and perceived product quality. The mixed prior exposure of consumers to composite flour products suggests that acceptance is not solely dependent on familiarity but also on the sensory performance of the product itself. Positive acceptance across attributes indicates that well-formulated composite biscuits can meet consumer expectations even when non-wheat flours are used. The favourable perception of the red rice-cassava biscuit is consistent with previous studies showing that composite flour biscuits can achieve high sensory acceptance when appropriate flour combinations are applied. For example, rice-green gram-potato composite biscuits achieved acceptability ratings ranging from “like moderately” to “like very much” compared to controls [71]. Likewise, wheat-millet biscuits fortified with Moringa oleifera and Camellia sinensis leaf powders also received strong sensory ratings, highlighting the growing consumer openness toward functional bakery products made with local or alternative flours [72].

These observations suggest that successful composite formulations can balance flavour and texture to overcome potential biases against non-traditional ingredients. In this context, the sensory appeal of the F3 formulation indicates that composite flour biscuits can be positioned as acceptable and potentially attractive alternatives to conventional wheat-based products. This supports the broader notion that incorporating local flours into bakery products can be feasible from a consumer acceptance standpoint, provided that sensory quality is maintained.