Effects of the addition of xanthan gum and rice flour to maize starch on quality of gluten-free biscuit

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Effects of the addition of xanthan gum and rice flour to maize starch on quality of gluten-free biscuit

Marko Jukic1, Daliborka Koceva Komlenic1, Gjore Nakov2, Matea Begic1, Sandi Keresturi1, Jasmina Lukinac1

Faculty of Food Technology, Josip Juraj Strossmayer University of Osijek, Croatia

College of Sliven, Technical University of Sofia, Sliven, Bulgaria



Abstract

Introduction. The aim of this research was to study the interaction of xanthan gum and a mixture of rice flour and corn starch, its effects on the quality of gluten-free cookies, as well as optimization of the gluten-free cookie recipe to produce high- quality products.

Materials and methods. The effects of addition of rice flour to maize starch in ratio 6:4, 8:2 and 10:0 and the xanthan gum in amounts of 0, 2 and 4% were analysed. Texture parameters (breaking force), dimensions (spread factor), colour (browning index), water activity and total sensory score were determined as response variables. xanthan gum gluten cookie

Results and discussion. An increase in xanthan concentration led to an increase in hardness, browning index and water activity while reducing the spread factor of the gluten- free biscuits. Increased amounts of maize starch in the gluten- free mixture contributed in particular to a lower hardness and a lower browning index of the gluten-free biscuits, while at the same time the spreading of the biscuits increased.

An increase in the addition of xanthan gum led to an increase in hardness, browning index and water activity as well as a reduction in the spread factor of gluten-free biscuits. Meanwhile, increasing the amount of maize starch in the gluten- free mixture in combination with rice flour contributed significantly to a reduction in the hardness and browning index of gluten-free biscuits, while at the same time increasing the spread of the biscuits. The curvilinear effects of the addition of xanthan gum and maize starch on the sensory score showed that the optimal amounts were between the minimum and maximum addition values in the experiment.

Conclusions. The optimal conditions to obtain gluten-free biscuits of high quality with total sensory score 7.7 were as follows: ratio of rice flour to maize starch 88.9:11.1 in combination with an addition of 1.1% xanthan gum.



Introduction

It is believed that wheat grain can be an allergen and, according to statistics, about 1% of the world population is gluten sensitive (Wang et al., 2017). The rising prevalence of gluten-related disorders, including coeliac disease and non-celiac gluten sensitivity, has led to an increasing demand for gluten-free products worldwide (Ivanov et al., 2021; Stabnikova et al 2021). To produce gluten-free bakery products, partial replacement of wheat flour with gluten-free cereal flours, such as rise (Sciarini et al., 2010), soy (Taghdir et al., 2017), and sorghum (Marston et al., 2016) flours has been proposed. Among gluten-free bakery products, gluten-free biscuits are a popular choice due to their versatility and palatability (Di Cariano et al., 2018).

However, unlike traditional wheat flour biscuits, gluten-free biscuits require careful consideration of alternative ingredients to achieve the desired texture, flavour, and appearance. Rice flour, maize starch, and xanthan gum are often used in gluten-free baking recipes due to their functional properties (Di Cariano et al., 2018). Wheat biscuits made from short dough with high fat and sugar content usually rely on the gelatinisation of starch rather than the development of gluten. Therefore, the use of gluten-free flours offers the potential to produce gluten-free biscuits with customised texture and spread (Mancebo et al., 2015a; Xu et al., 2020).

Rice flour, a main ingredient in many gluten-free recipes, serves as a corn base due to its unique properties. Rice flour has a neutral flavour and light texture, making it an ideal substitute for traditional wheat flour in gluten-free baking (Mancebo et al., 2015b). Its widespread use is due to its versatility and its ability to mimic the texture and structure of gluten-containing products, ensuring a satisfying sensory experience for people with gluten- related disorders (Benkadri et al., 2018).

Maize starch, another key component in gluten-free formulations, plays a crucial role in improving the structural integrity and mouthfeel of baked goods. Maize starch acts as a thickening agent and adds a smooth and desirable texture to various foods. In gluten-free baking, maize starch contributes to the viscosity of the dough, facilitating proper handling and shaping of the dough while improving the overall palatability of the final product (Perez- Carrillo et al., 2019).

Xanthan gum, a microbial high molecular weight exopolysaccharide synthesised by Xanthomonas campestris strains, serves as a multifunctional additive in gluten-free baking. Xanthan gum is known for its unique thickening and stabilising properties. It increases the viscosity and elasticity of dough, improving their handling and contributing to the desired texture of baked goods. In addition, xanthan gum helps to prevent ingredient separation and syneresis, resulting in products with improved storage stability and longer freshness (Benkadri et al., 2020).

The combined use of rice flour, maize starch and xanthan gum in gluten-free formulations enables the production of products that are very similar to their gluten- containing counterparts in terms of texture, flavour and appearance. By carefully adjusting the ratio of these ingredients, it is possible to achieve optimal results by balancing factors such as texture, structural integrity, and sensory properties to meet consumer expectations.

Overall, the use of rice flour, maize starch and xanthan gum represents a fundamental approach to gluten-free formulation allowing the development of high-quality, palatable products. Through continuous research and innovation, the gluten-free industry is able to offer a wide range of products that meet the nutritional needs and culinary preferences of consumers worldwide. Therefore, recipe optimisation is crucial for the production of gluten- free biscuits that meet consumer expectations and preferences (Lisovska et al., 2020).

The aim of this research was to study the interaction of xanthan gum and a mixture of rice flour and corn starch, its effects on the quality of gluten-free cookies, as well as optimization of the gluten-free cookie recipe to produce high-quality products.



Materials and methods

Materials

The following raw materials were used in this study: rice flour with 6.5% protein content (Galleria Internazionale Ltd., Zagreb, Croatia); maize starch (Dr. August Oetker KG, Bielefeld, Germany); xanthan gum powder (Doves Farm Foods Ltd., Berkshire, UK); shortening (Zvijezda plus Ltd., Zagreb, Croatia); granulated sugar (sucrose); table salt (NaCl), and sodium bicarbonate (NaHCO3).

Methods

Design of experiment

The experimental design was based on the Response Surface Methodology and was created using the statistical software Statistica (ver. 14.0.0.15, TIBCO Software Inc., Palo Alto, USA) in a three-level full factorial design (32). The independent variables included the ratio of rice flour to maize starch in the GLUTEN-FREE flour blend and the amount of xanthan gum added. The experimental design included nine experiments with three replicates of the central point (Table 1). The actual values were set at 60:0, 80:20 and 100:0 for the rice flour to maize starch ratio and at 0, 2 and 4% of the xanthan gum addition.

Production of gluten-free biscuits

The biscuits were prepared according to the experimental design (Table 1).

Table 1

Design of experiment

N	Ratio: Rice flour : Maize starch	Xanthan gum,%
1	60	0
2	60	2
3	60	4
4	80	0
5	80	2
6	80	4
7	100	0
8	100	2
9	100	4

After weighing all ingredients (expressed in baker's percentages, where the flour mixture consisted of rice flour and maize starch), shortening, 40%; sucrose, 42%; salt, 1.25%, and sodium bicarbonate, 1.1%, were mixed for 3 minutes at low speed using an electronic mixer (Gorenje MMC800W, Velenje, Slovenia). After the addition of water, 24%, mixing was continued at low speed for another minute, followed by another minute at medium speed.

The gluten-free flour mixture (100%) was then added and mixed at low speed for 2 minutes. The resulting dough was then rounded and refrigerated at 8 °C for 30 minutes (covered with plastic wrap). The dough was then rolled out to a thickness of 7 mm and pieces of dough with a diameter of 60 mm were cut out using a cylindrical mould. Baking took place in a convection oven (Wiesheu Minimat Zibo, Wiesheu GmbH, Grohbottwar, Germany) at 205 °C for 12 minutes. The biscuits were baked in double batches. After a cooling time of one hour, the gluten-free biscuits were analysed.



Physical analysis

The dimensions of the gluten-free biscuits were assessed according to the International Method 10-50.05 protocol (Cereals and Grains Association, 2010). First, the total width of six biscuits stacked side by side was measured, then each biscuit was rotated 90° and measured again. The average width was calculated and divided by six to determine W, cm. Six biscuits were then stacked vertically and their combined height was measured. After the biscuits were randomly rearranged, the height was measured again. The average height of the six biscuits was divided by six to obtain T, cm. The spread factor was then calculated as W/T multiplied by ten. Measurements were taken on six sample biscuits from each batch.

The texture of the gluten-free biscuits was assessed using the TA.XTplus Texture Analyser (Stable Microsystems Ltd., Surrey, UK) by the three-point bend test. The knife blade moved towards the biscuit, which was positioned between two lower supports 30 mm apart. The test was performed at a blade speed of 1 mm/s until the breaking point was reached. The peak force (N) was recorded, giving an indication of the softness/hardness of the biscuit. The evaluation was carried out on three biscuits from each batch.

The water activity (aw) in the ground samples of the gluten-free biscuits was determined using the Hygropalm AW1 device (Rotronic AG, Bassersdorf, Switzerland).

Color evaluation

The colour of the surface of the gluten-free biscuits was assessed using the CR-400 Chromameter (Konica Minolta, Japan). The CIELab colour model was used, in which the L* value (ranging from 0 to 100) represents the lightness or luminance, the b* value (ranging from -128 to 127) represents the blue-yellow axis and the a* value (ranging from -128 to 127) represents the green-red axis within the colour space. The CIELab values were used to calculate the browning index (BI) according to the following equations (Dadali et al., 2007):

Sensory analysis

The sensory analysis of gluten-free biscuits was carried out by a panel of fifteen trained persons (students and employees of the Faculty of Food Technology in Osijek, Croatia). Before starting the analysis, all panel members gave informed consent in accordance with the European Union guidelines for ethics and food-related research (Alfonsi et al., 2012). The sensory panel consisted of nine women and six men, with a median age of 24 years. Inclusion criteria for panel members included the absence of health conditions that could affect sensory evaluation (such as anosmia or colour blindness) and a typical preference for eating similar types of biscuits. The sensory evaluations were carried out in a dedicated tasting room equipped with individual test booths. Participants were given a brief introduction to familiarise them with the study and the samples to be evaluated. All gluten-free biscuit samples were presented to the tasters simultaneously and they were instructed to rinse their mouths with water between tastings. Sensory properties were rated using a 9-point hedonic scale, with scores ranging from 1 to 9 as follows: extremely dislike (1), very much dislike (2), moderately dislike (3), slightly dislike (4), neither dislike nor like (5), slightly like (6), moderately like (7), very much like (8), and extremely like (9). The sensory properties assessed were the external appearance, texture, flavour and taste. To optimise the recipe, the total sensory score was used as one of the response variables, which was determined by calculating the average of the ratings of the above-mentioned sensory properties.

Modelling the data and optimising the gluten-free biscuit recipe

The optimisation of the gluten-free biscuit recipe was carried out using Response Surface Methodology, with texture parameters (breaking force), dimensions (spread factor), colour (browning index), water activity and total sensory score as response variables. Regression analysis was used to determine the relationships between the independent input variables and each dependent output variable defined by the second-degree polynomial response surface model.

The analysis of variance (ANOVA) determined the statistical significance of each regression coefficient and the mathematical models were evaluated using the coefficient of determination (R2). On the basis of the mathematical models obtained, response surfaces were created, which represent 3D diagrams that visually illustrate how changes in the ratio of rice flour to maize starch and the proportion of xanthan gum affect various quality parameters of gluten-free biscuits.

In the final step, the optimum ratio of rice flour to maize starch and the optimum proportion of xanthan gum were determined using the desirability function. The optimisation procedure involved converting all monitored responses into individual desirability functions with values between 0 and 1, with the overall desirability function determined as their geometric mean. The responses were maximised (browning index and total sensory score), minimised (water activity) or set to a target value (peak force at 50 N and spread factor at 54.3). The optimisation of the gluten-free biscuit formulation was performed using the statistical software Statistica (ver. 14.0.0.15, TIBCO Software Inc., Palo Alto, USA).



Results and discussion

In the field of gluten-free baking, achieving the desired texture, appearance and overall quality of products is a major challenge, especially due to the absence of gluten, an important structural component in conventional baked goods. Therefore, researchers and food technologists are constantly exploring different ingredients and recipes to develop high- quality gluten-free products that meet consumer expectations. Rice flour and maize starch are commonly used ingredients in gluten-free baking, while xanthan gum is an important additive that mimics the functional properties of gluten. In order to optimise the recipes of gluten-free biscuits, it is important to understand how these ingredients interact and influence the final product. In this context, the present study investigated the influence of different ratios of rice flour to maize starch as well as different levels of xanthan gum on the quality of gluten-free biscuits. According to the experimental design (Table 2), baking tests were carried out and the quality of the gluten-free biscuit samples was examined in order to optimise the recipe with regard to the ratio of rice flour to maize starch and the proportion of xanthan gum. The ratio of rice flour to maize starch in the gluten-free mix was 60:40, 80:20 and 100:0, while the proportion of xanthan gum was 0%, 2% and 4%. The total amount of rice flour and maize starch mixture was constant in all samples. The actual samples of baked gluten-free biscuits are shown in Figure 1.

Figure 1. Produced gluten-free biscuits with different ratios of rice flour to maize starch and different proportions of xanthan gum

The results of testing the influence of different formulations on the peak force required to break gluten-free biscuits during the 3 -point band test are shown in Figure 2 and in Tables 2 and 3.

Quality parameters of gluten-free biscuits

Table 2

Run	Rice flour : Maize starch ratio	Xanthan gum,%	Dependent variables
			Peak force (N)	Spread factor	Browning index	Water activity	Total sensory score
1	60:40	0	15.7	60.9	38.6	0.407	6.0
2	60:40	2	37.6	54.0	39.0	0.452	6.9
3	60:40	4	47.2	53.2	43.7	0.505	6.8
4	80:20	0	17.0	57.8	43.6	0.424	7.2
5	80:20	2	56.5	53.1	44.2	0.465	7.5
6	80:20	4	59.4	52.4	44.6	0.482	7.3
7	100:0	0	28.1	55.5	45.3	0.445	6.6
8	100:0	2	62.6	53.0	47.2	0.477	7.5
9	100:0	4	67.8	52.3	47.3	0.515	7.2
10	80:20	2	55.2	53.6	43.5	0.483	7.3
11	80:20	2	58.0	52.9	45.0	0.471	7.8
12	80:20	2	55.1	52.5	44.4	0.484	7.8
Ratio of rice flour to maize starch	Xanthan gum addition
	0%	2%	4%

Table 3

Analysis of variance (ANOVA) and regression model of the influence of the different xanthan gum content and the ratio of rice flour to maize starch on biscuit hardness

Source of variation	SS	DF	MS	F	P
Xx - Rice flour : Maize starch ratio	560.7	1	560.7	40.1	0.001*
X2	14.2	1	14.2	1.0	0.352
X2 - Xanthan gum content	2148.6	1	2148.6	153.7	< 0.001*
X 2	537.8	1	537.8	38.5	0.001*
X, * X 2	17.0	1	17.0
Error	83.9	6	14.0
Total	3496.8	11
Model: Y = -45.602 + 1.305Xi + 19.538X2 - 0.006X12 - 3.550X22 + 0.052XiX2
R2 = 0.976

Of the formulations tested, the biscuits without maize starch (100% rice flour) and 4% xanthan gum had the highest hardness (67.8 N). The main effect of the ratio of rice flour to maize starch in the flour blend was statistically significant (F = 40.1, p = 0.001). The results showed a clear trend of decreasing hardness with increasing maize starch content. This observation can be attributed to the ability of maize starch to interfere with the formation of a strong protein network, resulting in lower biscuit hardness. Fitzgerald et al. (2003) suggested that rice flour may lead to the development of a gel matrix of denatured proteins when heated, which may provide specific mechanical reinforcement and help to stabilise the overall structure. The increasing hardness of gluten-free biscuits with increasing proportion of rice flour in the mixture with maize starch can be attributed to the fact that the protein content in the flour mixture increases with the higher addition of rice flour. The quadratic effect of the ratio of rice flour to maize starch was not statistically significant (F = 1.0, p = 0.352), suggesting that the relationship between maize starch content and peak force may have been linear rather than curvilinear in this context.

Figure 2. Response surface for the effect of different xanthan gum content and the ratio of rice flour to maize starch on biscuit hardness

The main effect of xanthan gum content was also highly significant (F = 153. 7 P < 0.001), indicating its significant influence on the increase in hardness of the biscuits. When the amount of xanthan gum was increased from 0% to 4%, there was a consistent increase in biscuit hardness at each ratio of rice flour and maize starch. Xanthan gum is known for its binding properties and its ability to improve the texture of gluten-free products. The increase in hardness with higher xanthan gum concentrations supports its role in improving the structural integrity of gluten-free biscuits (Benkadri et al., 2020). The quadratic effect of xanthan gum content was also statistically significant (F = 38.5, p = 0.001), indicating a curvilinear relationship between xanthan gum content and peak force. The interaction between the ratio of rice flour to maize starch in the flour mixture and xanthan gum content was not statistically significant (F = 1.2, p = 0.312), indicating that the combined effect of these factors on peak force was not significantly different from what would have been expected based on their individual effects. The regression model provided coefficients for each predictor variable and their interactions. The model had a high coefficient of determination (R2 = 0.976), suggesting that the model explained 97.6% of the variance in peak force, indicating a very good fit.

Several other studies have also investigated the effect of xanthan gum on the quality of gluten-free biscuits (Devisetti et al., 2015; Gul et al., 2018; Shahzad et al., 2021). Their studies showed that the addition of xanthan gum significantly changed the texture characteristics of gluten-free biscuits, particularly by increasing their hardness. Gul et al. (2018) attributed this effect to the branched structure of xanthan gum, along with its interactions with other ingredients in the biscuits. However, it is worth noting that Benkadri et al. (2018) found an opposite effect of xanthan gum on biscuit hardness. This discrepancy could be due to differences in the methodology of texture analysis. While our study employed a 3-point bend test, Benkadri et al. (2018) used the Volodkevich bite upper jaw probe method.

The results presented in Table 2 and Figure 3 show that the spread factor of gluten-free biscuits tends to decrease with the addition of xanthan gum and increases with the increase of maize starch content in the gluten-free flour mixture. The highest biscuit spread factor (60.9) was observed in a sample without added xanthan gum and with a 60:40 rice flour to maize starch ratio, and the lowest spread factor (52.3) was observed in the sample with 4% xanthan gum without maize starch in the biscuit recipe. This decrease in spread factor could be due to the higher viscosity caused by the combination of rice flour and xanthan gum, which inhibits the spread of the biscuit dough during baking.

The analysis of variance (ANOVA) and the regression model revealed significant effects of both the ratio of rice flour to maize starch and the xanthan gum content on the spread factor of gluten-free biscuits. The linear effect of maize starch ratio was found to be statistically significant (F = 24.4, p = 0.003), indicating that changes in the ratio of rice flour to maize starch significantly affected the spread factor of the biscuits. In addition, the quadratic effect of rice flour to maize starch ratio was not statistically significant (F = 1.4, p = 0.279), indicating that the relationship between maize starch ratio and spread factor was mostly linear (Table 4).

Table 4

Analysis of variance (ANOVA) and regression model of the influence of the different xanthan gum content and the ratio of rice flour to maize starch on biscuit spread

Source of variation	SS	DF	MS	F	P
Xx - Rice flour : Maize starch ratio	8.6	1	8.6	24.4	0.003*
X2	0.5	1	0.5	1.4	0.279
X2 - Xanthan gum content	44.9	1	44.9	127.4	< 0.001*
X 2	10.9	1	10.9	30.8	0.001*
X, * X 2	5.0	1	5.0	14.3	0.009*
Error	2.1	6	0.4
Total	75.1	11
Model: Y = 73.985 - 0.289X - 5.630X2 + 0.001X? + 0.504X22 + 0.028X2X2
R2 = 0.972

On the other hand, the linear effect of xanthan gum content was highly significant (F = 127.4, p < 0.001), indicating that variations in xanthan gum content significantly affected the spread factor of gluten-free biscuits. In addition, the quadratic effect of xanthan gum content was also statistically significant (F = 30.8, p = 0.001), highlighting the non-linear relationship between xanthan gum content and spread factor. The interaction effect between the ratio of rice flour to maize starch and xanthan gum content was significant (F = 14.3, p = 0.009), indicating that the combined effect of these factors on the spread factor differs from their individual effects. As in the case of gluten-free biscuit hardness, the resulting regression model showed a high degree of agreement (R2 = 0.972).

Figure 3. Response surface for the effect of different xanthan gum content and the ratio of rice flour to maize starch on biscuit spread

Comparing these results with previous studies, they are consistent with the studies by Shahzad et al. (2021), Gul et al. (2018), Kaur et al. (2015) and Devisetti et al. (2015), who also reported a significant decreasing effect of xanthan gum content on the spread factor of gluten-free biscuits. Xanthan gum, which is known for its thickening and stabilising properties, could contribute to the lower spread factor by increasing the dough viscosity. However, in contrast to the influence of xanthan gum, the addition of maize starch causes an increase in the spread factor of biscuits, which, similar to the effect on biscuit hardness, can be explained by the general decrease in the amount of protein in the gluten-free mixture. This weakening of the biscuit structure leads to increased spread. This conclusion is also supported by Schroder et al. (2003), who found that low protein and high starch ingredients in gluten- free mixes contribute to weak structural stability. These results underline the importance of both the rice flour to maize starch ratio and the xanthan gum content in determining the spread factor of gluten-free biscuits, which has implications for optimising gluten-free biscuit formulations to achieve the desired texture properties.



Table 5

Analysis of variance (ANOVA) and regression model of the influence of the different xanthan gum content and the ratio of rice flour to maize starch on biscuit browning index

Source of variation	SS	DF	MS	F	P
X1 - Rice flour : Maize starch ratio	57.0	1	57.0	50.0	< 0.001*
X12	1.6	1	1.6	1.4	0.281
X2 - Xanthan gum content	10.9	1	10.9	9.6	0.021*
X 2	0.1	1	0.1	0.1	0.743
Xi * X 2	2.4	1	2.4	2.1	0.197
Error	6.8	6	1.1
Total	78.8	11
Model: Y = 15.183 + 0.503 X + 2.000X2 - 0.002X? + 0.056X22 - 0.019X2X2
R2 = 0.913

Figure 4. Response surface for the effect of different xanthan gum content and the ratio of rice flour to maize starch on browning index of biscuits

It is generally known that gluten-free bakery products often have a less pronounced colour than their wheat flour counterparts. This applies in particular to products that contain certain amounts of different starch preparations in their composition. The reason for this is that different starches are often used in the production of gluten-free products, including gluten-free biscuits, due to their techno-functional properties. However, large amounts of starch preparations in the recipe can lead to a pale colour of the product, which is less accepted by consumers. Their use reduces the total protein content in the flour blend, which is involved in the Maillard reactions, known to be one of the most important mechanisms in the formation of product colour (Mancebo et al., 2015b). Therefore, in our study we also considered the browning index of biscuits as an important parameter in the evaluation of the quality of gluten-free biscuits.

The results obtained showed that increasing the maize starch content tends to reduce the browning index of gluten-free biscuits. For example, with a xanthan gum content of 0%, the browning index increases from 38.6 to 45.3 when the maize starch content in gluten-free blend decreases from 40% to 0% (Table 2 and Figure 4). Conversely, the effect of the xanthan gum content on the browning index appears to be less pronounced compared to maize starch. Although the browning index increases slightly with a higher xanthan gum content, the effect is not as significant as that of maize starch. The influence of xanthan gum on the increase in the browning index of gluten-free biscuits is probably only due to the fact that the samples with higher levels of xanthan gum also had a higher moisture content and higher water activity, which could have influenced the slightly darker appearance of the biscuits. The availability of water molecules can play an important role in facilitating Maillard reactions. Higher water activity provides a more favourable environment for these reactions as it increases the mobility of reactants and promotes their interaction. Consequently, biscuits with higher water activity are more prone to extensive Maillard browning, resulting in darker and more intensely coloured crusts and surfaces (Lund et. al., 2017).

According to the ANOVA results (Table 5), both the ratio of rice flour to maize starch (F = 50.0, p < 0.001) and the xanthan gum content (F = 9.6, p = 0.026) significantly influenced the browning index of gluten-free biscuits, with only a linear relationship with the browning index. However, the interaction between the ratio of rice flour to maize starch and xanthan gum showed no significant effect on the browning index. The model had a high coefficient of determination (R2 = 0.913).

A comparison of these results with previous studies showed similar trends regarding the influence of maize starch and xanthan gum content on the browning index of gluten-free biscuits. The study by Mancebo et al. (2015) also reported significant effects of the influence of increased maize starch addition on the decrease in brightness of gluten-free biscuits made from rice flour and maize starch, confirming the consistency of the current results. Similar results were obtained in the gluten-free bread samples, where the replacement of rice flour with starch also resulted in a lighter crust colour compared to the gluten-free bread with rice flour in the control (Mancebo et al., 2015; Minarro et al., 2010). The darker colour of gluten- free biscuits with added xanthan gum was also observed in the study by Shahzad et al. (2021).

Table 6

Analysis of variance (ANOVA) and regression model of the influence of the different xanthan gum content and the ratio of rice flour to maize starch on biscuit water activity

Source of variation	SS	DF	MS	F	P
Xx - Rice flour : Maize starch ratio	0.0009	1	0.0009	5.6	0.055
X2	0.0000	1	0.0000	0.1	0.815
X2 - Xanthan gum content	0.0085	1	0.0085	54.1	< 0.001*
X 2	0.0002	1	0.0002	1.6	0.257
X, * X 2	0.0002	1	0.0002	1.2	0.307
Error	0.0009	6	0.0002
Total	0.0108	11
Model: Y = 0.377 + 0.0002X + 0.043X2 + 0.000005X? - 0.002X22 - 0.0002XrX2
R2 = 0.913



Figure 5. Response surface for the effect of different xanthan gum content and the ratio of rice flour to maize starch on biscuit water activity (aw)

Water activity (aw) is an important parameter in gluten-free biscuits as it influences various aspects of their quality, including texture, shelf life and microbial stability (Cervenka et al., 2006). Therefore, maintaining an optimal water activity level is crucial for the desired quality of gluten-free biscuits. As previously mentioned, water activity also influences Maillard browning reactions, which contribute to the development of colour, flavour and aroma in baked goods (Lund et al., 2017). Therefore, understanding and controlling water activity are important aspects in the formulation of gluten-free biscuits to achieve optimal texture, shelf life and sensory quality.

The water activity of the gluten-free biscuits, as shown in Table 2 and Figure 5, varied in the different formulations, which were characterised by different contents of maize starch and xanthan gum. Increasing the xanthan gum content in particular led to a significant increase in water activity, as can be seen from the rising values when the xanthan gum content was increased from 0% to 4%. The influence of maize starch content on water activity was not statically significant, with marginal differences observed between the different maize starch contents. These results indicate that xanthan gum plays a greater role than maize starch in determining the water activity of gluten-free biscuits.

A further investigation using the regression model shown in Table 6 confirmed these observations. The model showed a significant positive coefficient for xanthan gum content (F = 54.1, p < 0.001), indicating its strong influence on water activity. In contrast, the coefficient for maize starch was relatively small and not statistically significant (F = 5.6, p =

055), supporting the assumption that variations in maize starch content have minimal effects on water activity. In addition, the lack of significance of the interaction term indicates that the combined effect of maize starch and xanthan gum on water activity was not significantly different from their individual effects. The coefficient of determination was the same as in the model for the browning index (R2 = 0.913).

These results emphasise the importance of xanthan gum in influencing the water activity of gluten-free biscuits, which is probably due to its hygroscopic properties and its ability to interact with water molecules. Comparing these results with the existing literature, our findings are in agreement with the studies of Gul et al. (2018), Benkadri et al. (2018), Benkadri et al., 2020) and Shahzad et al. (2021), who also reported a significant influence of xanthan gum content on the increased water activity in gluten-free biscuits.

Table 7

Analysis of variance (ANOVA) and regression model of the influence of the different xanthan gum content and the ratio of rice flour to maize starch on total sensory score

Source of variation	SS	DF	MS	F	P
Xx - Rice flour : Maize starch ratio	0.427	1	0.427	7.8	0.032*
X2	0.667	1	0.667	12.1	0.013*
X2 - Xanthan gum content	0.375	1	0.375	6.8	0.040*
X 2	0.540	1	0.540	9.8	0.020*
X, * X 2	0.010	1	0.010	0.2	0.685
Error	0.330	6	0.055
Total	2.949	11
Model: Y = -2.333 + 0.216X + 0.675X2 - 0.001 X? - 0.113X22 - 0.001XrX2
R2 = 0.888

The total sensory score of the gluten-free biscuits, as shown in Table 2 and Figure 6, showed variability between the different recipes, characterised by different ratios of rice flour to maize starch and xanthan gum content. These variations indicate that both maize starch and xanthan gum content play an important role in influencing the overall sensory perception of gluten-free biscuits. The lowest sensory rating (6.0) was found for the samples with no added xanthan gum and the 60:40 ratio of rice flour and maize starch in the flour blend. The samples with an addition of 2% xanthan gum, regardless of the ratio of rice flour to maize starch, received the highest total sensory scores (6.9-7.5).

Figure 6. Response surface for the effect of different xanthan gum content and the ratio of rice flour to maize starch on total sensory score

A further investigation using the regression model shown in Table 7 provided information on the individual and combined effects of the ratio of rice flour to maize starch and the xanthan gum content on the total sensory score. The model showed significant linear and quadratic effects for both maize starch and xanthan gum content, indicating that they influence the total sensory score of gluten-free biscuits. In addition, the F values for the quadratic effects (F = 12.1, p = 0.013 and F = 9.8, p = 0.020) were higher than for the linear effects (F = 7.8, p = 0.032 and F = 6.8, p = 0.040), suggesting that the optimal amount of maize starch and xanthan gum lies between the minimum and maximum addition levels. In contrast to the linear and quadratic effects observed for the ratio of rice flour to maize starch and xanthan gum content, the interaction term was not statistically significant, indicating that the combined effect on sensory score does not deviate significantly from the individual effects.

Comparing these results with the existing literature, our findings are consistent with a previous study by Mancebo et al (2015a), which also reported that a high addition of maize starch reduces the sensory acceptability of gluten-free biscuits. Benkadri et al. (2018) concluded that the addition of xanthan gum in an amount of 0.5% to 1.5% did not significantly affect sensory acceptability. Similar to our results, Gul et al. (2018) found that the addition of xanthan gum improved the sensory quality of gluten-free biscuits, but this improvement was only noticeable up to an addition level of 3%, after which the values decreased slightly. Shahzad et al. (2021) found that the addition of xanthan gum in the recipe for the production of biscuits led to a decrease in sensory ratings, but in their study, they added xanthan gum at a level of 5%, as opposed to the maximum of 4% in our study.

The final step in this study was the optimisation of the gluten-free biscuit recipe. From the results presented above, it can be seen that the quality of gluten-free biscuits was influenced by several factors and not by a single main factor. Each independent variable played a significant role in shaping the characteristics of the gluten-freE biscuits. All effects resulting from the response surface plots were taken into account during the optimisation process, as the ideal solution requires a balance between the different responses. In this study, the responses were maximised (browning index and total sensory score), minimised (water activity) or fixed at a target value (peak force at 50 N and spread factor at 54.3).

The optimisation process resulted in the following optimal conditions to achieve the desired response values: the ratio of rice flour to maize starch 88.9:11.1 and an addition of 1.1% xanthan gum. The predicted response values based on these conditions were: peak force 47.7, spread factor 54.3, browning index 44.7, water activity 0.458 and total sensory score 7.4. A desirability score of 0.72 was calculated for the gluten-free biscuits using the optimal recipe (Figure 7). After identifying the best solution, the gluten-free biscuits were produced according to the optimised recipe. These biscuits were analysed for all five responses to validate the predictive capability of the models and to compare the theoretical predictions with the experimental results. The experiments carried out under optimal conditions showed a high degree of agreement between the predicted and experimental values for all responses (Table 8). The total sensory score obtained (7.7) was even higher than the score predicted by the model (7.4), placing the biscuit samples between a score of 7 (moderately liked) and 8 (very much liked) on the hedonic scale used, indicating a high preference for the gluten-free biscuits produced.

Figure 7. Response surface for the effect of different xanthan gum content and the ratio of rice flour to maize starch on desirability of gluten-free biscuits



Table 8

Optimal formulation of gluten-free biscuits with predicted and experimental values of response variables

Parameter	Predicted	Experimental
Peak force (N)	47.7	49.1
Spread factor	54.3	53.4
Browning index	44.7	44.1
Water activity	0.458	0.449
Total sensory score	7.4	7.7
Optimal rice flour to maize starch ratio - 88.9:11.1
Optimal xanthan gum content - 1.1%



Conclusions

This study emphasised the significant influence of the ratio of rice flour to maize starch in the flour blend and the xanthan gum content on the quality characteristics of gluten- free biscuits. The observed effects underlined how important it is to optimise the recipe of gluten-free biscuits in order to achieve the desired sensory properties and consumer acceptance.

An increase in the addition of xanthan gum led to an increase in hardness, browning index and water activity as well as a reduction in the spread factor of gluten-free biscuits.

Increasing the proportion of maize starch in the gluten-free mixture in combination with rice flour contributed significantly to a reduction in the hardness and browning index of gluten-free biscuits, while at the same time increasing the spread of the biscuits. However, the effect of maize starch on water activity was inconclusive.

The curvilinear effects of the addition of xanthan gum and maize starch on the sensory score showed that the optimal amounts were between the minimum and maximum addition amounts in the experiment.

Through the optimisation process, the optimal conditions were determined to achieve the desired response values: a ratio of rice flour to maize starch of 88.9:11.1 in combination with an addition of 1.1% xanthan gum.



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