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. 2024 Apr 2;89(5):2645–2658. doi: 10.1111/1750-3841.17054

Exploitation of Aureobasidium pullulans NRRL Y‐2311‐1 xylanase in mulberry and rice flours–based gluten‐free cookie formulation: Effects on dough properties and cookie characteristics

Sirma Yegin 1,✉, Burak Altinel 1, Kubra Tuluk 2
PMCID: PMC13281140  PMID: 38563094

Abstract

Xylanases are mainly utilized in bakery industry for the hydrolysis of dietary fiber–based fractions. Their applications in gluten‐free products have not been considered before. In the present study, the xylanase produced by Aureobasidium pullulans NRRL Y‐2311‐1 was utilized in a mulberry and rice flours–based gluten‐free cookie formulation for the first time. Effects of various xylanase concentrations on gluten‐free dough rheology and cookie characteristics were elucidated. Only rice flour–based cookie and only wheat flour–based cookie formulations were also prepared as comparison. Incorporation of xylanase into all cookie recipes resulted in softer cookie doughs with lower absolute stickiness. The hardness and absolute stickiness of the cookie doughs prepared by the mixture of mulberry and rice flours decreased by the addition of the enzyme into the formulation in a concentration‐dependent manner. Enzyme concentrations above 100 U/100 g flour did not provide statistically significant further changes on gluten‐free cookie doughs. Incorporation of xylanase into the cookie recipes resulted in increased baking loss and spread ratio in an enzyme concentration–dependent manner for all cookie types. Hardness values of both types of gluten‐free cookies decreased by xylanase incorporation. Different effects on fracturability were observed depending on the cookie type and enzyme concentration. Enzyme concentration of 100 U/100 g flour provided mulberry and rice flours–based cookies with a more flexible and softer structure. No significant effects on color parameters of cookies were observed by xylanase incorporation.

Keywords: Aureobasidium pullulans, cookie, gluten free, mulberry flour, xylanase

1. INTRODUCTION

Cereals have been possessing a crucial role in human nutrition for thousands of years. They had also a central effect on human civilization. The most consumed cereal grains in the world are rice, wheat, and maize. About 50% of the daily calorie intake of the world has been supplied by the consumption of cereals. They are an indispensable part of human diet as they contain many of the nutrients required for the proper body functions. Among cereals, wheat is the principal dietary protein source (Awika, 2011). Gliadins and glutenin are the main protein fractions of wheat gluten that is known as a unique structure‐forming component in bakery products. The wheat flour exhibits superior characteristics over other flour types due to its ability to form gluten‐based three‐dimensional viscoelastic doughs. Gluten has effects on several dough properties, such as extensibility, elasticity, mixing tolerance, and gas‐holding capacity.

Celiac disease is a systemic immune‐mediated disorder caused by the gliadin fraction of wheat gluten and prolamins of rye (secalins) and barley (hordeins) in people with a genetic susceptibility (Gallagher et al., 2004). Digestion of gluten and/or gluten counterparts results in inflammation of small intestines, leading to reduced absorption of nutrients in individuals with celiac disease. It can be seen at any age, and lifetime gluten‐free diet is the only available treatment for these patients. There are other types of allergic disorders that occur because of gluten, such as wheat allergy. Moreover, there is another controversial disorder named non‐celiac gluten sensitivity. Its causes are mentioned as neither auto‐immunogenic nor allergic (Henggeler et al., 2017). The demand for gluten‐free food products is growing not only because of those individuals who are having such disorders but also because of the people who are intentionally excluding gluten from their diet (Pellegrini & Agostoni, 2015). However, it is important to emphasize that gluten‐free products are mainly designed by considering the people with celiac disease.

When gluten is eliminated from the composition of different cereal‐based products, various technological problems occur, especially dough handling and moulding become very difficult. In bread making, the absence of gluten leads to a liquid batter rather than dough. Gas holding, which is one of the most important quality criteria in bread making, becomes more difficult (Collar, 2019). The final baked breads usually have poor color and crumbling texture (Gallagher et al., 2004). The main problem faced during the production of gluten‐free pasta and noddle is product dissolution due to the weak network development. High cooking loss, extreme firmness, and springiness are other problems that appear depending on the chosen flour types for pasta making (Padalino et al., 2016). In cake making, gluten contributes to the formation of desirable cake volumes and prevents collapse (Wilderjans et al., 2008). However, gluten network development is relatively limited in cookie making since it has a negative effect on cookie diameter, which is accepted as the most critical quality parameter for cookies (Pareyt & Delcour, 2008). Therefore, the elimination of gluten from cookie recipes does not generate a serious issue from this perspective. Nevertheless, it should be noted that the handling properties of gluten‐free cookie dough (e.g., sheetability and moulding) are much more difficult as compared to wheat flour–based cookie dough. Gluten‐free cookies usually have gritty texture and mouthfeel.

Gluten‐free cookies are highly demanded products by celiac patients (da Silva & Conti‐Silva, 2018; Valitutti et al., 2017). Rice flour is one of the most preferred flour types in gluten‐free cookie production. However, different flour types have also been used in combination with rice flour in order to improve the technological properties (e.g., texture, mouthfeel, color) and nutritional values of the final products (Yen et al., 2023). A variety of oligosaccharides (da Silva & Conti‐Silva, 2018), gums (Hamdani et al., 2020), and fruit pomaces as a source of fiber and antioxidants (Saric et al., 2016) are the other ingredients that have been incorporated into the gluten‐free cookie formulations.

Xylanases are an important group of enzymes utilized in bakery industry, targeting the hydrolysis of dietary fiber. Incorporation of xylanases can provide several benefits in bread making, such as better dough stability and machinability, higher loaf volume, and longer shelf life (Yegin et al., 2018). The industrially available xylanases are mainly used in bread making. There are very few studies focusing on the utilization of commercial xylanase preparations in cookie formulation (Bilgicli & Levent, 2014; Jia et al., 2011; Uysal et al., 2007). In these studies, the effects of different enzyme concentrations on dough and cookie properties were not tested. Moreover, all these studies considered only gluten‐containing cookies. Different types of fiber sources were tested in wheat flour–based cookie formulations in the presence and absence of the xylanase. Uysal et al. (2007) tested apple, lemon, wheat, and wheat bran individually as the fiber source, whereas almond skin flour and lupin flour/bran were tested by Jia et al. (2011) and Bilgicli and Levent (2014), respectively. To the best of our knowledge, there is no previous study focusing on the elucidation of the potential of xylanases in gluten‐free cookie formulations. Incorporation of appropriate types and concentrations of xylanases targeting the hydrolysis of fiber fractions can alter the gluten‐free dough handling properties since fibers are effective ingredients with high water‐holding capacity. Adjustment of water‐holding capacity can have profound effect not only on gluten‐free dough properties but also on final product characteristics. Modification of fiber fractions by xylanases has significant potential on the improvement of final cookie characteristics, such as spread ratio, texture, and mouthfeel.

In the light of the abovementioned shortcomings, the objective of the present study is the development of a novel mulberry and rice flours–based cookie formulation using xylanase produced by Aureobasidium pullulans NRRL Y‐2311‐1. Mulberry flour was used in gluten‐free cookie formulation for the first time as a source of dietary fiber. Application of mulberry flour was also advantageous from the nutritional point of view, because it is rich in several vitamins, minerals, antioxidants, proteins, and lipids. Effects of various concentrations of xylanase on gluten‐free dough rheology and cookie characteristics were elucidated. Only wheat flour–based cookie formulation and only rice flour–based cookie formulation were also prepared as comparison. Furthermore, the effects of xylanase on dough rheology and cookie characteristics for these formulations were tested.

2. MATERIALS AND METHODS

2.1. Materials

Beechwood xylan was purchased from BOC Science (Shirley, NY, USA), yeast nitrogen base was from Difco (BD Detroit, MI, USA), and all other chemicals used were analytical grade, which were purchased from either AppliChem (Darmstadt, Germany) or Sigma‐Aldrich (Steinheim, Germany). All the ingredients utilized in cookie making were supplied from local supermarkets, and they were stored at appropriate conditions depending on their individual requirements.

2.2. Microorganism and propagation

A. pullulans NRRL Y‐2311‐1 was kindly supplied by the US Department of Agriculture, Agricultural Research Service (Peoria, IL, USA). The strain was propagated on yeast‐mold (YM) media containing (g/L): yeast extract 3.0, malt extract 3.0, peptone 5.0, glucose 10.0, and agar 20.0. The YM plates were incubated at 24°C for 3 days before use in enzyme production.

2.3. Xylanase production

Xylanase production by A. pullulans NRRL Y‐2311‐1 was carried out under the conditions as described before (Yegin et al., 2017). Seed culture was prepared by inoculation of two loops of A. pullulans NRRL Y‐2311‐1 cells from YM plates into 250 mL Erlenmeyer flasks containing 50 mL of seed medium (pH 5.0). The composition of seed medium was (g/L): xylose 10.0, yeast nitrogen base 6.7, asparagine 2.0, KH2PO4 5.0. Seed culture was incubated at 28°C and 150 rpm for 24 h on an orbital shaker. Xylanase production was performed in 250 mL Erlenmeyer flasks containing 50 mL nutrient medium with the following composition (g/L): xylose 10.0, yeast extract 1.0, (NH4)2SO4 2.5, KH2PO4 5.0. The pH of the medium was adjusted to 4.24 with HCl and NaOH before sterilization (121°C for 15 min). The flasks were inoculated with 2% of the inoculum. They were incubated at 30°C and 200 rpm on an orbital shaker for 126 h. At the end of the bioprocess, the biomass was harvested by centrifugation (21,500 g) at 4°C for 15 min, and the supernatant was referred to the crude enzyme.

2.4. Xylanase assay

Xylanase activity was determined by quantifying the liberated reducing sugars after enzymatic hydrolysis of beechwood xylan using 3,5‐dinitro salicylic acid (DNS) reagent (Bailey et al., 1992; Yegin et al., 2017). The substrate solution (beechwood xylan, 0.5%, w/v) was prepared in 0.05 M citrate buffer (pH 5.2). For the hydrolysis reaction, 0.1 mL of suitably diluted enzyme preparation was mixed with 0.9 mL of substrate solution, and the mixture was incubated at 50°C for 5 min. After incubation, the reaction was terminated by the addition of 1.5 mL DNS reagent. The content was further boiled at 100°C for 5 min followed by cooling in ice‐cold water for 1 min. A specific control for each sample was included simultaneously, which contained all the reagents where the reaction was terminated prior to the addition of the enzyme. The amount of liberated reducing sugars was calculated using the absorbance measurements at 540 nm. One unit of xylanase activity was defined as the amount of enzyme required to liberate 1 µmol of xylose equivalent per min under the assay conditions (50°C and pH 5.2).

2.5. Cookie production

The amounts of ingredients (in relation to the total flour amount) utilized in cookie formulation are provided in Table 1. The amount of xylanase required for each batch was calculated according to the xylanase activity of the liquid enzyme preparation. Xylanase at concentrations of 50, 100, 150 U per 100 g of flour were tested. The required enzyme amount (mL) was mixed with the water included in the recipe before use. The total amount of liquid in the recipe was kept constant by this way. This has been preferred in order to prevent extra volume generation and to provide homogenous distribution of the enzyme within the cookie dough. A domestic blender equipped with a flat beater (Kitchen Aid, Model 5KSM125, St. Joseph, MI, USA) was used in the preparation of the cookie dough. The details of cookie making procedure are shown in Figure 1. As it can be observed from Figure 1, there is a dough resting step, which has been performed in a proofing cabinet (Inoksan FGM 100, Bursa, Turkey) at 30°C for 1 h. This step has been intentionally included in order to provide suitable conditions for xylanase to act. Each batch has been produced using 350 g of flour and/or flour mixture. The effect of xylanase (0–150 U/100 g flour) on cookie dough and final cookie characteristics were tested in three different cookie formulations: (i) rice and mulberry flours–based (80:20) cookies, (ii) only rice flour–based cookie, (iii) only wheat flour–based cookie. All cookie recipes were prepared in duplicate.

TABLE 1.

Cookie formulation.

Component Amount
Flour and/or flour combination a 100 g
Granulated sugar 45 g
Shortening 50 g
Skim milk powder 10 g
Sodium bicarbonate 2.0 g
Salt 1.5 g
Water 18 mL
Xylanase (0–150 U /100 g flour)
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a

Rice flour:mulberry flour (80:20).

FIGURE 1.

FIGURE 1

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Cookie production scheme.

2.6. Determination of dough characteristics

The cookie dough characteristics were elucidated using a TA.XTPlus texture analyzer (Stable Microsystems, Surrey, UK) by applying a modified compression test. The parameters used in the instrumental analyses were; P/35 cylindrical aluminum probe, pretest speed: 2.00 mm/s, test speed: 2.00 mm/s, post‐test speed: 10.00 mm/s, strain (penetration percentage): 40%, trigger force: 5.0 g (5 kg load cell). The maximum peak force (hardness) and dough stickiness were recorded. Twelve cookie samples (shaped & unbaked cookie) from each trial (24 samples in total for each recipe) were used in the determination of the dough characteristics.

2.7. Determination of cookie characteristics

2.7.1. Baking loss

Baking loss was measured gravimetrically by weighing cookies before and after baking.

2.7.2. Spread ratio

The spread ratio of the cookies was determined by using the method of AACC International Methods (2000) (Method 10‐52). According to the mentioned protocol, six cookie samples were laid edge to edge, and the total diameter of the six samples was measured by a scale. Afterward, the samples were rearranged in the same manner, and another measurement was taken. The average of two total diameter measurements was noted. In order to determine the thickness of the cookies, six cookie samples were laid one over the other. The total thickness of these samples was measured by a digital caliper. The samples were repositioned, and another thickness measurement was taken. The average of two thickness measurements was noted. Finally, the spread ratio was calculated by dividing the average diameter of samples to their average thickness value.

2.7.3. Texture profile analysis

The textural properties of the cookie samples were determined using a TA.XTPlus texture analyzer (Stable Microsystems) equipped with a three‐point bending rig (HDP/3 PB). The parameters used in the instrumental analyses were; pretest speed: 1.00 mm/s, test speed: 3.00 mm/s, post‐test speed: 10.00 mm/s, distance: 15 mm, trigger force: 50 g (5 kg load cell). The maximum peak force (hardness) and fracturability values were recorded.

2.7.4. Color parameters

The color parameters of the cookies were determined on the basis of CIE (L*; lightness, a*; redness–greenness, b*; yellowness–blueness) color system using a Konica Minolta Chroma Meter CR‐400 (Tokyo, Japan). Measurements were taken at the center of the top surface of each cookie sample.

2.8. Statistical analysis

Statistical analyses were performed by using SPSS 15.0 Software (SPSS Inc., Chicago, IL, USA). All statistical analysis of the data was performed by analysis of variance. The mean values were compared using the Duncan test (p < 0.05).

3. RESULTS AND DISCUSSION

3.1. Xylanase production by A. pullulans NRRL Y‐2311‐1

The xylanase preparation utilized in different cookie formulations was produced by A. pullulans NRRL Y‐2311‐1 using xylose as the carbon and energy source after 126 h of cultivation at optimum bioprocess conditions (Yegin et al., 2017). The harvested crude enzyme preparation possessed 119.44 ± 4.71 U/mL xylanase activity.

In our previous studies (Yegin, 2017a, 2017b; Yegin et al., 2017), the detailed characterization studies were performed for the elucidation of the crude and purified enzyme properties. Both enzymes exhibited almost identical characteristics, except that the crude xylanase exhibited higher thermostability than the purified enzyme. The enzyme exhibited optimum activity at pH 4.0 and at temperature values of 30–50°C. It was stable over a wide pH range (pH 3.0–8.0). The thermostability studies implied the stability up to 65 and 70°C for the crude and the purified enzymes, respectively. More detailed comparative information about the mentioned enzymes can be acquired in Yegin (2017a). The most functional xylanases for baking industry have been described as the xylanase varieties acting at low and moderate temperatures because dough preparation and proofing steps usually take place at temperature values below 35°C (Collins et al., 2005). The xylanase used in the present study exhibited its maximum activity at a wide temperature range of 30–50°C, in contrast to many other commercial fungal xylanase preparations (45–65°C) (Yegin et al., 2018). This indicates the more usefulness of A. pullulans NRRL Y‐2311‐1 xylanase over most of the other commercial xylanase preparations.

It is important to note that the preliminary studies resulted in very similar effects of the crude and purified enzymes in cookie making. Therefore, the utilization of crude enzymes in cookie making was preferred. It is well known that the purified enzymes are more expensive as compared to the crude counterparts. If it is possible to achieve the similar effect with both enzymes, the utilization of crude enzyme systems is recommended in order to decrease the overall process cost.

3.2. Dough properties

The rheological properties of gluten‐free doughs are important because they profoundly affect the dough machinability and, consequently, the quality of the final products. In the present study, the cookie dough characteristics were measured in terms of hardness and stickiness by applying a modified compression test using a texture analyzer. It is noteworthy to emphasize that texture analyzers are preferred for elucidation of the cookie dough properties by other researchers as well (Benkadri et al., 2018; Laguna et al., 2011; Mamat & Hill, 2014; Mancebo et al., 2015). The cookie dough characteristics are shown in Table 2. The lowest values of cookie dough hardness and absolute stickiness were obtained for the cookies prepared by the mixture of mulberry and rice flours. Wheat flour–based cookie doughs had the highest hardness and absolute stickiness values. Incorporation of xylanase into all three cookie recipes resulted in softer cookie doughs with lower absolute stickiness. The hardness and absolute stickiness of the cookie doughs prepared by the mixture of mulberry and rice flours decreased by the addition of the enzyme into the formulation in a concentration‐dependent manner. Increasing the enzyme concentration to 150 U/100 g flour did not provide statistically significant further changes. Therefore, the enzyme concentration of 100 U/100 g flour seemed appropriate. Similar trend was also valid for the other gluten‐free rice flour–based recipe. In the case of wheat flour–based cookie formulation, an enzyme concentration of 150 U/100 g flour provided the lowest absolute stickiness and hardness values.

TABLE 2.

Effects of xylanase concentration on dough properties: (a) rice and mulberry flours–based cookies, (b) only rice flour–based cookies, (c) only wheat flour–based cookies.

Cookie sample Hardness (N) Stickiness (N s)
(a)
RMF—Control 18.55 ± 1.46a −7.20 ± 0.75c
RMF—50 U 18.06 ± 0.98a −6.85 ± 0.39b
RMF—100 U 16.16 ± 1.02b −6.66 ± 0.37ab
RMF—150 U 15.71 ± 0.76b −6.50 ± 0.36a
(b)
RF—Control 52.57 ± 2.99a −11.32 ± 0.93c
RF—50 U 41.82 ± 3.06b −8.42 ± 1.57b
RF—100 U 42.06 ± 2.19b −7.60 ± 0.75a
RF—150 U 42.43 ± 2.50b −7.38 ± 1.02a
(c)
WF—Control 62.22 ± 3.31a −17.65 ± 1.52d
WF—50 U 52.08 ± 4.95b −16.17 ± 1.05c
WF—100 U 44.51 ± 8.06c −13.65 ± 0.79b
WF—150 U 42.97 ± 6.85c −13.00 ± 0.82a
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Note: Means ± standard deviations (n = 24) followed by different superscripts within the same column indicate significant differences (p < 0.05).

Abbreviations: RF, rice flour–based cookies; RMF, rice and mulberry flours–based cookies; WF, wheat flour–based cookies.

Studies considering the effect of xylanases on cookie dough characteristics are extremely scarce. No previous study was come across with regard to the xylanase incorporation into gluten‐free cookie dough formulation. In a study performed by Jia et al. (2011), almond skin flour incorporated wheat flour was utilized in cookie formulation together with a commercial xylanase. The dough characteristics were determined by using a Mixolab. It was revealed that incorporation of almond skin flour (5%–25%) as a dietary fiber source caused a decrease in dough stickiness values when no xylanase was included in the formulations. Dietary fiber has been indicated as a stickiness‐reducing ingredient. In the present study, incorporation of mulberry flour into rice flour–based cookie formulation also resulted in decreased absolute stickiness values. However, in contrast to the results of the present study, when xylanase was incorporated together with the almond skin flour into the recipe, increase in dough stickiness was observed by the same researchers. It is noteworthy to mention that the cookie recipe in the study was a wheat flour–based recipe, and the effects of different enzyme concentrations were not tested. It is well experienced that gluten‐free doughs are stickier and more fluidic than the wheat‐based doughs due to the lack of gluten (Lazaridou & Biliaderis, 2009). The present study has shown that incorporation of an appropriate concentration of xylanase in cookie formulation is an effective strategy for reduction of dough stickiness, which is a serious problem for gluten‐free dough handling.

3.3. Cookie properties

3.3.1. Baking loss

Effect of different xylanase concentrations on baking loss for the cookies formulated by different flours are shown in Table 3. As it can be clearly observed from Table 3, the baking loss was remarkably high for the cookies formulated with a combination of rice and mulberry flours as compared to the cookies formulated only with rice flour and only with wheat flour. It was generally indicated that the addition of the flours with higher protein content to the cookie recipe led to lower baking loss, although there are some contradictorily reported results about this issue in the literature. For example, Yen et al. (2023) tested the effect of different amounts of germinated mung bean flour substitution to the rice flour in cookie formulation. They observed that increasing the amount of protein‐rich bean flour in the recipe resulted in lower baking loss. On the other hand, the same researchers also observed that the baking loss values for only rice flour–based cookies were lower than the only wheat flour–based cookies. However, it is well known that wheat flour has higher protein content than the rice flour. Therefore, there might be different effects of non‐wheat proteins. Schmelter et al. (2021) found that the baking losses for the cookies prepared by using different varieties of whole fava bean flours were in the range of 14.6%–15.7%. These values were lower than the baking loss value obtained for the reference whole wheat flour cookies (16.2%). When the wheat flour was replaced by different varieties of fava bean flour at a concentration of 50%, some bean varieties resulted in lower baking loss, whereas others led to higher baking loss than the reference whole wheat flour cookies. In the same study, the reduced baking loss due to the incorporation of bean flours has been linked to the water‐binding capacity of the proteins, which was indicated as an effective factor in prevention of evaporations. In the present study, the protein contents of the flours utilized are wheat flour > rice flour > mulberry flour, according to the manufacturer's instructions. When the baking losses of the controls (without xylanase) of each cookie recipe were compared, it was observed that the reduced protein content resulted in higher baking loss. Although the result of the present study supports the hypothesis of negative correlations between the protein content and baking loss, it will not be legitimate only considering the protein content. The different components of composite flours other than proteins, such as dietary fiber, starch, fat, and sugar, can significantly affect the water retention capacity of the cookies (Yen et al., 2023). A special emphasis should be given to dietary fiber due to its high water‐holding capacity. The water‐holding capacities of dietary fibers strongly depend on the source of fibers. The dietary fibers from fruits and vegetables have greater affinity for water than the cereal dietary fiber (Elleuch et al., 2011). From this perspective, in the present study, lower baking loss should be expected for the cookies prepared with mulberry and rice flours mixture as compared to cookies made by only rice flour. In contrast, the highest baking loss was noted for the cookies prepared with mulberry and rice flours mixture. Similar to our findings, Chakraborty et al. (2021) observed that incorporation of banana pseudostem flour into wheat flour–based cookies resulted in increased baking loss. It is also important to emphasize that dietary fibers consist of soluble and insoluble fractions. It has been demonstrated by Mancebo et al. (2018) that the incorporation of insoluble fibers into cookie recipes results in cookies with a higher moisture content, which implies lower baking loss. The proportions of soluble and insoluble fractions in plant‐based foods vary depending on the botanical origin and the maturity of the fruits/seeds (Zhou et al., 2021). As mentioned by Mancebo et al. (2018), the reason for contradictory results might be the differences in the cookie formulations, but also the variety of dietary fibers utilized. Moreover, the interactions between the components may also generate such types of contradictory results because bakery products have very complex matrices.

TABLE 3.

Effects of xylanase concentration on baking loss, diameter, thickness, and spread ratio of the cookies: (a) rice and mulberry flours–based cookies, (b) only rice flour–based cookies, (c) only wheat flour–based cookies.

Cookie sample Baking loss (%) Diameter (mm) Thickness (mm) Spread ratio
(a)
RMF—Control 10.82 ± 0.37a 433.50 ± 1.83b 60.17 ± 0.36a 7.21 ± 0.03b
RMF—50 U 11.14 ± 0.83a 438.13 ± 3.42ab 59.37 ± 0.74ab 7.38 ± 0.15ab
RMF—100 U 11.26 ± 0.28a 438.50 ± 1.96a 59.41 ± 0.59ab 7.38 ± 0.10ab
RMF—150 U 11.42 ± 0.57a 439.38 ± 1.93a 58.81 ± 0.47b 7.47 ± 0.08a
(b)
RF—Control 7.32 ± 0.97b 373.00 ± 1.58a 67.32 ± 0.85a 5.51 ± 0.14a
RF—50 U 7.78 ± 0.57ab 375.38 ± 1.80a 68.12 ± 0.84a 5.52 ± 0.09a
RF—100 U 7.81 ± 0.50ab 375.75 ± 1.32a 68.19 ± 0.59a 5.51 ± 0.07a
RF—150 U 8.03 ± 0.34a 375.88 ± 1.75a 67.52 ± 0.85a 5.57 ± 0.09a
(c)
WF—Control 7.49 ± 0.38c 393.75 ± 3.52a 73.24 ± 1.59a 5.38 ± 0.10a
WF—50 U 7.62 ± 0.39c 395.00 ± 2.08a 73.28 ± 0.14a 5.39 ± 0.03a
WF—100 U 8.07 ± 0.36b 394.50 ± 1.68a 73.06 ± 0.93a 5.40 ± 0.07a
WF—150 U 8.71 ± 0.42a 398.75 ± 2.90a 73.28 ± 0.67a 5.45 ± 0.07a
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Note: Means ± standard deviations followed by different superscripts within the same column indicate significant differences (p < 0.05). Baking loss, n = 12; diameter, n = 4; thickness, n = 4; spread ratio, n = 4.

Abbreviations: RF, rice flour–based cookies; RMF, rice and mulberry flours–based cookies; WF, wheat flour–based cookies.

In the present study, the incorporation of xylanase into the cookie recipes resulted in increased baking loss in a concentration‐dependent manner for all cookie types (Table 3). The increase was not statistically significant for mulberry and rice flours–based cookies. However, the increase in baking loss for wheat flour–based cookies was statistically significant. Xylanases hydrolyze the hemicellulosic fractions of dietary fiber. Therefore, they are commonly utilized in bread formulations to increase the amount of water‐soluble pentosans (Hilhorst et al., 1999). Because the incorporation of xylanase can increase the water‐soluble fractions, reduction in water‐holding capacity should be expected. As indicated before, Mancebo et al. (2018) have shown that insoluble fiber incorporation provides higher water‐holding ability. Therefore, the hydrolysis of such insoluble fractions into soluble ones by xylanases leads to lower water holding, implying higher baking loss. This might be a possible explanation for the increased baking loss by the xylanase addition. In our previous study (Yegin et al., 2018), the same enzyme was utilized in bread formulation comparatively with the commercial xylanase preparations. In the mentioned study, increases in baking loss for all types of xylanases were also observed. However, reduction beyond certain enzyme concentration (100 U/100 g flour) for the xylanase from A. pullulans NRRL Y‐2311‐1 was noted (Yegin et al., 2018).

3.3.2. Spread ratio

The highest cookie diameter, the lowest thickness, and consequently, the highest spread ratio values were observed for the cookies formulated with mulberry and rice flours mixture (Table 3). The highest spread ratio was obtained for these cookies when the enzyme concentration of 150 U/100 g flour was utilized. The spread ratios of the rice flour–based cookies and wheat flour cookies were similar to each other but considerably lower than the mulberry and rice flours–based cookies. The wheat flour–based cookies exhibited the lowest spread ratio.

Spread ratio is a key parameter for the evaluation of the cookie quality. Pareyt and Delcor (2008) described the most desirable cookies as the ones having high spread ratio and narrow cracks. Several factors have influence on cookie spread ratio. Increased fiber concentration has been indicated as a predominant factor leading to decreased spread ratio due to the higher water absorption (Pareyt & Delcor, 2008). The studies, including the incorporation of fruit‐based dietary fiber into cookie recipes, mostly focused on wheat flour–based cookies. Broad variations can be seen in the results of these studies. For example, Naknaen et al. (2016) figured out that the incorporation of watermelon rind powder into wheat flour–based cookie formulation (10%–30%) reduced the spread ratio of the cookies significantly. As the concentration of fiber increased, the spread ratio of the cookies got decreased. Ajila et al. (2008) incorporated mango peel (5%–20%) into wheat flour–based cookies as a source of fiber. Slight reduction in spread ratio was observed for some of the concentrations tested. However, the changes in spread ratio were not statistically significant. Incorporation of banana pseudostem flour (10%–30%) (Chakraborty et al., 2021) and goji berry by‐product (10%–40%) (Bora et al., 2019) in wheat flour–based cookies led to increase in spread ratio in a concentration‐dependent manner, whereas the incorporation of kadam fruit powder (2.5%–10%) into wheat flour–based cookies did not have effect on spread ratio (Osama et al., 2023). There are few studies considering the incorporation of such fruit‐based fibers into gluten‐free cookie formulations. Ayo‐Omogie (2023) prepared sorghum flour–based gluten‐free cookies incorporated with various amounts of unripe banana flour (30%–65%) and sesame meal (5%). Similar to our findings, increased banana flour concentration in the recipe led to increase in spread ratio values. In contrast, Stoin et al. (2020) found that incorporation of coconut flour (15%–60%) into rice flour–based cookie formulation caused decrease in cookie spread ratio.

There is no previous study focusing on the utilization of xylanases in gluten‐free cookie formulations. Uysal et al. (2007) utilized xylanase enzyme (0.4%) in wheat flour–based cookie formulations that were supplemented with different fibers (apple, lemon, wheat, and wheat bran) individually. However, the effects of different enzyme concentrations on the characteristics of cookies were not tested. It was found that increasing fiber content (15%–30%) in cookie formulations without enzyme addition decreased the spread ratio for all types of fiber except wheat bran. When wheat bran was included, increase in spread ratio was observed. The addition of xylanase to the main wheat flour–based cookie formulation without any fiber incorporation increased the spread ratio significantly. When both fiber and xylanase were incorporated into the recipe, decrease in spread values as compared to control cookies was observed. As the concentration of fiber increased in the enzyme‐added cookie recipes, the spread ratio got decreased. However, wheat bran as a fiber source exhibited opposite behavior in enzyme incorporated cookies as well. In this case, increase in spread ratio was observed when the wheat bran concentration was increased in the presence of the xylanase enzyme. The effect of wheat bran as a fiber source was different than the other fiber sources. Wheat bran is the richest fiber source in arabinoxylan fraction among the other sources tested in the mentioned study. Therefore, it might be exhibiting different behaviors than the other fibers. The enzyme specificity has the utmost importance. The substrate preference of xylanases may differ from each other depending on the xylanase family to which they belong. In the present study, it was obvious that incorporation of mulberry flour (20%) into the rice flour–based cookie formulation increased the spread ratio of the cookies at a great extent as compared to the cookies prepared only with rice flour and only with wheat flour (Table 3). The same trend was observed by Uysal et al. (2007) when they used wheat bran as a fiber source. It is important to indicate that the xylanase types and cookie formulations are different in this study. Comparisons should be made by considering these issues. In the present study, increasing the xylanase concentration led to increase in spread ratio values for all types of cookies, although statistical significance was observed only for rice and mulberry flours–based cookies. On the other hand, Jia et al. (2011) observed that the addition of xylanase into the cookie recipes, prepared by the mixture of almond skin flour (20%) and wheat flour, led to decrease in the spread ratio of the cookies. It was also noted in the same study that no significant change was observed in the spread ratio values when the almond skin flour concentration was increased from 20% to 23% with the addition of xylanase.

The protein content of the flours has also been mentioned as a critical factor effecting the spread ratio values of cookies. It has been indicated that non‐wheat high‐protein flours can absorb more water than wheat flour, thereby limiting the sufficient water amount to dissolve the sugar during baking. This can cause increase in dough viscosity and decrease in spread ratio (Bilgicli & Levent, 2014; Kissell & Yamazaki, 1975). In accordance with this claim, the lowest spread ratio values were noted for wheat flour–based cookies in the present study. Interestingly, both the baking loss and the spread ratio were highest for mulberry flour–incorporated rice flour–based cookies. High baking loss implies less water absorption, which in turn should promote low cookie spread, according to above‐mentioned claim. It seems that non‐wheat proteins also exhibit different behaviors. It has been shown in some studies that incorporation of seed and legume flours generally decreased the spread ratio of the cookies due to their high water absorption capacities (Gerzhova et al., 2016; Li et al., 2020; Yen et al., 2023).

3.3.3. Textural properties

The textural properties are one of the most critical factors utilized in the evaluation of the gluten‐free cookies. Gluten‐free cookies usually have gritty texture and mouthfeel as compared to the gluten‐containing counterparts. Hardness and fracturability are significant parameters utilized in the evaluation of textural characteristics of cookies, with hardness being the most commonly utilized one. “Hardness” or “firmness” or “breaking strength” is the maximum force required to break a cookie sample (Hadnadev et al., 2013). In the present study, the textural properties of the cookies are shown in Table 4. As it can be clearly observed from Table 4, the highest hardness values were obtained for the wheat flour–based cookies. Incorporation of mulberry flour into rice flour led to significant decrease in hardness values of the cookies when no enzyme was included in the formulations. Incorporation of xylanase promoted reduction in hardness values of both types of gluten‐free cookies in a concentration‐dependent manner. Application of xylanase at a concentration of 50 U/100 g flour provided more profound decrease in hardness values of cookies prepared by only rice flour. When 50 U/100 g flour xylanase was utilized in only wheat flour–based cookie formulation, reduction in hardness values was also observed. Increase of enzyme amount beyond this concentration led to increase in hardness values, but the hardness values were still lower than control, which did not have any enzyme.

TABLE 4.

Effects of xylanase concentration on textural parameters (a) rice and mulberry flours–based cookies, (b) only rice flour–based cookies, (c) only wheat flour–based cookies.

Cookie sample Hardness (N) Fracturability (mm)
(a)
RMF—Control 22.83 ± 2.19a 0.95 ± 0.09c
RMF—50 U 20.75 ± 4.80a 1.07 ± 0.07b
RMF—100 U 19.90 ± 5.45a 1.21 ± 0.06a
RMF—150 U 19.76 ± 3.70a 0.66 ± 0.07d
(b)
RF—Control 28.40 ± 1.86a 1.06 ± 0.12c
RF—50 U 21.02 ± 2.44b 1.72 ± 0.18a
RF—100 U 20.67 ± 2.60b 1.69 ± 0.13a
RF—150 U 19.15 ± 1.26b 1.53 ± 0.13b
(c)
WF—Control 31.37 ± 1.57a 1.42 ± 0.08b
WF—50 U 28.36 ± 1.69b 0.97 ± 0.12c
WF—100 U 29.24 ± 1.91b 1.53 ± 0.13a
WF—150 U 30.04 ± 2.14ab 1.34 ± 0.14b
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Note: Means ± standard deviations (n = 24) followed by different superscripts within the same column indicate significant differences (p < 0.05).

Abbreviations: RF, rice flour–based cookies; RMF, rice and mulberry flours–based cookies; WF, wheat flour–based cookies.

Similar to our results, individual incorporation of unripe banana flour (30%–65%) and sesame meal (5%) into the sorghum flour–based gluten‐free cookies provided reduction in cookie hardness values (Ayo‐Omogie, 2023). Incorporation of watermelon rind powder (10%–30%) (Naknaen et al., 2016) and incorporation of kadam fruit powder (2.5%–10%) (Osama et al., 2023) into wheat flour–based cookie formulations also reduced the hardness of the cookies significantly. On the other hand, Torra et al. (2021) utilized partially substituted chickpea flour with chestnut flour in gluten‐free cookie formulation. Substitution of 25% chestnut flour generated reduction in hardness values of the cookies, whereas the addition of chestnut flour at concentrations above 50% provided increase in cookie hardness. In contrast, incorporation of raspberry and blueberry pomace powder mixtures (30%) into commercial gluten‐free flour mixture provided significant increase in cookie hardness (Saric et al., 2016). Substitution of rice flour with different concentrations of alfalfa seed flour (15%–45%) led to increase in cookie hardness in a concentration‐dependent manner (Giuberti et al., 2018). Similarly, incorporation of banana pseudostem flour (10%–30%) into wheat flour–based cookies also led to increase in hardness values of the cookies in a concentration‐dependent manner (Chakraborty et al., 2021). As is clear from the results of above‐mentioned previous studies, the types and concentration of the additional fiber source have effect on final cookie hardness.

In the case of evaluation of the effect of xylanase enzyme incorporation on cookie hardness, Uysal et al. (2007) showed that the addition of commercial xylanase preparation (0.4%) into wheat flour–based cookie recipes that were supplemented with various fiber sources (apple, lemon, wheat, and wheat bran) individually reduced the hardness of the cookies. Xylanase addition caused reduction in cookie hardness when no fiber was added as well. It can be concluded that xylanase incorporation into the cookie recipe provides softer cookies. It is important to note that the effect of fiber concentration on cookie hardness varied depending on the fiber source in the mentioned study. Incorporation of lemon and wheat fiber (10%–30%) increased the cookie hardness in a concentration‐dependent manner. However, the effect of fiber concentration on cookie hardness exhibited broad variations for apple and wheat bran fibers. In the present study, incorporation of xylanase into both types of gluten‐free cookies reduced the cookie hardness in a concentration‐dependent manner. Utilization of 50 U/100 g flour xylanase in wheat flour–based cookie formulation also reduced the cookie hardness. However, higher enzyme concentration increased the cookie hardness values, but they were still lower than the control of wheat flour–based cookies prepared without any enzyme addition.

Fracturability is also a critical parameter in the objective evaluation of the textural properties of cookies, which has often been neglected. Fracturability is the distance traveled by the breaking blade through the cookie in millimeters just before a cookie breaks that can be measured by a texture analyzer device (Hadnadev et al., 2013). Therefore, the greater fracturability value implies the more flexibility (Hwang et al., 2016). In other words, lower fracturability value indicates a more fragile structure. In the present study, when the cookie recipes without enzyme incorporation were compared, the highest fracturability values (more flexibility) were obtained for the cookies prepared by only wheat flour. This is an expected result attributed to the existence of gluten in this recipe. Gluten provides more flexibility due to its elastic nature. The fracturability value was the lowest for rice and mulberry flours–based cookies when no enzyme was included in the formulations. Cookies prepared by only rice flour exhibited better flexibility (higher fracturability value) than the cookies made by the mixture of rice and mulberry flours when no enzyme‐incorporated recipes were compared. In contrast, increased cookie fracturability values due to the replacement of wheat flour with pumpkin flour (Kulkarni & Joshi, 2013) and banana pseudostem flour (Chakraborty et al., 2021) were reported in the literature. However, it is important to emphasize that these formulations include gluten.

In the present study, when xylanase was incorporated into the cookie formulations, different effects on fracturability were observed depending on the cookie type and enzyme concentration (Table 4). Fracturability values got increased for the rice and mulberry flours–based cookies when the enzyme concentration was increased up to 100 U/100 g flour. A sharp decrease was observed when the enzyme concentration was increased to 150 U/100 g flour for these cookies. The results implied that enzyme concentration of 100 U/100 g flour provided cookies with a more flexible and softer structure since the change in the hardness values between the enzyme concentrations of 50 and 100 U/100 g flour was also not statistically significant. Cookies prepared with only rice flour also exhibited partially similar trend in terms of fracturability to the cookies prepared by rice and mulberry flours mixture. The fracturability values got increased up 100 U/100 g flour enzyme utilization. A slight decrease was noted when the enzyme concentration was further increased to 150 U/100 g flour. There was also slight difference between the fracturability values obtained by applications of 50 and 100 U/100 g flour enzyme concentrations, but it was not statistically significant. The application of xylanase in gluten‐containing wheat flour–based cookies resulted in lower fracturability values at a concentration of 50 U/100 g flour as compared to the control cookies without enzyme incorporation. Utilization of 100 U/100 g flour enzyme for the same type of cookies led to higher fracturability values than the control cookies have. Slight reduction was observed at fracturability values above this enzyme concentration. The most profound effect of xylanase addition on fracturability values can be seen for the gluten‐free cookies prepared by only utilization of rice flours.

3.3.4. Color parameters

Color is an important quality aspect that has a critical influence of consumer decision. In the present study, the color parameters obtained for each type of cookie recipe are shown in Table 5, and the images of all types of cookies are shown in Figure 2. As it can be clearly observed from Table 5, the incorporation of xylanase into the all three types of the cookie recipes did not exhibit any statistically significant effect on the color parameters as compared to the controls of each recipe that did not include any xylanase. Incorporation of mulberry flour into rice flour resulted in cookies with darker color as implied by lower L* values. The lightness values of the wheat flour and rice flour cookies were similar to each other. However, the L* values of the rice flour cookies were slightly higher than the wheat flour cookies as expected because rice flour itself has slightly lighter color. The highest a* values (redness–greenness) and b* values (yellowness–blueness) were noted for cookies prepared by the mixture of rice and mulberry flours. The lowest a* and b* values were observed for cookies prepared by only rice flour.

TABLE 5.

Effects of xylanase concentration on color parameters: (a) rice and mulberry flours–based cookies, (b) only rice flour–based cookies, (c) only wheat flour–based cookies.

Cookie sample L* a* b*
(a)
RMF—Control 55.60 ± 0.93a 10.29 ± 0.53a 32.15 ± 0.87a
RMF—50 U 54.95 ± 1.22a 10.67 ± 0.80a 31.96 ± 0.61a
RMF—100 U 56.07 ± 1.36a 10.38 ± 0.82a 32.01 ± 0.40a
RMF—150 U 55.27 ± 0.79a 10.87 ± 0.42a 32.22 ± 0.82a
(b)
RF—Control 71.12 ± 0.86a 2.41 ± 0.37a 24.17 ± 1.31a
RF—50 U 71.57 ± 1.18a 2.53 ± 0.14a 24.04 ± 0.97a
RF—100 U 71.04 ± 0.83a 2.49 ± 0.21a 23.95 ± 0.96a
RF—150 U 71.28 ± 0.73a 2.66 ± 0.30a 24.46 ± 0.98a
(c)
WF—Control 70.35 ± 0.60a 4.36 ± 0.71a 27.02 ± 1.29a
WF—50 U 70.02 ± 1.75a 4.18 ± 0.08a 27.28 ± 0.80a
WF—100 U 70.29 ± 1.38a 4.29 ± 0.77a 27.12 ± 0.85a
WF—150 U 70.14 ± 1.11a 4.06 ± 0.44a 27.36 ± 1.56a
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Note: Means ± Standard deviations (n = 24) followed by different superscripts within the same column indicate significant differences (p < 0.05).

Abbreviations: RF, rice flour–based cookies; RMF, rice and mulberry flours–based cookies; WF, wheat flour–based cookies.

FIGURE 2.

FIGURE 2

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Images of the cookies formulated with different xylanase concentrations (0–150 U/100 g flour): (a) RMF: rice and mulberry flours–based cookies, (b) RF: rice flour–based cookies, (c) WF: wheat flour–based cookies.

It has been indicated in many of the studies (Giuberti et al., 2018; Mancebo et al., 2015; Naknaen et al., 2016) that the color of the cookies can be effected by two main factors: (i) the original color and/or pigment content of flour types utilized; (ii) the degree of Maillard reactions that can occur during baking. The Maillard reaction that occurs between the reducing sugar and protein during baking has effect on the development of the final cookie color (Naknaen et al., 2016). Therefore, higher protein content flours are more likely to produce cookies with darker color because the products of Maillard reactions are typically known for their brownish colors. As indicated before, the protein contents of the flours utilized in the present study are: wheat flour > rice flour > mulberry flour according to manufacturer's instructions. The darker color of the cookies prepared by mulberry and rice flours mixture was mainly due to the darker color of the mulberry flour. Although these cookies had the lowest protein content, they still had the darkest color because of the mentioned reason. The sugar contents and the sugar types that take role in Maillard reactions also influence final cookie colors. High sugar content of the cookies may promote caramelization reactions as well (Torra et al., 2021), leading to the production of caramel flavor and brownish color. Mulberry flour also provides different fruit‐based sugar varieties as compared to the wheat and rice flours. This possibly also effected the final cookie color. Incorporation of different fruit‐based powders/flours into the cookie formulations had significant effects on final cookie color, depending on the fruit source. Most of them produced cookies with darker color as compared to their controls, such as mango peel (Ajila et al., 2008), watermelon rind powder (Naknaen et al., 2016), and banana pseudostem flour (Chakraborty et al., 2021).

4. CONCLUSION

Application of xylanase enzymes in gluten‐free cookie formulations has not been considered before. Xylanases are commonly utilized in bread making to improve the dough and bread quality attributes. The present study explored that xylanases could also be useful aids in cookie making, either gluten‐free or gluten‐containing. Incorporation of xylanase from A. pullulans NRRL Y‐2311‐1 into cookie formulations decreased the dough stickiness, thereby facilitating the gluten‐free dough handling. Reduction in cookie hardness and increase in spread ratio were the other improvements regarding the final cookie characteristics. Application of appropriate enzyme concentration resulted in the production of mulberry and rice flours–based cookies with a more flexible and softer structure.

AUTHOR CONTRIBUTIONS

Sirma Yegin: Conceptualization; methodology; supervision; investigation; writing—original draft; project administration; funding acquisition. Burak Altinel: Supervision; investigation; formal analysis. Kubra Tuluk: Investigation; formal analysis

CONFLICT OF INTEREST STATEMENT

The authors report no conflicts of interest.

ACKNOWLEDGMENTS

This work was financially supported by “Ege University, Scientific Research Projects Coordination Unit” (Project ID: FGA‐2021‐23243 & Project Number: 23243). Sirma Yegin is a working group member in Cost Action CA18101 entitled “Sourdough Biotechnology Network Towards Novel, Healthier and Sustainable Food and Bioprocesses (SOURDOMICS)”. The technical supports from “Ms. Kibar Yeğin” for project design were gratefully acknowledged. We would also like to thank “Mr. Adem Göküş” who assisted in TA.XTPlus texture analyzer usage.

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