Antistaling properties of encapsulated maltogenic amylase in gluten‐free bread

Abstract Staling of bakery products especially gluten‐free products is a challenge on the development of these products. For retarding staling of gluten‐free bread, maltogenic amylase (MAase) at concentrations of 8.2, 45, and 82 mg/ml was encapsulated into beeswax (BW) at 1%, 2.5%, and 4% levels. Results showed the treatment with 8.2 mg/ml MAase and 2.5% beeswax had the highest encapsulation efficiency (42.04%) and chosen for subsequent experiments. The size of encapsulated particles was 362.70 nm and had a zeta potential of −15.35 mV. Surface morphology of encapsulated MAase was almost spherical with layered appearance. The free and encapsulated MAase with the activity of 5.2 µmol/min were used in gluten‐free batter and breads, respectively. In the rheological tests, batters containing free and encapsulated MAase showed lower cross over point than control batter (without enzyme or wall material) (59 and 53 Hz, respectively). Encapsulated MAase contained bread had darker crust, whiter and softer crumb, and more aerated structure in comparison with free MAase loaded one. Both breads containing MAase as free or encapsulated had higher moisture content and water activity in crust and crumb than control bread. However, bread with free MAase had softer crumb after four days of storage, and bread with encapsulated MAase had higher sensorial acceptability than other breads after 2 and 4 days of storage.


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HAGHIGHAT-KHARAZI eT Al. not able to further hydrolysis of starch and producing soluble dextrins, which is the cause of gumminess in prepared bread (Gerrard, Every, Sutton, & Gilpin, 1997). In comparison with enzymes, MAase is unique in yielding significant softness to bread and maintaining a high level of crumb elasticity during storage. It is extensively used in the baking industry to retard bread staling (Gomes-Ruffi et al., 2012;Li et al., 2018).
Lipid-based wall materials in enzyme encapsulation for application in the baking process have been done by some scientists. Dusterhoft et al. (2006) have reported the encapsulation of α-amylase into hydrogenated stearin fraction of palm kernel oil by spray chilling technique for application in bread formulation. Plijter and Meesters (2000) also investigated the application of coated lipase into maltodextrin and other materials on the quality of baked products. Waxes have good stability at various moisture content and pH. They have no immunogenicity for human because of their insoluble nature. Moreover, producing technique of waxes microparticles does not need complex devices, organic solvents, and heating for a prolonged time (Hassan, Eshra, & Nada, 1995;Ranjha, Khan, & Naseem, 2010). Beeswax is a permitted additive used in the European Union (E901-903) and is frequently used for encapsulation of drugs and bioactive compounds (Chitprasert & Sutaphanit, 2014;Mellema, Van Benthum, Boer, Von Harras, & Visser, 2006;Ranjha et al., 2010). In our previous work, we have optimized the encapsulation of α-amylase into beeswax using RSM and applied the optimized one into the gluten-free batter and bread (Haghighat-Kharazi, Milani, Kasaai, & Khajeh, 2018). Here in this work, we aimed to investigate the application of beeswax for encapsulating MAase and its application into gluten-free batter and bread. After determination of efficiency and physicochemical characteristics of encapsulated MAase, we studied the application of encapsulated enzyme into gluten-free rice-based batter and bread and assayed the qualitative, sensorial, and staling features of prepared batter and bread. 11.87% protein, 2.76% fat; 0.28% ash) was acquired from a local farmer (Mazandaran, Iran). Chickpea flour (Seity, Iran); diacetyl tartaric acid esters of monoglycerides (DATEM) (Pars Behbood Asia, Iran); hydroxypropyl methylcellulose (HPMC) (Fluka, Switzerland), instant yeast (Razavi Yeast, Iran), vegetable oil (Behpak Industrial company, Iran), salt, and sugar were also used for the preparation of gluten-free batter and bread.

| Encapsulation of MAase into beeswax
The encapsulation of MAase into beeswax was done using the method described by (Haghighat-Kharazi et al., 2018;Kheradmandnia, Vasheghani-Farahani, Nosrati, & Atyabi, 2010) with minor modifications. In this method, encapsulated MAase was prepared by emulsion-congealing technique. Beeswax was melted in a water bath at 90°C. Then, the melted beeswax (1, 2.5, or 4 g) was poured into 100 ml of stirring phosphate buffer solution (50 mM, pH 7), which had been previously heated to a temperature higher than the melting point of beeswax (˃+5°C). Next, Tween 20 and MAase (8.2, 45, or 82 mg/ml) were added to the molten lipid and buffer mixture. After maintaining the stirring for 2 min using a magnetic stirrer (Heidolph, Kelheim, Germany), the emulsion was immediately cooled down using the same volume of cold phosphate buffer solution (0-2°C) under mechanical stirring to produce spherical solid particles. Finally, the obtained solid spheres were collected and filtered through Whatman no.3 filter paper and rinsed by distilled water to remove any surfactant and enzyme residues. The air-drying process was performed at room temperature (25°C) for 24 hr to produce single and free-flowing solid spheres. Final products were stored at 4°C for further experiments.

| Determination of enzyme encapsulation efficiency
The efficiency of MAase encapsulation was evaluated by determining the amylolytic activity of free and encapsulated enzymes.
MAase activity was assayed according to the method of Bernfeld (1955) using spectrophotometer (Perkin-Elmer, Lambda 25, UVvis) at 540 nm for determining the amount of produced maltose.
One unit of α-amylase is expressed as the amount of enzyme producing 1 μmole of maltose per min. To determine the encapsulation efficiency, bellow equation was used (Amid, Manap, & Zohdi, 2014): The encapsulation efficiency of various formulations is presented in Table 1. The highest encapsulation efficiency (42.04%) was attributed to the treatment code of BW 4 -MAase (beeswax = 2.5% and enzyme concentration = 8.2 mg ml −1 ) as the optimized one.

| Particle size and zeta potential measurements
Particle size and zeta potential of encapsulated enzyme were estimated by a dynamic light scattering technique using a Malvern Zetasizer Nano-Series (Nano-ZS, Malvern Panalytical Instrument, England).

| Scanning electron microscopy (SEM)
The morphology of the encapsulated enzyme was determined by SEM instrument (FE-SEM, Hitachi S4160, Japan), under an acceleration voltage of 20 kV. The microstructure of bread was also determined using SEM (Electric Systems, Cambridge, Model 1455VP, UK). The bread samples were air-dried. Each sample of encapsulated enzymes or bread was placed on a copper grid and was coated with a thin layer of gold. Ten loaves of each bread were made for each treatment.

| Rheological properties of gluten-free batters
The rheological properties of gluten-free batters were meas-

| Physicochemical parameters of bread
Weight loss percent (WL%) during baking was measured as described by Demirkesen et al. (2013). Loaf specific volume was evaluated using the rapeseed displacement as described in the approved method of AACC, 10-05 (AACC, 2000). Volume, symmetry, and uniformity indexes were measured as described in AACC, 10-91 (AACC, 2000). Crumb/crust weight ratio was calculated based on the weight of crumb to the weight of crust (Curic et al., 2008). Crumb porosity of loaves was assayed using a flatbed scanner (Model Scan jet

TA B L E 1 Effect of beeswax and
MAase concentrations on encapsulation efficiency of prepared particles 2410, HP, Cupertino, USA) with a resolution of 300 dpi and the data were processed using an Image-Pro plus 4.5 (Media Cybernetics Inc., USA) (Esteller, Zancanaro, Palmeira, & da Silva Lannes, 2006). The crumb porosity features chosen were mean cell area (mm 2 ), mean diameter (mm), minimum diameter (mm), maximum diameter (mm), and nonuniformity of gas cells.

| Estimation of crumb and crust color
The

| Texture analysis
Texture profile analysis (TPA) of samples was carried out using a tex-

| Evaluation of bread staling
Breads were stored in the sealed polyethylene bags at room temperature (25°C). At one, two, and four days after baking, breads were subjected to the following tests. Crumb and crust moisture contents were determined by an air oven gravimetric method of AACC, 44-15 (AACC, 2000). Water activity (a w ) of crumb and crust was measured Sensory evaluation of staling was performed by 5 panelists to evaluate the loaves for overall acceptability at first, second, and fourth days. Acceptability was determined by scoring the samples from 1 to 5 (1 for an unacceptable and 5 for a high satisfactory).

| Statistical analysis
All experiments were performed in two replicates. Data were statistically analyzed using SPSS V16.0 for analysis of variance (ANOVA), followed by Duncan's multiple range test (p ˂ 0.05) for determining significant differences.

| Encapsulation of MAase into BW
The effects of BW and MAase concentration on the encapsulation efficiency of encapsulated MAase are presented in Table 1 respectively. According to these results, we can say that the efficiency of enzyme encapsulation into lipidic or carbohydrate-based wall materials depends on both the kind of enzyme and wall materials.

| Particle size and zeta potential characterization
The particle size of the encapsulated MAase into BW was 96.85 µm.
The negative zeta potential of −15.35 ± 0.35 mV was obtained for encapsulated MAase into BW. Encapsulation of α-amylase into BW also yielded in a low negative zeta potential (Haghighat-Kharazi et al., 2018). Colloidal dispersions with potentials within +30 to −30 mV tend to coagulate, whereas colloids with potentials greater than +30 mV or smaller than −30 mV are electrically stabilized (Honary & Zahir, 2013). Therefore, the aggregation of this encapsulated enzyme in an aqueous medium is expected (Kheradmandnia et al., 2010).

| Morphology of encapsulated enzyme
Surface morphology of encapsulated MAase into BW measured by SEM is illustrated in Figure 1.

| Rheological features of gluten-free batters
The variations of G′ and G″ as a function of frequency for different gluten-free batters were illustrated in Figure 2a. were obtained rather than BW and control batters (90 and 98 Hz, respectively). As beeswax has high hydro repellency nature due to long-chain fatty acids, a delayed release of the enzyme in the batter is possible (Chitprasert & Sutaphanit, 2014). Some research also proved plate-like crystals in waxes, organizing the labyrinth effects that were effective in preventing the diffusion of small compounds (Mellema et al., 2006). Nevertheless, in our study, batter contained BW-MAase showed cross over point in lower frequencies rather than a batter containing MAase.
The effect of temperature on the structure of batters (complex modulus, G*, versus temperature) was illustrated in Figure 2b.
Generally, all batters mostly exhibited a similar trend and the same starch gelatinization temperatures.

| Physicochemical features of gluten-free breads
Various physicochemical features of different gluten-free bread formulations are illustrated in . Gluten-free batters require more hydration than wheat dough to achieve an appropriate consistency. Higher moisture retention (for breads after the baking), yielded in an improvement in the quality of gluten-free bread via a reduction in firmness (Furlán, Padilla, & Campderrós, 2015). Values of weight loss obtained in this study were higher than the results reported by de la Hera, Martinez, and Gómez (2013) which could be related to a higher water content in our batters formulation (125 g/100 g versus 110 g/100 g of flour), and a higher baking temperature (220°C versus 190°C). The lower weight loss of BW-MAase bread than BW bread is due to its higher moisture retention, which causes to lower specific volume of BW-MAase bread (2.58 against 2.78 cm 3 /g) ( Table 2). Although the volume index of BW-MAase bread is higher than BW, this difference like uniformity index was not significant (p > .05) ( Table 2).
Control bread showed the highest crumb/crust ratio than other breads (

| Color of gluten-free breads
Color is a significant property of breads, which is related to their texture and aroma, and consumer preference (Esteller & Lannes, 2008).
However, in most cases of crust color parameters in Table 3 Negative values for a were obtained for all gluten-free breads; that is, no red color for crumb was observed. Crumb yellowness was also observed for all breads in comparison with control (Table 3).

| Textural parameters of gluten-free breads
Texture profile analysis was done on gluten-free breads and the results are shown in Table 3 including hardness, resilience, cohesiveness, springiness, gumminess, and chewiness. The mechanical features of bread are a function of the crumb structure and in particular, crumb hardness has been studied widely due to its high correlation to sensory evaluations. As can be seen in Table 3, although there were no significant differences in hardness and chewiness of control, and bread with MAase and bread with BW (p > .05), breads with BW-MAase showed the lowest hardness and chewiness than other breads.

| Microstructure of gluten-free breads
The SEM was also used to investigate cellular structure of bread crumb of different gluten-free breads (Figure 3). In general, breads with formulations of control and BW resulted in a rough structure  Tables 2 and   3, particularly lower weight loss, higher volume index, darker crust, whiter crumb, less hardness in crumb and also more aerated microstructure, one can conclude that breads made from the encapsulated MAase with BW exhibited a better quality in comparison with other formulation breads.

| Gluten-free bread staling
The variation of moisture content, water activity (a w ) for the crust and crumb, hardness and sensorial acceptability of different glutenfree breads during four days of storage are presented in Figure 4.
Water is the main plasticizer in foods. Plasticizers make foods softer via embedding between polymer chains, reducing attraction forces between them and reducing T g . The plasticizing effect has a great importance in shelf life and sensorial properties of foods (Furlán et al., 2015). When bread started cooling, the difference in vapor pressure between crumb and crust resulted in the migration of moisture from crumb to crust of loaf, which ultimately leading to a decrease in moisture content of crumb and an increase in moisture content of the crust (Sabanis & Tzia, 2011). According to Figure 4a, b, c, and d, breads with BW-MAase and MAase formulations had higher moisture content and water activity in crust and crumb than control and BW bread, keeping the structure of crumb softer than other breads after 4 days of baking.
Hardness for all bread samples increased with an increase in the storage time. Among bread samples, the bread with BW-MAase formulation had softer crumb structure than other breads after 2 days of storage time (p < .05), which could be attributed to its higher moisture content in comparison with the other bread samples. However, the hardness of BW-MAase bread on the fourth day of storage reached the highest in comparison with other breads, and still, its sensorial acceptability is higher in both 2 and 4 days after baking.

| CON CLUS ION
In this study, MAase, which is a kind of antistaling enzyme, was encapsulated into beeswax and its application on gluten-free batter and bread quality and staling properties was determined. Results showed that this encapsulation technique had relatively good enzymatic efficiency, and its application in gluten-free bread could lead to maintaining more water content, higher volume index, darker crust, and whiter crumb and reduce the hardness of baked product. However, this encapsulation system increases hardness of gluten-free bread after 4 days of baking, and prepared bread still has a good sensorial acceptability. Therefore, application of beeswax in enzyme encapsulation for the baking industry can be considered as a good wall material.

INFORMED CONS ENT
Written informed consent was obtained from all study participants.

ACK N OWLED G M ENT
This work was supported by Sari Agricultural Sciences and Natural Resources University (SANRU).

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.