Recent practical researches in the development of gluten-free breads


  • 1 Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8642 Japan.
  • PMID: 31304279
  • PMCID: PMC6550274
  • DOI: 10.1038/s41538-019-0040-1

Wheat bread is consumed globally and has played a critical role in the story of civilization since the development of agriculture. While the aroma and flavor of this staple food continue to delight and satisfy most people, some individuals have a specific allergy to wheat or a genetic disposition to celiac disease. To improve the quality of life of these patients from a dietary standpoint, food-processing researchers have been seeking to develop high-quality gluten-free bread. As the quality of wheat breads depends largely on the viscoelastic properties of gluten, various ingredients have been employed to simulate its effects, such as hydrocolloids, transglutaminase, and proteases. Recent attempts have included the use of redox regulation as well as particle-stabilized foam. In this short review, we introduce the ongoing advancements in the development of gluten-free bread, by our laboratory as well as others, focusing mainly on rice-based breads. The social and scientific contexts of these efforts are also mentioned.

Keywords: Nutrition; Technology.

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  • Published: 14 July 2021

Physico-chemical properties of an innovative gluten-free, low-carbohydrate and high protein-bread enriched with pea protein powder

  • Monika Wójcik 1 ,
  • Renata Różyło 1 ,
  • Regine Schönlechner 2 &
  • Mary Violet Berger 2  

Scientific Reports volume  11 , Article number:  14498 ( 2021 ) Cite this article

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Metrics details

  • Biomaterials – proteins
  • Chemical engineering

The study aimed to determine the effect of pea protein powder on the pasting behavior and physico-chemical properties including the composition of amino and fatty acids of gluten-free bread with low-carbohydrate content. The control bread recipe was based on buckwheat flour (50 g) and flaxseed flour (50 g) as main flours. Additionally, the improving additives for this control bread such as psyllium husk (4 g), potato fiber (2 g), and guar gum (2 g) were used. The mixture of base flour was supplemented with the addition of pea protein powder (PPP) in the amount ranging from 5 to 25%. The results of Visco analyzes measured by RVA apparatus showed that the addition of 10% PPP to the control bread did not significantly differentiate peak viscosity and pasting temperature which was at the level 3115 cP and 3149 cP and 50 °C, respectively. Supplementation of low-carbohydrate bread with 10% of PPP was acceptable and significantly increased the content of all analyzed amino acids, as well as the amount of α-linolenic acid concerning the control bread. The lowest value of chemical score was observed for leucine. The EAAI (essential amino acid index) value increased from 34 to 40 when the optimal protein supplement was added. The developed gluten-free, low-carbohydrate, and high protein bread was characterized by contents of carbohydrate of 16.9%, protein of 17.1%, fiber of 13.7%, fat of 3.3% and its calorific value was 194 kcal/100 g.


Bread and flour-based foods are an important part of the diet of most people around the world. These products provide energy, protein, and minerals 1 , 2 . Recently, consumers are looking for functional breads 3 , among them gluten-free, high-protein, or low-carbohydrate bread. Although gluten-free bread recipes have been improved increasingly 4 , 5 , only a little research has been done on high-protein or even less on low-carbohydrate bread 6 . Low-carbohydrate bread can be proposed for people suffering from diabetes.

Concerning high protein bread, wheat bread was mainly enriched with high-protein flour from legumes. Such flours are often characterized by a high content of protein, fat, vitamins, fiber, and usually lower content of carbohydrates than wheat flour 7 , 8 . Also, these flours are characterized by a high content of lysine, and also improve the balance of essential amino acids in baked products 9 . Coda et al . 10 studied the effects of the substitution of wheat flour with faba bean flour (30%) on the properties of obtained bread. These authors obtained an improvement in the quality of the bread protein through faba bean sourdough addition. In addition to those mentioned lupin flour was incorporated into wheat-based bread by Villarino et al . 8 and another study at producing white wheat bread with increased protein, fiber, resistant starch, and decreased carbohydrate contents by partially substituting wheat flour with soy protein isolate, oat bran, and chickpea flour 6 . A low carbohydrate bread formula was also prepared using hard red spring wheat flour, soy protein, and vital gluten 11 .

An increasing part of the human population is intolerant to gluten, including the storage proteins found in wheat, rye, and barley. Therefore, scientists are looking for alternative cereals 12 . Most gluten-free raw materials are characterized by a low protein content, which affects the nutritional value of bread. Some authors studied the possibilities of substituting gluten-free bread with chickpea flour, pea isolate, carob germ flour, or soy flour 13 . According to these authors, chickpea bread had the best physico-chemical characteristics, and therefore could be a good alternative to soy proteins. In other studies, chickpea protein together with tiger nut flours was proposed as alternatives to emulsifier, and shortening in gluten-free bread 14 .

As mentioned above, there have been only a few attempts to create low carbohydrate bread, and they were mainly based on wheat flour. There are no clear reports where gluten-free bread with reduced carbohydrates, and increased protein content was studied. The aim of the study was therefore to determine the effect of varying protein contents after pea powder addition on the pasting behavior, and properties of a low-carbohydrate bread. Besides, the amino acid composition, and fatty acid content in an optimized bread were measured.

Materials and methods

In the present study, the following raw materials were used to make the control dough: buckwheat flour (Helcom, Poland), flaxseed flour (Bio Planet, Poland), psyllium husk (Dimica, Slovakia), potato fiber (Spiegel Hauer, Germany), guar gum (NatVita, Poland). All raw materials were purchased from a health food store. Dried yeast (Saf Instant, France), and Himalayan salt (Intenson, Poland) were also added. Also, the following high-protein ingredient was used: pea protein powder (Bio Planet, Poland) with protein contents of 78%.

Determination of basic chemical compositions of materials and bread

The chemical compositions of flours (buckwheat, and flaxseed), and pea protein powder, such as protein content 15 , fat content 16 , ash content 17 , moisture content 18 and dietary fiber content 19 were investigated. Carbohydrates were calculated by subtraction of protein, fat, moisture, and dietary fiber. The calorific value (per 100 g of bread) was calculated according to Costantini et al . 20 using Atwater coefficients.

Bread-making procedure

The control bread dough consisted of buckwheat flour (50 g), and flaxseed flour (50 g), psyllium husk (4 g), potato fiber (2 g), guar gum (2 g), salt (2 g), yeast (1 g), and tap water (130 ml). Buckwheat flour, and flaxseed flour were basic flour (100%), and were used in equal proportions (1:1) in the amount of 50 g each flour. Other additives were treated as technological improvers, and were additionally added to 100 g of base flour (according to baking practice—the amount of flour is given as 100%, and the ratio of the other components are converted to the weight of flour). The addition of pea protein powder (PPP) was used in the range of 5–25% as a substitute for the base flour. For example, if 10% protein was added, the percentage of buckwheat flour, and the same flaxseed flour was 45% or 45 g, together 90% of the base flour, and 10% of the added pea flour. The addition of water was the same in all the analyzed samples. Raw materials with a low carbohydrate content were selected for the basic bread recipe. The recipe composition has been selected as a result of numerous laboratory baking to obtain a good quality bread, without crumbling (disintegrate due to the lack of gluten). The bread was made according to the straight dough method earlier had been used for gluten-free bread (Ziemichód et al . 27 ) with slight modifications. All dry ingredients were combined with water. The dough was mixed to optimum development (6 min) in a laboratory spiral mixer type GM-2 (Sadkiewicz Instruments, Bydgoszcz, Poland), and was then divided into 120 g pieces, gently rounded, and then transferred into loaf tins (95 × 60 mm top; 80 × 50 mm bottom; 40 mm deep). Fermentation was performed at 30 °C, and 80% relative humidity for 60 min, afterward the bread was baked at 210 °C for 35 min. The obtained bread was cooled to room temperature, packed in polyethylene bags, and stored for 24 h until analysis. The bread baking experiments were done in three replicates.

Analysis of pasting properties of flour mixtures

A Rapid Visco Analyzer (RVA-4500, PerkinElmer, USA) was used to analyze the pasting properties of the control sample (C), which was the same mixture of flours (buckwheat, flaxseed), and improvers (psyllium husk, potato fiber, guar gum) as those used in baking, and described above. This mixture with varying pea protein (PPP) supplementation (5%, 10%, 15%, 20%, and 25%) was also tested the same as baking. Samples with different PPP additives were named 5CP, 10CP, 15CP, 20CP, and 25CP. On the other hand, we were also interested in examining the characteristics of buckwheat flour (BW) itself, and the mixture (BF) of buckwheat flour, and flax flour (1:1). These flours themselves did not give good quality bread, but studies of their properties are lacking in the available literature. It was useful to further explain the overlapping relationships. The measurements were made according to the approved method 22-08 (AACCI, 2000) 22 . In the beginning, we prepared flour mixtures, then we tested their moisture content, which was in the range from 11.1% to 13.7%. Based on this moisture content, the program calculated the appropriate weight of the mixture corresponding to 3.5 g of flour with 14% moisture. After weighing a sample was transferred directly into a metal RVA canister, and filled with 25 ml of distilled water. Samples (stirred at the speed of 160 rpm) were heated from the temperature of 50 °C to 95 °C for 5.5 min, maintained at 95 °C for 5 min, cooled to 50 °C in 5 min, and kept at this temperature for 5 min. The RVA software was used to evaluate the curve characteristics (Thermocline for Windows v2.2, Newport Scientific Pty. Warriewood NSW, Australia). Paste viscosity parameters recorded were peak viscosity (cP); trough (cP); final viscosity (cP), and pasting temperature (°C). The tests were replicated thrice.

Basic properties of bread

The volume of low-carbohydrate bread was measured 24 h after baking using the millet seeds displacement method 22 . Values were calculated for 100 g of bread. The pH of the crumb of bread was tested using the pH meter 206-ph2 (Testo, Pruszków, Poland). The baking loss was calculated by measuring the weight of the dough piece before baking, and weight after baking.

Colour measurements of bread

The colour change of the bread crumb as a result of the addition of pea protein was assessed using a colorimeter CR30-16 (Precise Color Reader, 4Wave, Tychy, Poland). The measurement was based on the CIE L*a*b* system where L* defines lightness from 0–100 (black to white), a* denotes red( +)/green(−)value, and b* the blue (−)/yellow ( +) coordinate. Three replicates of each bread sample were analyzed.

Texture profile analysis of bread

The analysis of the texture parameters 24, and 48 h after baking was performed using the ZWICK Z020/TN2S (Zwick Roell Group, Ulm, Germany) strength testing machine with a round measuring head with a diameter of 25 mm. Bread crumb slices were cut directly before the measurement (15 mm of thickness) using a square cutter (20 mm × 20 mm). The samples were subjected to double compression to 60% of their thickness at speed of 20 mm s -1 , which allowed the calculation of texture parameters such as hardness, springiness, cohesiveness, and chewiness 23 . The analysis was conducted in eight replicates.

Sensory evaluation of bread

The sensory analysis of the obtained bread was carried out 24 h after baking by seventy panelists (18–70 years, 40 females, and 30 males). For the tests, square-shaped samples with dimensions of 20 × 20 mm were prepared, which were cut with a special cutter from a slice of bread of 1 cm thickness. They were then coded, and submitted for evaluation in a closed odor-free room. The following quality indicators were assessed: taste, colour, texture, odor, and overall acceptability. The degrees of liking for the low-carbohydrate bread were based on a seven-point hedonic scale (1: dislike very much, 4: neither like nor dislike, 7: like very much) 24 .

Amino acid composition of bread

The amino acid composition was determined after the execution of protein hydrolysis. The acid hydrolysis was performed according to Davis and Thomas 25 . The hydrolysis procedure according to Schramm et al . 26 was used to determine the sulfur amino acids, and tryptophan. The content of the amino acids with tryptophan was measured using the acid analyzer AAA 400 (Ingos, Prague, Czech Republic) following the methodology described by Ziemichód et al . 27 . Additionally, the chemical score (CS) of essential amino acids, and the EAAI index were calculated 28 .

Fatty acid composition of bread

Gas chromatography was used to determine the qualitative, and quantitative composition of the mixture of fatty acid methyl esters (FAME) in the sample of bread prepared by ISO 12966-2:2017-05 29 . Chromatographic separation was performed using Varian 450-GC gas chromatograph with Galaxie Chromatography Data System software.

Statistical analyses

Statistical analysis of the final results was carried out in Statistica 12.0 considering a significance level α = 0.05. Analysis of variance (ANOVA) was performed, and Tukey’s test was used to compare the mean values.

Human participants

Authors declare that research involving human research participants have been performed in accordance with the Declaration of Helsinki. Peoples were informed and informed consent was obtained from the patients. All experimental protocols were approved by a University of Life Science in Lublin institutional committee.

Experimental research on plants/seeds

The collection of plant material complied with relevant institutional (University of Life Science in Lublin), national (Poland), and international guidelines and legislation.

Results and discussion

Basic chemical composition of flours.

Buckwheat flour, and flaxseed flour used to produce low-carbohydrate bread contained, respectively: 13.0 ± 0.05%, and 40 ± 0.22% protein, 3.1 ± 0.07%, and 8.8 ± 0.02% fat, 63.1 ± 0.1%, and 3.9 ± 0.07% carbohydrates, 4.1 ± 0.2%, and 34.0 ± 2.1 fiber, and 1.20 ± 0.02%, and 6.9 ± 0.4% ash content. Pea protein powder (PPP) was characterized by protein content of 78.4 ± 0.41%, carbohydrates content of 7.2 ± 0.3%, and fat content of 6.8 ± 0.09%. Buckwheat, and flaxseed flour were selected for the bread recipe based on the available literature, and chemical analysis as raw materials with a low carbohydrate content. For example, white wheat flour usually contains more than 70% carbohydrates 30 , and rice flour at most 80% 22 , 30 , while corn flour has usually even more than 80% carbohydrates 30 .

Pasting behavior of flours and bread mixtures

The pasting behavior of buckwheat, blend of flaxseed, and buckwheat flours, and control bread mixture with a different percentage of the PPP is shown in Fig.  1 . The RVA curves obtained from the measurements are also presented.

figure 1

Pasting properties of studied flours, and blends with different concentration of pea protein powder: BW—buckwheat, BF—blend of buckwheat, and flaxseed flours; C—control sample, 5CP—blend of flours with 5% pea protein powder added, 10CP—blend of flours with 10% pea protein powder added, 15CP—blend of flours with 15% pea protein powder added, 20CP—blend of flours with 20% pea protein powder added, 25CP—blend of flours with 25% pea protein powder added . *Values in the same column marked with different letters are significantly (α = 0.05) different.

The control sample with the addition of psyllium husk, potato fiber, and guar gum (C) had a significantly higher peak viscosity, and lower pasting temperature compared to the blend of buckwheat, and flaxseed flours (BF). The control bread (C) recipe included technological enhancers which resulted in a significant improvement in these parameters, and the addition of protein had no negative effect. As reported by Casas et al. 35 the apparent viscosity of guar gum solutions increased with guar gum concentration, and they showed pseudoplastic behavior. In another study by Harasztos et al . 36 addition of arabinoxylans, the major components of dietary fiber wheat flours showed a constant increase in all measured viscosity parameters. The authors assumed that arabinoxylans had a significant impact on viscosity despite their low concentration. The effect of dietary fiber concentration on RVA rheological properties of wheat starch/fiber systems was evaluated by Yildiz et al . 37 , and authors showed that peak, trough, break down, final, and setback viscosity increased; however, pasting temperature decreased as fiber concentration increased. The same results were found in our study, using a mixture of guar gum, potato, and psyllium fiber, peak, and final viscosity increased, pasting temperature decreased.

The addition of protein did not change the pasting temperature, which for these mixtures was at 50 °C. The highest viscosity values were observed in the control sample (C) with 5%, and 10% of PPP, but a PPP addition of 15%, and more resulted again in a significant reduction in the viscometric parameters. It is known that differences in the protein composition can affect pasting viscosity, as found for example by Ragaee et al . 34 . Xie et al . 38 explained that this decrease in paste viscosity was probably due to hydrolysis of the protein rather than the starch components. Although in our studies it may have been caused by starch dilution.

In the case of buckwheat flour alone or a mixture of buckwheat flour, and flaxseed flour, the peak viscosity values were much lower, and the temperature was higher than control bread. Also, the results showed a decrease in peak viscosity by adding flaxseed flour to buckwheat flour, which could be mainly attributed to the change of carbohydrate (starch) content in the final blend. In our study flaxseed flour used in a mixture with buckwheat flour was characterized by a very low content of carbohydrates, which significantly reduced the content of the resulting blend, thus reduced its peak viscosity. Similar to our study the final viscosity of a barley-flaxseed composite blend (1:1) presented by Inglett et al . 31 was lower than that of barley flour, and other composites. As these authors explained it may be due to the low viscosity contributed by the ground flaxseeds. As reported by Kaushal et al . 32 , flaxseed flour due to higher protein content, showed a lower swelling ability because of stronger bonding in this flour, which directly influences the peak viscosity of its blend. In other studies 33 , the addition of potato starch to wheat flour increased the peak, and final viscosity in the mixtures of wheat flour with potato starches. Also, Ragaee and Abdel-Aal 34 explained that the high content of starch in wheat flours compared to wholegrain meals may contribute, to some extent, to the higher pasting viscosity. The peak viscosities increased significantly with an increase in the starch content in the mixtures.

Physical properties of low carbohydrate bread with pea protein

The addition of PPP caused significant changes in the basic properties of the resulting bread (Table 1 ). With the increase of protein, bread moisture increased from 53.4% in the control sample to 57.2% in the bread with 25% of PPP. It was also observed that bread with 5%, and 10% of PPP had a lower baking loss (23%), while at higher PPP amounts it increased again. On bread volume, PPP addition had a negative impact, a decrease was noticed. Ziobro et al . 38 reported that the volume of the bread baked with pea protein was smaller than control bread. Kamaljit et al . 40 noticed the same trend in the case of pea flour addition to wheat bread. The addition of PPP resulted in a slight but significant increase in the pH value of the bread crumb.

Colour parameters analysis showed that the addition of PPP up to 10% did not cause any significant change in the colour of the crumb of the low-carbohydrate bread (Table 3 ). Only a higher addition caused a slight but significant increase in lightness(L*), and a decrease in the a*-, and b*-value.

Figure  2 presents the textural parameters of the bread crumb supplemented with PPP after 24 h, and 48 h of storage. It was noticed that the amount of PPP up to 10% did not cause changes in hardness, cohesiveness, and chewiness of bread crumbs (Fig.  2 a,b,d), even after a long storage time. Only the crumb of the sample with 20% PPP addition was characterized by lower hardness after 48 h of storage compared to the sample stored for 24 h (decrease of about 2 N). However, in general, a higher share of the PPP brought about an increase in the bread crumb hardness, and chewiness, and a decrease in cohesivness. In contrast, the springiness of bread crumb (Fig.  2 c) increased already with small amounts of PPP addition, as compared to control bread.

figure 2

Changes of textural crumb properties of low-carbohydrate bread as a result of PPP addition: ( a ) hardness, ( b ) cohesiveness, ( c ) springiness, ( d )chewiness; mean values in the same figure marked with different letters are significantly (α = 0.05) different.

Sensory evaluation of low-carbohydrate bread with pea protein

The results of the sensory evaluation of the low-carbohydrate bread enriched with pea protein powder are presented in Fig.  3 .Sensory evaluation showed that the control bread, and the bread with PPP addition at the levels of 5, and 10% had the highest liking score. Similarly, Ziobro et al . 41 demonstrated in the sensory evaluation of gluten-free bread with the addition of non-gluten proteins (i.e. albumin, soy, pea, lupine, collagen) that the bread supplemented with pea protein was the best assessed in case of structure, and porosity, and obtained the highest number of points for taste, and smell among the evaluated bread. The addition of higher levels of PPP (above 15%) caused an unpleasant aroma, and bitter taste. Furthermore, the low-carbohydrate bread generally received slightly low notes for the texture. It was caused by higher water addition, and the lack of gluten in the composition of flours used in the production of bread. It was also found that a higher percentage of pea protein caused crumbling of the crumb, and generally, the texture was not compact. The addition of PPP did not significantly change the bread crumb colour, which had a dark-brown colour that was generally acceptable for the evaluators.

figure 3

Overall view, and results of sensory evaluation of low-carbohydrate bread with the addition of 5% -25% pea protein powder (PPP) . The author of the photo in this figure is Monika Wójcik.

Amino-acid and fatty acid composition of low carbohydrate bread with optimal pea protein

Supplementation of low-carbohydrate bread with 10% of pea protein increased the content of all analyzed amino acids (Table 2 ). The chemical score (CS) for each essential amino acid has increased. The lowest value of CS was observed for leucine. EAAI value increased from 34 to 40 after the addition of the optimal protein supplement (10% PPP).

According to Gorissen et al . 42 pea as a plant-based protein source is rich in essential amino acids like lysine, and leucine, and non-essential amino acids like arginine, alanine, proline, and glutamic acid. The authors pointed out that pea has essential amino acid contents that meet the requirements as recommended by the WHO/FAO/UNU, and that the amount of essential amino acid in pea is higher than in plant materials, such as corn, soy, hemp, lupine, oat or brown rice. In the case of the content of glycine, cysteic acid, methionine sulfone, and histidine, only a slight increase in their content was detected in the composition of bread with 10% PPP addition, which was not statistically significant.

In the analyzed bread the following fatty acids were identified above 0.100 g/100 g: palmitic acid, octadecanoic acid, oleic acid + elaidic acid, linoleic acid + trans-9,12-octadecadienoic acid, and α-linolenic acid (Table 3 ). A significant increase of α-linolenic acid content in relation to the control bread occurred in the bread with 10% pea protein.

Caloric value of low-carbohydrate bread with optimal pea protein amount

The addition of PPP had a significant effect on the chemical composition of the low-carbohydrate bread (Table 2 ). In the case of the bread with the optimum amount of pea protein at the level of 10% significant increases in the protein content (from 14.7% to 17.1%), and decrease in the carbohydrates content (from 18.4% to 16.9%) was noticed. According to García-Segovia 43 the addition of 10% of pea protein increased protein content (to 19.3%), compared with the control wheat bread, and in the case of supplementation of wheat bread with pea protein concentrate (at the same level), as reported by Des Marchais 44 , even up to 20%. In other wheat bread studies, the protein content was 8.9%, carbohydrates were at 45.3% and after the addition of lupine isolate the protein content increased to almost 14.0%, and the carbohydrate content decreased to 37.9% 45 . The addition of faba bean flour (30%) wheat bread increased the protein content from 11.6 up to 16.5% 10 . The use of the addition of various types of lupine in the amount of 20% to wheat bread increased the protein content from 13.4% to about 19%, and the reduction of the carbohydrate content from 71% to about 60%. The fiber content increased from 9.2% to 15–16% 46 . In our study the fiber content of the protein-enriched bread was equal to 13.7%, there were no significant differences in the fiber, and fat content between the control bread, and the 10% PPP-based bread. The resulting low-carbohydrates, and high protein bread have a low-calorific value, compared to other breads whose calorific value in various publications were at a level of more than 200 kcal/100g 20 , 30 . Conventional wheat bread presents low protein, high carbohydrate, and small amounts of dietary fibre 6 . Our recipe, on the other hand, made it possible to obtain bread with increased protein content, and significantly reduced carbohydrate content.


The pasting properties showed that mixing buckwheat flour with low-carbohydrate flaxseed flours significantly reduced peak viscosity, while technological additives (e.g. hydrocolloids, dietary fiber) significantly increased peak viscosity and reduced pasting temperature from 70 °C to 50 °C. The addition of pea protein (PPP) up to 10% did not significantly change pasting behavior, only higher amounts of proteins reduced viscosity parameters.

The results of volume, texture, and sensory evaluation indicated that enrichment of low-carbohydrate bread with pea protein powder addition up to 10% gave satisfactory results. However, the higher addition of this protein negatively influenced the volume, texture, and also taste, and odor of bread (crumbling of crumb, unpleasant aroma, and bitter taste). Pea protein-supplemented bread contained significantly higher amounts of amino acids (lysine, leucine, arginine, alanine, proline, glutamic acid, and tryptophan). The lowest value of chemical score was observed for leucine. EAAI value increased from 34 to 40 after the addition of the optimal protein supplement (10% PPP). Developed low-carbohydrate bread with increased protein was characterized by a carbohydrate content of 16.9%, protein content of 17.1%, the fiber content of 13.7%, and a calorific value of 194 kcal/100 g. This bread could be consumed for physically active people because of its role in the prevention of various human diseases. Besides, this kind of bread contains no gluten, and can be consumed by patients with celiac disease.

Data availability

All the data generated or analyzed during this study are included in this published article.

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The research was financed by the ‘Excellent science’ program of the Ministry of Education and Science as a part of the contract no. DNK / SP / 465641/2020. The role of the agricultural engineering and environmental engineering in the sustainable agriculture development. In addition, support was received from the Erasmus + grant (STT - Staff Mobility for Training).

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research paper on gluten free bread

Characteristics of gluten-free bread: quality improvement by the addition of starches/hydrocolloids and their combinations using a definitive screening design

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  • Hayat Bourekoua 1 , 2 ,
  • Renata Różyło 2 ,
  • Leila Benatallah 1 ,
  • Agnieszka Wójtowicz 3 ,
  • Grzegorz Łysiak 2 ,
  • Mohammed Nasreddine Zidoune 1 &
  • Agnieszka Sujak 4  

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To establish factors affecting the quality of gluten-free bread based on rice semolina supplemented with field bean semolina and improving its final quality, a new study with definitive screening design was conducted after an appropriate choice of six factors: agar–agar, water, two types of gums gum arabic and locust bean gum, and two types of starches tapioca starch and corn starch. We investigated the effect of the aforementioned parameters on specific volume, hardness, chewiness, and springiness of breads. The results showed that specific volume of gluten-free breads increased significantly ( p  < 0.05) with the addition of gum arabic, tapioca and corn starches, and water; addition of agar–agar, gum arabic, tapioca starch and water affected the hardness. With regard to chewiness, the results showed that gum arabic and water and also the interaction between them had a significant effect. Gum arabic, tapioca and corn starches, and water affected the springiness. In addition, we observed the interactions among the additives. For all the tested parameters, water and gum arabic had statistically significant ( p  < 0.0001) effect and affected all the properties of examined breads. These factors were retained for process characterization of optimized gluten-free bread. The final optimum formulation of rice/field bean contained 1.5% of gum arabic and 71.5% of water. The optimum gluten-free bread with gum arabic showed high volume, good textural, structural, and sensory qualities with high acceptability compared to the gluten-free control bread without any improver.

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Celiac disease is currently one of the most common gastrointestinal diseases. It affects about 1% of the world’s population [ 1 , 2 ]. Because the digestive system of patients with celiac disease is sensitive to gluten present in wheat and other prolamin containing cereals such as rye, barley, and triticale, they have to exclude gluten from their diet [ 3 , 4 ]. Hence, there is an urgent need to develop gluten-free products for patients with celiac disease.

In Algeria, patients with celiac disease suffer due to the nonavailability of gluten-free products, which makes it difficult for them to follow their diet restrictions. One of the methods to improve their situation to tackle the disease is the development of traditional gluten-free products [ 5 ].

Making high-quality bread requires the presence of gluten, a protein which is responsible for the final structure of bread and also helps to retain gas bubbles and imparts a pleasing volume and texture to the bread dough system [ 6 ]. Therefore, elimination of gluten from the diet of patients with celiac disease implies greater difficulties in the bread making process such as lack of cohesion and elasticity and low gas retention capacity of the gluten-free dough. Thus, a bread without gluten displays properties such as low volume, friable texture, poor flavor, and rapid firming compared to popular wheat breads [ 7 , 8 , 9 ]. Use of bread quality improvers has become an unavoidable element in improving the quality of bakery products [ 10 ].

Recently, various gluten-free formulations have been developed with the help of nongluten components such as starches and hydrocolloids to mimic the viscoelastic properties of gluten and to improve the final quality of bread [ 6 , 11 ]. With respect to the ingredients in the bread making process, rice is the most commonly used ingredient, followed by corn, as these are the two most productive cereals around the world. Furthermore, supplementation of gluten-free dough with legumes has also been previously performed [ 5 , 12 ]. Corn starch and starch from tubers such as potato and tapioca are most commonly used in the manufacture of gluten-free bread [ 8 ].

Due to their functional properties, thickening agents, stabilizers and enhancers of water retention, enhancers of textural properties, and various hydrocolloids are frequently used in the formulation of gluten-free breads to improve their structural properties as well as their acceptability. Among them, hydroxypropyl methylcellulose, xanthan gums, cellulose gums, pectin, guar gum, or gum arabic are most commonly used [ 4 , 13 , 14 , 15 , 16 ].

As starches and hydrocolloids are most frequently used in the formulation of bakery products, their combinations have also been investigated by many authors [ 17 , 18 , 19 ]. In addition, synergistic interactions between starches and gums have also been studied in recent years [ 20 , 21 , 22 ].

Most gluten-free breads manufactured with rice still have weaker physical and textural qualities than those manufactured with traditional wheat breads. Therefore, supplementation of gluten-free rice bread formulation with hydrocolloids and/or additives is often required [ 4 ]. Thus, further research in the development of gluten-free rice breads with acceptable textural and sensory properties is highly warranted.

Design of experiment is a statistical model that efficiently examines multiple parameters in a minimum number of runs, thereby helping us to optimize the factors and their interactions. During the analysis of factors, screening designs must be used to select the parameters that affect the response significantly; however, such parameters are limited and generally need a more detailed study to understand the effects of interaction between such factors [ 23 ]. Therefore, a new design called definitive screening design (DSD) with three levels has been proposed by Jones and Nachtsheim [ 24 ], which allows screening of factors to obtain information that can clarify details about their effects. In this design, secondary interactions are evenly estimated so that it provides more information about the combination effect between factors.

Thus, in this study, we used DSD approach to investigate the effect of two different starches (tapioca and corn), two different gums (gum arabic and locust bean gum), and agar–agar and their possible combinations on the textural and sensory parameters of gluten-free bread based on rice and field bean semolina.

Materials and methods

Rice semolina with particle size between 200 and 500 µm was obtained after grinding long grain white rice using a laboratory mill (LMN-100 Testchem, Radlin, Poland). The long grain white rice was purchased from Makro K&K Sp. z.o.o. (Cmolas, Poland). Rice semolina was characterized with 10.33% moisture content, 0.22% ash content, 0.50% lipid content, and 7.80% protein content. Field bean semolina ( Vicia faba ) (10.46% moisture, 0.50% ash, 1.03% lipid, and 30.86% protein) was obtained after grinding the dehulled bean seeds purchased from Al-Amir Company (Albehera, Egypt). Instant dry yeast was purchased from Saf-Instant (France); salt, commercial sunflower oil, and fresh eggs were purchased from a local market. Tapioca starch (extracted from cassava root— Manihot esculenta ) was obtained from Thailand (exotic food, Sriracha, Thailand); corn starch was obtained from Kraków (Bezgluten, Poland); agar–agar (derived from agarose–polysaccharide polymer material extracted from algae) and gum arabic (natural gum from various species of the acacia tree derived from Africa) were purchased from NatVita (Długołęka, Poland); locust bean gum (galactomannan vegetable gum extracted from carob seeds) was obtained from China (Samic Enterprise, China, Guangdong).

Thermal properties of starches and hydrocolloids by differential scanning calorimeter (DSC)

Thermal properties of tapioca and corn starches, agar–agar, gum arabic, and locust beangum were evaluated using DSC (DSC Mettler-Toledo AG, Greifensee, Switzerland). Measurements were controlled with STARe Software. Temperature was controlled by Huber high precision thermoregulation system TC100MT, with an accuracy of ±0.01 °C.

Measurements were conducted under nitrogen atmosphere. Experiments were performed in aluminum crucibles with pin (40 μL). Empty crucible was used as a reference. Samples were thermally equilibrated at 25 °C for 10 min and then heated up to 180 °C at a heating rate of 10 °C/min. Plots of heat flow versus temperature were registered. Thermal parameters (onset, peak position, final temperature, and enthalpy of transition) were calculated using evaluation mode from STARe system.

DSD was used to study the effect of six continuous factors ( k  = 6; X 1 agar–agar, X 2 gum arabic, X 3 locust bean gum, X 4 tapioca starch, X 5 corn starch, and X 6 water) and their possible interactions on quality characteristics of gluten-free bread presented by four responses: Y 1 specific volume (cm 3 /g), Y 2 hardness (N), Y 3 chewiness (N), and Y 4 springiness.

For making DSD model, three levels for each factor are necessary −1, 0, and 1 refer to the minimal, median, and maximal concentrations of factors (Table  1 ). For hydration, minimal and maximal water levels were selected based on our preliminary trials, which were finalized between 65 and 78 g/100 g of formula, respectively (65 g/100 g of formula is the minimum level of water necessary to make dough and 78 g/100 g of formula is the maximum level of water necessary for the bread to be with good volume and crumb after being baked). Concentrations of added starches, gums, and agar–agar ranged according to the preliminary tests of feasibility and data published previously [ 4 , 25 , 26 , 27 ] from 0 to 1% for agar–agar, from 0 to 3% for gum arabic, and from 0 to 2% for carob gum, tapioca starch (0–10% w/w), and corn starch (0–20% w/w) based on rice/field bean semolina weight.

For DSD, the minimum number of required runs is one more than twice the number of factors (2 k  + 1) included in one center point. For the DSD generated in this study, there were six factors and, therefore, 13 runs were required. To give more power to the design, four additional extra runs were added. Runs were conducted randomly to maximize specific volume and springiness and minimize hardness and chewiness according to the control bread without any improver. Optimization was generated by DSD using the desirability function approach.

According to Šimurina et al. [ 28 ], the desirability function approach is an optimization method useful to find the best compromise between several responses. Often there are multiple responses measured and the desirability of the outcome involves several or all of these responses. D  = ( d 1  ×  d 2  × d 3 …×  d n ) 1/ n where d i are the desirability indices for each response ( d i  = 0 least desirable; d i  = 1 most desirable according to the optimization method, for example, for specific volume, if the desirability indices is close to 1 or 100%, the specific volume is optimum) and n is the number of responses in the measure. The values of experimental design and levels for each factor are shown in Table  1 .

Baking tests

A formula with rice semolina and field bean semolina in a ratio of 2:1 for 100 g of formula (66.66 g of rice semolina/33.33 g of field bean semolina) was used in this study, aiming to offer a better nutritional balance in amino acids [ 3 ].

The gluten-free bread making process was performed according to Bourekoua et al. [ 5 ]. A control gluten-free bread made without any additives with 75 g water/100 g of formula, fixed according to our preliminary trials, was used. Gluten-free breads were prepared using 2% salt, 2% instant dry yeast, 10 g fresh egg, and 20 mL of sunflower oil based on rice/field bean semolina weight. Water and the additives were added according to the experimental design data (Table  1 ). In the first step, all the ingredients were mixed (1 min) with the exception of fresh egg and additives and left to rest for 10 min. After resting, fresh egg, additives, and rest of the water were added. The mixture was kneaded for 15 min at 25 °C. The resulting dough was weighted to 80 g on four baking molds and then subjected to proofing for 45 min at 37 °C with a relative humidity of 75–80% in a fermentation cabinet. Breads were baked in an oven for 20 min at 230 °C (Sadkiewicz Instruments, Bydgoszcz, Poland). The baked breads were allowed to cool for 1 h at room temperature prior to quality evaluation process.

Quality evaluation of gluten-free bread

Properties of the gluten-free breads were measured approximately 1 h after baking. For each analysis, four samples of bread were used.

Volume of bread was determined by millet seed displacement method according to the AACC approved method 10.05 [ 29 ], and specific volume (cm 3 /g) of the bread was calculated by dividing its volume by weight.

Moisture content was evaluated based on ICC 110/1 method [ 30 ].

Texture profile analysis of bread crumb was performed using a texture analyzer (ZWICK Z020/TN2S strength tester, Germany). Samples of bread crumb collected from the center of the loaf with a dimension of 30 × 30 × 20 mm were double compressed using a head equipped with a 30 mm penetrator until a 50% depth at a crosshead speed of 1 mm/s was achieved [ 31 ] and then following parameters were recorded: hardness, springiness, and chewiness.

Color of bread crumb was measured using a colorimeter 4Wave CR30-16 (Planeta, Tychy, Poland) under the following conditions light D 65; space Lab; diameter 16 mm; style 8/d. Color was determined in CIE-Lab system, where L * indicated lightness. The redness +/greenness− and the yellowness+/blueness− are denoted by a * and b * values, respectively. Data from three slices per loaf were averaged.

Analysis of crumb cells was performed by Image J software according to Gonzales-Barron and Butler [ 32 ]. 10-mm thick central slices were made on four bread loaves, and their images were captured using a scanner (HP ScanJet 3530c). The number of cells and their average size were calculated.

Thermal parameters of gluten-free breads’ crumb were measured using DSC according to the methods described above. In this instance, scanning temperatures varied from 25 to 200 °C.

Structural analysis of optimum gluten-free bread obtained by analysis of desirability function and control bread was performed using a scanning electron microscopy on dry samples. Samples of bread were freeze dried prior to analysis. Dried samples were mounted on carbon disks using a silver tape and sprayed with gold in a vacuum sublimator K-550X (Emitech, RC, Ashford, England). The VEGA LMU microscope (Tescan, Warrendale, PA, USA) operating at 30 kV was used to examine the cross-section of samples at different magnifications (100× and 400×).

For sensory evaluation, the samples were sliced mechanically (1 cm thick) and divided into eight parts [ 33 ]. The panel for sensory evaluation consisted of 52 untrained consumers (23–48 years old; 28 females and 24 males) who were habitual consumers of bread. According to a nine-point hedonic scale (1 dislike extremely, 5 neither like nor dislike, 9 like extremely), the taste, aroma, texture, and the overall acceptability of gluten-free breads were evaluated [ 34 ].

Statistical analysis

The design and analysis of experiments were performed with JMP statistical software, version 13 (SAS Institute, Cary, NC, USA). p value was used to determine if a factor is significant; as a rule, this component was compared to α value of 0.05. If the value of p was less than 0.05, the factor was significant. Data were averaged and means were compared and evaluated using one-way analysis of variance followed by the Tukey’s significant differences post hoc test, performed using STATISTICA 7.0 software (StatSoft, Inc., Tulsa, OK, USA). A statistical difference at p  < 0.05 was considered significant.

Results and discussion

Thermal properties of additives.

DSC parameters of the starches and hydrocolloids are shown in Table  2 . For corn and tapioca starches, DSC parameters were almost similar considering their values of onset, peak, end set, and enthalpy of transition. No statistical difference between the two starches was found ( p  > 0.05).

Considering hydrocolloids, locust bean gum and agar–agar exhibited the lowest and the highest transition temperature and enthalpy. Agar–agar demonstrated the highest enthalpy (336.96 J/g) and highest transition temperature (97.32 °C) than locust bean gum, which showed an enthalpy of 223.99 J/g and transition temperature of 94.48 °C. For gum arabic, enthalpy value and transition temperature were found to be 261.03 J/g and 94.95 °C, respectively. Gum arabic demonstrated the highest end set temperature (165.52 °C) than that of other gums ( p  < 0.05). DSC parameters of gums were highly variable and depended on the natural source of the gums. The values were also affected by sample preparation and operation status of the instrument. Consequently, it is often difficult to compare data obtained from various DSC studies [ 26 ].

Comparing thermal properties of starches and hydrocolloids, we observed that hydrocolloids exhibited higher thermal properties.

Effect of factors on gluten-free bread characteristics and DSD results

DSD model used in this study was found to be efficient because the coefficients of determination ( R 2 ) were found to be 0.97, 0.93, 0.85, and 0.94 for Y 1 , Y 2 , Y 3 , and Y 4 , respectively, and the regression explains the phenomenon studied since the significance of the risk ( p  < 0.0001) is less than 0.05 for all responses.

While studying the effect of factors, a null hypothesis considering lack of difference between control and substituted bread was assumed so no observed effect on parameters. An alternative hypothesis was considered when there was a significant effect of the factors on parameters being tested. Effects were evaluated at p  < 0.05. All factors that were not included in the model ( p  > 0.05) were screened out. The explanation for some is difficult and as it might relate to the other components of the traditional Algerian bread such as whole egg and oil that can mask the effect of some factors for different responses.

Influence of factors on specific volume

Bread loaf volume is an important parameter used in the determination and assessment of quality of bread [ 38 ]. Results in Table  3 confirm that agar–agar with a negative sign demonstrated a main significant effect ( p  < 0.05) on specific volume, whereas gum arabic, tapioca starch, corn starch, and water demonstrated a positive effect on specific volume. The main effect with a positive sign for a factor indicates that a high concentration of this factor is nearly optimum, and a negative sign for a factor indicates that a low concentration of this variable is nearly optimum. Gum arabic and corn starch were involved in a significant negative two-way interaction. This means that the combination of small levels of gum arabic and corn starch positively affected the specific volume of gluten-free bread.

Gum arabic showed the highest positive main effect (0.189) on specific volume indicating that the presence of this component in high concentrations in gluten-free bread can improve its volume. Asghar et al. [ 25 ] indicated that loaf volume of bread was significantly affected by the addition of gums; the maximal volume of bread was recorded at 3% of gum arabic. Agar–agar increased specific volume of bread with low levels, as indicated by Collar et al. [ 39 ]. Hydrocolloids when used in small quantities [<1% (w/w) in formula] are expected to increase water retention and loaf volume. Collar et al. [ 39 ] and Mir et al. [ 40 ] reported that the quality of gluten-free breads is primarily affected by the nature, content, and properties of hydrocolloids as these components increase dough foam stability by increasing viscosity and coalescence, preventing effects on the aqueous phase of dough and thus affecting the stability of the liquid film surrounding gas bubbles, preventing gas retention.

Positive effect of tapioca and corn starches indicates that their addition to gluten-free bread can increase its volume [ 4 , 41 ]. Addition of starches to bread could help in gas retention as well as the expansion of gas bubbles during proofing and baking, and contribute to the structural architecture and mechanical strength of gluten-free bread [ 4 ].

Water showed a positive effect on specific volume of gluten-free bread. Many authors concluded that water positively affects the volume of gluten-free bread [ 5 , 31 , 42 ].

The combination of gum arabic and corn starch could increase the specific volume of bread significantly. Many studies referring to Mollakhalili Meybodi et al. [ 6 ] reported that the starches/gums are used in combination to improve gluten-free bread quality including volume.

Influence of factors on bread crumb texture

Hardness of bread crumb is considered as a very important quality of bread. The data from Table  4 show a range of negative effects and two-way interaction values on hardness accompanying the presence of gum arabic, tapioca starch, water at low levels, and the combination of agar–agar and gum arabic. Agar–agar and tapioca starch at low concentrations had positive effect on hardness of gluten-free bread. The only positive effect was observed for agar–agar (1.44). The presence of low levels of gum arabic and high levels of agar–agar can decrease the hardness of bread. Similarly, the presence of low levels of tapioca starch can improve the hardness of bread at high levels of agar–agar.

Mir et al. [ 40 ] reported that hydrocolloids could improve the texture of gluten-free bread. Toufeili et al. [ 16 ] also reported that incorporating gum arabic at low concentrations could decrease hardness of bread.

An interaction between gum arabic and agar–agar at low levels affects the hardness of gluten-free bread. This can be difficult to explain because as indicated by Collar et al. [ 39 ], there is a lack of information on the influence of mixtures of hydrocolloids on dough parameters and baking quality of bread. But, the interaction between agar–agar and tapioca starches can be explained by the effect of gums on rheological parameters and swelling power of tapioca, which improve bread quality [ 11 ].

According to the results presented in Table  4 , only gum arabic (−1.24) and water (−0.75) showed a significant effect on chewiness of bread but with a negative sign, and the two-way interaction of both showed a positive effect which involves both the presence of gum arabic and water at high concentrations to improve chewiness of bread. Mir et al. [ 40 ] reported that hydrocolloids interact with water, reducing its diffusion and stabilizing its presence. The interaction between different dough components and water is important to the property of dough and, therefore, quality of the corresponding bread needs to be evaluated [ 4 ].

Springiness is related to aeration and elasticity of bread and high values are desired [ 33 ]. In our gluten-free bread, springiness showed different effect (Table  4 ). A negative effect of gum arabic indicates that improving springiness in bread requires the presence of gum arabic in small amounts. But, a positive effect of tapioca starch, corn, and water was observed which involve the addition of these factors at high concentrations to improve springiness of gluten-free bread.

For all crumb properties (hardness, chewiness, and springiness), water showed a significant effect as reported by numerous authors [ 5 , 31 ].

Optimal conditions

According to the results of DSD and the results presented in Tables  3 and 4 , gum arabic and water only showed a significant effect on specific volume, hardness, chewiness, and springiness at the same time. Of all the tested factors, gum arabic and water significantly affected the quality of gluten-free bread.

The desirability function in DSD model in the Fig.  1 shows the optimum levels of gum arabic and water to maximize specific volume and springiness and to minimize hardness and chewiness, with a desirability of 80%. Medium concentrations of these two factors were selected to improve gluten-free bread quality: 1.5% of gum arabic and 71.5 g/100 g of water.

Optimal conditions of gluten-free bread

Characteristics of optimum gluten-free bread

Characteristics of optimum gluten-free bread (specific volume, textural parameters, moisture content, and color of crumb and image analysis) are shown in Table  5 against the control bread without improver.

Specific volume of optimum gluten-free bread was higher (2.87 cm 3 /g) than that of control bread without improver (2.50 cm 3 /g). Table  5 shows that optimum gluten-free bread had an average moisture content of 28.09% which was found to be less than the control bread (32.15%). This indicates that gum arabic decreases the moisture content of bread. Textural properties of the bread supplemented with gum arabic demonstrated hardness of 14.94 N, chewiness of 4.26 N, and springiness of 0.791 for optimum gluten-free bread; however, control bread demonstrated hardness of 25.3 N, chewiness of 6.391 N, and springiness of 0.72 ( p  < 0.05).

Considering the color of the crumb of gluten-free optimum and control bread (Table  5 ), control bread appeared to be more bright because the value of L * was higher (63.37) than that of the optimum gluten-free bread (60.27) ( p  < 0.05). Higher value of b * of the control bread crumb may be attributed to the addition of eggs in bread recipe, thereby improving the yellow tint.

Image analysis revealed that bread with gum arabic was characterized with higher number of pores with big size compared to control bread without improvers. Optimum bread exhibited an aerated crumb structure.

Thermal properties of gluten-free breads

The effect of addition of gum arabic on thermal properties of gluten-free bread is shown in Table  6 . Regarding DSC results, control bread exhibited the highest DSC values than that of optimum bread ( p  < 0.05). The values of onset for control bread were higher than that of optimum bread, which means that more energy was necessary to start the gelatinization process. Lowering of onset temperature in optimum bread is important, since it implies an earlier beginning of starch gelatinization. This fact can be related to the specific interactions that take place between the different components of the optimum bread. Gum arabic as new structure-forming additive could interact with amylopectin and retard its recrystallization.

Control bread showed the highest transition temperature (115.85 °C) and the highest enthalpy (824.55 J/g) than that of optimum bread (110.61 °C transition temperature and 618.93 J/g enthalpy). The addition of gum arabic to gluten-free bread caused a decrease in enthalpy in comparison to control.

The addition of gum arabic had a significant impact on the thermal properties of gluten-free bread ( p  < 0.05). These results are consistent with those previously published [ 35 , 36 ]. These studies reported that the addition of gums tended to lower enthalpy values. Shinoj et al. [ 37 ] reported that high transition temperatures could result from a high degree of crystallinity, which gives the starch granules a stable and more resistant structure during gelatinization. Addition of gum arabic caused a decrease in transition temperature. This phenomenon can explain the improvement of technological parameters required to prepare an optimum bread.

Microstructure of bread

Figure  2 a shows the image of control bread at a magnification of 100×. Control bread demonstrated the characteristic porous structure of a baked bread with internal empty pores. Internal surface of the pores of control bread was rough and uneven as compared to smooth surface of the bread supplemented with gum arabic (Fig.  2 c). Gum arabic imparted a homogenous structure to the bread (Fig.  2 d), whereas control bread showed dense and compact structure (Fig.  2 b). Control bread was characterized with harder structure according to the presence of visible starch particles less integrated with bread matrix than that in the optimum sample with the addition gum arabic. Swollen and ungelatinized singular starch granules were visible on rugged surface of the tested control sample (Fig.  2 b) as opposed to the unified structure of the optimum bread with more amorphous and homogenous structure (Fig.  2 d).

figure 2

Microstructure of gluten-free breads a , b control bread; c , d optimum bread, at magnification ×100 ( a , c ) and ×400 ( b , d )

Sensory evaluation

Sensory evaluation (Table  7 ) showed no significant difference ( p  > 0.05) with respect to taste and aroma between gluten-free control bread and optimum gluten-free bread with gum arabic. Results showed that the optimum gluten-free bread showed the highest scores for appearance, texture, and overall evaluation significantly than that of the control gluten-free bread. Thus, an acceptable bread was prepared with the application of gum arabic in basic gluten-free bread recipe.

In this study, the effect of combination of starches/hydrocolloids on the quality of rice/field bean in gluten-free bread was evaluated using an efficient method of screening, that is, DSD. After performing statistical validation of the obtained model, we analyzed the effects of factors. DSD approach allowed the estimation of optimal conditions with significant factors such as gum arabic and water. Optimum gluten-free bread with 1.5% of gum arabic and 71.5 g/100 g of water was manufactured and tested for its characteristics. Based on the results of loaf volume, textural, structural, color, DSC, microstructure, and sensory analysis of the optimum gluten-free bread, it can be concluded that of all the selected factors (starches and hydrocolloids), gum arabic was found to be the best additive for making gluten-free rice-based bread for patients with celiac disease.


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This research was funded by the exceptional National Program. H. Bourekoua acknowledges the Financial support of Institut de la Nutrition, de l’Alimentation et des Technologies Agro-Alimentaires (INATAA). Experiments were also supported by statutory funds of Life Science University in Lublin, Poland.

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Bourekoua, H., Różyło, R., Benatallah, L. et al. Characteristics of gluten-free bread: quality improvement by the addition of starches/hydrocolloids and their combinations using a definitive screening design. Eur Food Res Technol 244 , 345–354 (2018).

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Gluten-free products in celiac disease: Nutritional and technological challenges and solutions

Seyede marzieh hosseini.

Department of Food Science and Technology, National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Nafiseh Soltanizadeh

1 Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran

Parisa Mirmoghtadaee

2 Specialist in Community and Preventive Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Parisa Banavand

Leila mirmoghtadaie, saeedeh shojaee-aliabadi.

In celiac patient exposure to even only a small amount of gluten can lead to malabsorption of some important nutrients including calcium, iron, folic acid, and fat-soluble vitamins because of small-intestine inflammation. A strictly followed gluten-free (GF) diet throughout the patient's lifetime is the only effective treatment for celiac disease; however, elimination of gluten from cereal-based product leads to many technological and nutritional problems. This report discusses different substitutes to replace gluten functionality and examines the economic and social impacts of adherence to a GF diet. Better knowledge about the molecular basis of this disorder has encouraged the search for new methods of patient treatment. The new and common GF sources and different challenges encountered in production and consumption of these products and different solutions for improving their properties are discussed in this review.


Celiac disease is a chronic inflammatory disorder of the intestine which being asymptomatic to causing severe malnutrition.[ 1 ] The prevalence of celiac disease is <0.5%–1% worldwide.[ 2 ] Gluten is the storage protein of wheat and includes glutenin and alcohol-soluble gliadin. Gliadin and other prolamins in rye (secalins) and barley (hordeins) are toxic for patients with celiac disease.[ 3 ] A gluten-free diet (GFD) is the mainstay of celiac disease treatment.[ 3 ] Adherence to a GFD improves many clinical and serological symptoms[ 4 ] and reduces the incidence of malignancies.[ 5 ] Furthermore, it can prevent the development of many autoimmune diseases such as hematologic disorders, hepatitis, and inflammatory bowel and insulin-dependent diabetes mellitus diseases.[ 6 ] While a limited amount of gluten is permitted in a celiac patient's diet, the amount of tolerable gluten varies widely between 10 mg and 34–36 mg gluten per day.[ 7 ] This has led to confusion about labeling “GF” products. For example, in Canada, such products must meet standards of <20 ppm gluten (20 mg gluten/1 kg), whereas other countries specify a maximum of 200 ppm.[ 8 ] However, producing food that provides a daily gluten intake of <10 mg is acceptable.[ 7 ] Omitting or reducing gluten lowers the quality of end products; this could be overcome with gluten substitutes. This paper aims to review the current knowledge on different GF cereals and gluten substitutes used for the production of GF food and the recent advances in molecular knowledge of celiac disease which can help in the development of new methods for celiac therapy.


Hitherto, total lifelong avoidance of gluten ingestion has remained the primary treatment for celiac disease. The overall objective of the GFD is maintaining health through the adoption of a well-balanced diet without using gluten. Observing a strict GFD is not easy, not least because it contributes to the social isolation of patients with celiac disease. In addition, nutritional deficiencies in Vitamins D and B, iron, zinc, calcium, magnesium, and fiber may occur. Furthermore, developing good-quality GF products could be challenging due to the unique properties of gluten.[ 9 ]

Several significant properties of rice – it lacks gluten, has a bland taste, is colorless and hypoallergenic, has low levels of protein, sodium, fat, and fiber, and contains high amounts of easily digested carbohydrates – make it suitable for making flour that can be used to prepare GF products. As rice contains a relatively small amount of prolamin, it is necessary to combine it with some sort of gum, emulsifier, enzymes, modified starch, or dairy products to obtain viscoelastic properties.[ 10 ] The color of the crust and texture characteristics of acidic extruded rice-flour bread is been found to be similar to those of wheat bread, but it has a low specific volume.[ 11 ] Rice–noodle products are important foods in many Asian countries. Since rice protein cannot participate in the forming of a cohesive dough structure, gelatinized starch plays a role as a binder.[ 12 ] Rice can also be formed into flakes: rice is cooked, coated with skim milk as a nutritious ingredient, and then partially dried, tempered, passed through flaking rolls, and toasted in an oven. Crackers can be also obtained using either nonwaxy or waxy rice.[ 13 ] Technological characteristics of rice-flour products could be improved by the addition of a protein source such as spirulina.[ 1 , 14 ]

The high protein, fat, and fiber content of pure oats make them a suitable choice for celiac patients.[ 15 ] However, the safety of oats in a GFD has been questioned in some studies due to possible contamination of the oats with gluten-containing cereals[ 16 , 17 ] during growing cycle in the farm, cleaning, transportation, storage, or processing. Therefore, it is necessary to extend strategies that would supply uncontaminated oats. The Professional Advisory Board of the Canadian Celiac Association in cooperation with Health Canada had reviewed the literatures on pure oat safety in celiac disease and had recommended the consumption of only limited amount of pure oats about 20–25 g/day (65 ml or ¼-cup dry-rolled oats) for celiac children and 50–70 g/day (125–175 ml or ½ to 3/4-cup dry-rolled oats) for celiac adults.[ 18 ] Fermented oat slurry provides a yoghurt-type product that can be used by patients with celiac disease, lactose intolerance, or a milk allergy.[ 19 ] Moreover, oat β-glucans are technologically feasible thickening agents in soups and have high acceptance among consumers.[ 13 ]


In contrast to the most common grains, pseudocereals are composed mainly of albumins and globulins and contain very little or no storage prolamin proteins;[ 18 ] thus, they are good substitutes for cereal in GF foods. The nutritional values of wheat and different important GF flour are compared in Table 1 .[ 18 ]

Certain mineral content of pseudocereals

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Amaranth consists of small seeds with a nutritional value better than that of any other vegetable, including cereals, and much higher amounts of fiber and minerals than any other GF grain. It has a high amount of lysine, arginine, tryptophan, and sulfur-containing amino acids.[ 20 ] Amaranth flour has already been used to enrich cereal-based foods, including GF pasta.[ 21 ] Amaranth bread, which has higher levels of protein, fiber, and minerals, is acceptable for celiac patients.[ 20 ] A mixture of popped and raw amaranth flour produces bread loaves with a higher specific volume and more homogeneous crumb than other kinds of GF bread.[ 21 ]

Quinoa protein is rich in lysine, methionine, and cysteine. Thus, it is a good complement for legumes, which have low methionine and cysteine. In addition, quinoa is a relatively good source of Vitamin E and B-group vitamins and has high levels of calcium, iron, and phosphorous. It also has a suitable fatty acid composition.[ 22 ] Dogan and Karwe demonstrated that quinoa could be used to make a novel, healthy, extruded snack product. Quinoa's high lipid and low amylase contents make it necessary to have a high shear in extrusion cooking.[ 23 ]

Buckwheat seeds contain fagopyritols, a type of soluble carbohydrates. Fagopyritols are a source of D-chiro-inositol, a compound that has shown efficiency in patients with noninsulin-dependent diabetes through improved glycemic control. Buckwheat has a low glycemic index and also shows a beneficial effect on human health, lowering blood pressure and helping cholesterol metabolism.[ 24 ] Replacement of cornstarch with buckwheat flour in GF bread has been shown to have a positive effect on bread texture and delays staling because of buckwheat flour's lower starch gelatinization enthalpy.[ 25 ] Utilization of buckwheat in the production of GF crackers leads to a product with acceptable sensory qualities.[ 26 ] Buckwheat and quinoa breads have a higher volume than other kinds of GF breads.

Schoenlechner et al . compared different characteristics of amaranth, quinoa, and buckwheat pasta. They found that the firmness and cooking time of amaranth pasta was lower than those for the other flours, while the cooking loss of quinoa pasta was greater than other flours. Decreasing the moisture content to 30% and using higher amount of egg white powder and emulsifier (distilled monoglycerides) led to a firmness that was more acceptable than that for the wheat pasta.[ 22 ]

Maize's high yields have made it a key crop in ensuring food availability and promoting food security.[ 27 ] It is recommended as a safe source for the production of GF pasta. In addition, products such as curls, puffs, and balls can be produced by extrusion cooking of maize grits or meal, and fried snack products such as tortilla chips can be made from alkaline-processed maize. Breakfast cereals such as flakes, shreds, granules, puffs, or other forms can also be produced from maize.[ 13 ]

One good source of nutrients, especially fiber, calcium, and other minerals, is millet.[ 28 ] Protein makes up about 7%–12% of the grain. Lysine is a limiting amino acid in millet, while tryptophan and threonine are not deficient.[ 9 ] The best-known flat breads produced from millet are injera, kisra (fermented), and roti (unfermented). Injera made from millet stales much more slowly than that made from sorghum or other cereals. Teff is a kind of millet that has protein content similar to the other cereals (10%–12%) and is a good source of minerals, particularly calcium and iron. The main use of teff grain in human food is in injera.[ 29 ] Teff starch has a slow retrogradation rate that delays bread staling.[ 13 , 30 ] Millet's lysine deficiency can be overcome by blending it with a lysine-rich flour such as legume flours. Baby foods, snack foods,[ 31 ] and breakfast cereals[ 32 ] are other products made from millet. Germinated, popped, and roasted millet flours have been used along with milk solids, legume flour, and other cereals for the production of complementary and infant foods.[ 33 ]

White, pleasant-tasting, and GF flour can be produced from sorghum.[ 34 ] The nutrition quality of sorghum protein is poor, as sorghum is deficient in essential amino acids. Malting can increase lysine and improve protein quality.[ 35 ] Breads produced from sorghum have lower volume than wheat bread.[ 36 ] For sorghum bread, soft batters rather than firmer dough are required to obtain sufficient rise and good elasticity without brittleness; thus, more water is generally required.[ 34 ] In GF products, gas cells should be surrounded by liquid films and stabilized by surface-active substances such as polar lipids, soluble proteins, and soluble pentosans; these are present in sorghum, making it suitable for producing bread without any additives. However, using hydrocolloids could improve sorghum bread's quality.[ 34 ] Various researchers have studied the effect of using different additives on sorghum bread quality. Some of these studies are presented in Table 2 . Sorghum flours have also been used to produce biscuits, granolas, infant food, and snack foods such as crisps and chips.[ 35 , 37 ]

Different gluten substitute used in different gluten-free food

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Chestnut flour contains high-quality proteins with 4%–7% essential amino acids, 20%–32% sugar, 50%–60% starch, 4%–10% dietary fiber, 2%–4% fat, and some vitamins and minerals, such as B-group vitamins and Vitamin E, phosphorous, magnesium, and potassium. Since the amounts of Vitamin B, iron, folate, and dietary fiber are not sufficient in most GF flour, the use of chestnut flour seems to be advantageous for improving nutritional value. Unfortunately, the qualities of chestnut bread, such as volume and color, are not suitable because of weak interactions between components of the chestnut dough,[ 1 ] inadequate starch gelatinization, and high amounts of sugar and fiber. This flour is more suitable for pastry making.[ 38 ] However, blending chestnut flour with other flours such as rice flour[ 38 ] and adding some hydrocolloids such as guar gum, xanthan gum, or hydroxypropyl methylcellulose (HPMC)[ 1 ] can help to overcome these problems.

The chia ( Salvia hispanica L.) seed and flour were one of the main staple foods in Central America. It attracts a great deal of interest due to its nutritional and functional potential in food and pharmaceutical industries. The chia seed is a good source of phenolic compounds, dietary fiber (20%–37%), protein (18%–25%), and oil (21%–33%) with approximately 60%–63% α-linolenic acid. Sandri et al . used chia flour, potato starch, and rice flour in a GF bread formulation by application of mixture design and response surface methodology to achieve the best sensory properties. They found no suitable physical and sensory properties when whole chia flour alone was used. After that, 5%, 10%, and 14% whole chia flour was added to GF bread-containing rice flour as a main ingredient that led to negligibly decrease in crumb moisture, crumb firmness, and loaf volume.[ 39 ] Huerta et al . observed no significant differences in replacing rice and soy flour with 2.5%, 5.0%, and 7.5% whole chia flour in specific volume, baking loss, and sensory acceptability (scores ranging from 4.5 to 5.5, on a 7-point hedonic scale) on GF bread in comparison to control.[ 40 ] In another study, 2.5%–7.5% whole chia flour was used in chestnut flour-based GF bread formulation. They found improved in the dough rheological properties of elasticity, viscosity, and stability up to using 7.5% chia flour.[ 41 , 42 ] Steffolani et al . found that replacing of rice flour with 15% whole chia flour reduced the specific volume, darkened the GFB color, and increased the bread hardness but does not have significant effect on overall acceptability.[ 43 ]

Breads produced from legumes such as pea isolate, chickpea flour, soya flour, or carob germ flour showed good sensory profiles and physicochemical characteristics. Carob germ flour produced batters with good rheological characteristics, but its bread had poor properties. However, chickpea flour and pea isolate kinds of bread obtained good results in all parameters.[ 44 ] In another study, Gularte et al . made GF cake using chickpea, pea, lentil, and bean flours along with rice in a proportion of 50:50. Application of legume flours, especially lentil, led to lower batter viscosity and consequently higher specific volume than in the control sample. In addition, lentil-enriched cakes showed similar crumb hardness and higher springiness than the control cake. In terms of nutritional quality, legumes have a higher protein content and protein availability than cereals; this makes legumes as a recommended flour for enriching GF cakes.[ 45 ] Tsatsaragkou et al . (2014) showed replacing 15% of rice flour with carob flour resulted in the production of GF bread with better crumb structure and color, and lower moisture loss but harder crumbs and lower specific volume than rice bread. The decrease in size of carob flour led to a slower rate of firming.[ 46 ]


A comparison between GF commercial foods and their gluten-containing counterparts shows that GF food is more expensive.[ 47 ] The price of one loaf of GF bread is two or three times that of regular bread. Activities such as baking celiac-specific cereal products, buying foods in large quantities with friends or support-group members, and choosing longer lasting products such as carrots, potatoes, and parsnips, seasonal products, and legumes could help patients to reduce food costs.[ 48 ]

Nutritional deficiencies

Between 20% and 38% of celiac patients show nutritional deficiencies: 12%–69% display iron deficiency and 8%–41% display Vitamin B 12 deficiency. In addition, damaged villi in celiac patients lead to lactose intolerance because of decreased lactase production, resulting in phosphorus, calcium, and Vitamin D deficiencies.[ 47 ]

Using starches and refined flours with low fiber content in GF products leads to inadequate fiber intake.[ 47 ] The incidence of anemia in newly diagnosed celiac patients was reported as 4% in the United States. Gluten-containing products have higher folate content than their GF counterparts. Therefore, fortification of GF products with folate is essential.[ 49 ] Immediately after diagnosis of a deficiency in these and other micronutrients, GF vitamins and minerals should be added to the patient's diet in therapeutic doses based on individual factors, including laboratory test results, age, overall eating habits, and compliance with the GFD.[ 8 ] Patients should be encouraged to use foods rich in Vitamin B 12 (such as meat, milk, fish, and poultry), folate (such as dried beans and legumes, flax seeds, dark leafy greens, and citrus fruit), heme iron (such as lean meats, poultry, and seafood), and nonheme iron (such as legumes, seeds, and nuts), as well as vitamin C-rich food to increase iron absorption. Pseudocereals such as amaranth, buckwheat, and quinoa are good sources of iron, fiber, and some B vitamins.[ 50 ]

Recent studies showed a high prevalence of obesity in some celiac patients.[ 51 ] Almost half of all adult patients with celiac disease have a body mass index of 25 or more;[ 52 ] however, obesity is more prevalent in celiac children, and it is, therefore, necessary to test for celiac disease in obese children.[ 52 ] Hyper caloric content of commercially available GF foods might be resulted to obesity and weight gain.[ 53 ] Furthermore, damage of intestinal villi can lead to problems in food digestion and absorption that result in obesity.

Bone disease

Consumption of calcium-rich and Vitamin D-rich foods should be recommended throughout patients’ lives, particularly those patients with osteopenic bone disease.[ 54 ] Calcium-rich foods include milk, cheese, and calcium-fortified beverages such as orange or apple juice, and enriched, GF soy, almond, or rice milk, GF yogurt, sardines, or canned salmon with bones.[ 55 ] Vitamin D-rich foods include fatty fish and fish oils, egg yolk, liver, Vitamin D-fortified milk, and some GF enriched beverages; additionally, patients should be encouraged to expose their skin to sunshine during late spring, summer, and early fall.

Lactose intolerance

A common problem for celiac is bloating, gas, and diarrhea; these may indicate lactose intolerance. Lactose consumption should be avoided and limited for one or more months in this situation until lactase enzyme production recovers. Different recommended strategies include using lactose-reduced or lactose-free products such as Lactaid® milk, aged cheese, and GF yogurt with live and active cultures, enriched dairy-free/GF beverages such as soy, almond, or rice milk, and supplementation with GF lactase enzyme supplements.[ 55 ]

Technological challenges

As mentioned before in detail, the quality, mouth-feel, and flavor of GF products are lower than those of conventional wheat products. The elasticity and extensibility of dough and the volume of the loaves are attributed to gluten.[ 56 ] Cereal products baked with different GF cereals (with the exception of oats) have been shown to have lower volume and an inferior physical texture but a slower staling rate than wheat containing samples.[ 57 ] Different additives, such as hydrocolloids, emulsifiers, starch, eggs, and other materials, have been used as improvers in the production of GF products. Some of these additives are discussed in [ Table 2 ].


Hydrocolloids can be applied as gluten substitutes in the production of GF food due to their polymeric structure.[ 32 ] The properties of hydrocolloids used as gluten replacers, such as network forming, film formation, thickening, and water-holding capacity, are useful in the formulation of GF products. Guar gum and xanthan gum are the two most common hydrocolloids used in GF-baked products.[ 9 ] Addition of xanthan to GF formulations leads to a farinograph curve typical of wheat flour dough.[ 58 ] This gum has a positive effect on bread volume and leads to a product with a higher volume than do pectin and guar gum.[ 59 ] Increased xanthan content reduces the hardness of bread.[ 59 ] In addition, when xanthan gum was applied as a network former in the preparation of cornstarch bread, the resulting product had a good specific volume but a coarse crumb texture, without flavor.[ 60 ]

HPMC is a cellulose derivative that has a positive effect on the reduction of cholesterol and has also been used in GF breads to increase loaf volume.[ 61 ] The use of HPMC as a substitute for gluten ensures good gas-retaining and structure-forming properties in the crumb of rice bread.[ 62 ] In fact, a comparative study using different gums (xanthan gum, guar gum, agar, carrageenan, locust bean gum, and HPMC) in a rice–bread formulation showed that HPMC gave the highest specific loaf volume.[ 63 ] The cellulose carboxymethyl cellulose (CMC) has been used as a gluten replacer in the production of bread. CMC can increase the porosity and crumb elasticity of bread as well as the overall acceptability of a GF formulation.[ 58 ] When this gum has been used for the production of rice-flour cake, better sensory properties in terms of uniformity, crust property, rupture, aroma, taste, and flavor were obtained in comparison with control rice-flour cake.[ 64 ] Furthermore, an appropriate amount of CMC and HPMC improved rice-cracker texture.[ 65 ]

Pectin,[ 59 ] agarose,[ 59 ] oat β-glucan,[ 58 ] psyllium,[ 66 ] Arabic gum,[ 67 ] konjac,[ 68 ] locust bean gum,[ 56 ] agar-agar,[ 69 ] and guar gum[ 38 ] are other hydrocolloids that have improved the texture, rheology, appearance, sensory perceptions, and general quality of GF formulations. Some authors have investigated the effect of mixture of hydrocolloids.[ 70 ] Sumnu et al . studied the effects of different concentrations of xanthan and guar gums and their blends on the staling of GF rice cakes. They found that a blend of xanthan and guar gum decreased hardness, weight loss, enthalpy of retrogradation, and the change in setback viscosity values of cakes during storage, thus retarding staling.[ 70 ] Using xanthan, CMC, xanthan-guar, xanthan-locust bean, and HPMC have been shown to yield the lowest porosity, the lowest average area of pores, and the highest number of pores; this, in turn, leads to a finer texture of these crumbs along with lower hardness and higher cohesiveness and springiness.[ 38 ]

Starch plays a key role in the texture of many kinds of food products. In some cases, native starch does not provide the functional properties, such as thickening and stabilization, for the production of some special foods. Therefore, starches used in the food industry are often modified to overcome undesirable changes in product appearance and texture caused by retrogradation or breakdown of starch during processing and storage.[ 71 ] The most widely used starches in the food industry are hydroxypropylated, acetylated, and cross-linked starches. Hydroxypropylated starch influences the viscoelastic properties of dough. One of the main factors that could modify the rheological properties of GF modified starch as a part of the dough is water-binding capacity. However, the application of hydroxypropylated starches has not been shown to have a significant impact on pasting characteristics.[ 72 ] Hydroxypropyl distarch phosphate enhances the volume of GF loaves. This is accompanied by a decrease in average cell size and an increase in average cell number.[ 73 ]

Acetylation of starch is an important substitution method used for thickening GF food products.[ 15 ] Like hydroxypropylated starch, acetylated distarch adipate could enhance the volume of GF bread. Addition of modified starch causes a more elastic crumb structure. A slight decrease in the hardness and chewiness of the crumb was also observable on the day of baking.[ 73 ] Application of acetylated starch in cake batter could increase batter viscosity, cake volume, and whiteness of crust.[ 15 ] When high and stable viscosity is required in food, cross-linked starches are used as the thickener. Cross-linked starches play an important role in increasing shear resistance and providing viscous batter.[ 74 ] Cross-linked cornstarch provides stronger and more stable dough and increases the loaf volume.[ 75 ] The use of resistant starch has been shown to elevate zero-shear viscosity and reduce both creep and recovery compliance. Modified starch has shown higher starch gelatinization temperatures and lower viscosity. It has been found that loaves baked with a proportion of resistant starch had a softer crumb than the control sample.[ 76 ] Hydrolysis of some proportions of starch into a low molecular weight using amylolytic enzymes is another method of starch modification. The resulting modified starch, called maltodextrin or dextrin, significantly increases pasting temperature and reduces the viscosity of the obtained pastes. Maltodextrins can attenuate structure and increase deformation sensitivity. The addition of maltodextrins with low dextrose equivalent (DE) decreases loaf volume and causes the deterioration of bread quality. Maltodextrins with the higher DE positively influence bread volume and have a beneficial effect on crumb hardening during storage. Maltodextrin with the highest DE also effectively reduces the recrystallization enthalpy of amylopectin.[ 77 ]

Phongthai and D’Amico (2017) studied the properties of rice-flour-based GF pasta enriched by whey protein concentrate (WP), egg albumen (EB), soy protein (SP) and rice bran protein concentrate, separately. Using WP caused decrease in optimal cooking time. The enrichment of 9% (w/w) EB led to prevent structure from disintegration, improved pasta firmness, and decrease in cooking loss of P < 0.05, whereas using rice bran protein concentrate caused highest cooking loss ( P < 0.05). The GF pasta enrichment with 6% SP concentrate had similar L* values in comparison with commercial sample. Among the four sources of protein tested, EB had the highest potential for improving cooking properties of rice-flour-based GF pasta.[ 78 ]

In addition, application of modified protein could improve the quality of GF products. Deamidated oat protein has been shown to cause lower viscosity, a higher volume, and a darker color.[ 15 ] The substitution of a combination of deamidated protein and acetylated starch could improve oat-flour cake properties.[ 79 ]

GF flour often tends to have reduced fiber compared with products containing gluten. Different fiber sources, such cereal bran, legume outer layer, modified cellulose and resistant starch, and by-products of apple and potato processing, have been used in producing GF products. The replacement of 20% rice flour with a mixture of oat fiber and inulin in GF layer cakes has been shown to increase the cakes’ specific volume and quality.[ 45 ] The degree of polymerization of inulin and the proportion of low-molecular-weight sugars in the recipe could influence dough properties. The incorporation of inulin to dough formulations causes a significant decrease in paste viscosity and an increase in gelatinization temperature. Inulin significantly reduces the enthalpy of retrograded amylopectin, resulting in slower staling.[ 80 ] Addition of rice bran containing a high amount of soluble dietary fiber produces better bread color, a higher specific volume, and softer crumb with a better porosity profile. Furthermore, sensory acceptance increases and shelf life extends in higher levels of soluble dietary fiber.[ 81 ]

Dairy ingredient

The incorporation of dairy ingredients has long been established in the baking industry due to their nutritional and functional benefits, including improved flavor and texture and longer shelf life. Dairy products may be used as a gluten substitute to increase water absorption and enhance the handling properties of the batter.[ 82 ] All powders derived from milk increase crumb hardness with the exception of demineralized whey powder. Sensory analysis has shown a preference for breads containing skim milk, sodium caseinate, and milk protein isolate.[ 56 ] Other novel ingredients, such as calcium-fortified caseinate, were found to be suitable for gluten replacement, where calcium bonds in caseinate played the same role as sulfur-sulfur bonds in gluten.[ 9 ] Another benefit of using dairy products is the doubling of the bread's protein content.[ 56 ]

The enzyme transglutaminase (TGase) (EC has been used in many industries, including dairy, bakery, and meat processing. TGase, a γ-glutamyltransferase, can catalyze the reaction between lysine residues (ε-amino group on protein bound) and glutamine residues (β-carboxamide group on protein bond), which cross-link proteins via covalent bonds, leading to the decrease in the number of free amino groups. TGase was found to have a severe effect on dough water absorption, modifying viscoelastic behavior and enhancing thermal stability.[ 83 ] Furthermore, TGase has a significant effect on the specific volume of bread. Application of skim milk protein with 10 unit of enzyme has been shown to lead to the most compact structure, as reflected in the crumb texture profile. This could be due to the formation of a protein network in GF bread with the addition of TGase.[ 84 ] Another enzyme that affects dough's rheological properties and bread's physical quality is protease. Protease-treated rice bread had better crumb appearance, high volume, soft texture, and slower staling rate, depending on the amount of enzyme added.[ 85 ] The aggregation of partially degraded storage proteins surrounding the starch granules and protein-starch interaction may improve gas retention before baking and increase specific loaf volume.[ 86 ] In another study, application of protease of Aspergillus oryzae on the rheological properties of rice dough showed an increase in batter viscosity and a decrease in flour-settling behavior because of the aggregation of flour particles after partial cleavage of storage proteins.[ 86 ]

The use of sourdough represents an alternative to increase the quality of both gluten-containing and GF breads. Acidification of flour by sourdough fermentation can replace the function of gluten to some extent and enhance the swelling properties of polysaccharides, leading to a better bread structure. It also improves bread volume and crumb structure, flavor, nutritional value, and mold-free shelf life. Sourdough lactic acid bacteria could break down nongluten proteins and starch components, thus increasing the dough elasticity and delaying staling.[ 87 ] Furthermore, long-chain sugar polymers called exo-polysaccharides can be produced by many lactic acid bacteria and act as prebiotics and hydrocolloids to improve the technological as well as nutritional properties of GF breads.[ 87 ] Rühmkorf et al . optimized homoexo-polysaccharide production by lactobacilli in GF sourdoughs to achieve high amounts of exo-polysaccharides.[ 88 ] The complementary peptidases located in the cytoplasm of lactobacilli hydrolyze gluten and reduce its amount to <10 ppm through routine sourdough fermentation.[ 89 ] On the other hand, the proteolytic system of lactic acid bacteria has the ability to hydrolyze α-gliadin fragments and reduce gliadin levels to some extent. Furthermore, the application of these peptidases seems to be a possible technological alternative to reduce the gliadin concentration in wheat dough without using living bacteria as a starter.[ 90 ] Lactic acid bacteria can also produce antifungal, antimycotoxigenic, bioactive, and aroma compounds that have the ability to improve overall bread quality.[ 87 , 91 ]

Other materials

So far, some studies have been conducted in this area using uncommon materials as gluten alternatives. For example, the study of replacing wheat flour with a mixture of GF flours and psyllium showed no change in the preference or acceptability of modified products compared with standard products. Healthful, tasty, and low-cost products could be made at home using this replacement.[ 66 ] Another material, which contains high amounts of protein, dietary fiber, calcium, and ω-3 fatty acids, is the pulpy by-product of soy milk named okara. It can play an important role as a gluten substitute, which develops proper product texture, mouthfeel, and volume after some reformation. Okara has large amounts of fiber that interferes with protein-starch interactions. Decreasing the fiber size can overcome this problem. In addition, in comparison with a commercial GF flour in batter formulations, okara has been suggested as a novel marketable ingredient for the formulation of a variety of GF products.[ 92 ]


As mentioned above, the traditional concept of celiac disease is a chronic inflammatory disorder that identified by malabsorption in human.[ 93 , 99 ] Although celiac disease is treatable by the total lifelong GFD,[ 94 , 100 ] due to mentioned problems, the use of other controlling methods can delay symptoms. Nutrigenomics can be used as a new method for celiac disease control. Nutrigenomics and nutrigenetics are two research fields that elucidate some interactions between diet, nutrients, and genes. Nutrigenomics studies the functional interactions of food with the genome. Some food ingredients such as plant flavonoids, carotenoids, and long-chain ω-3 fatty acids can modulate oxidative stress, gene expression, and production of inflammatory mediators; this modulation activity can preserve the integrity of the intestinal barrier and protect against the toxicity of gliadin peptides; thus, these ingredients can be used in nutritional therapy for celiac disease.[ 93 ] Vitamins C and E can modulate immune responses in several ways, such as via leukocyte function and lymphocyte proliferation. They have also antioxidant activity that leads to modulations of the inflammatory process. Vitamin E, especially γ-tocopherol, decreases the release of the pro-inflammatory cytokines IL-8 and PAI-1. In addition, Vitamin C can inhibit the augmented secretion of interferon-gamma, tumor necrosis factor-alpha, and IL-6 and increase the expression of IL-15 triggered by gliadin; this is beneficial in the treatment of celiac disease.[ 101 ] Other effective compounds on the intestinal epithelial cells are several polyphenols and carotenoids found in fruit and vegetables that have antioxidant and anti-inflammatory properties. Flavonoids reduce the concentration of prostanoids and leukotrienes through inhibiting the activity of eicosanoid-generating enzymes such as phospholipase A 2 and preventing the induction and expression of inducible nitric oxide synthase in different cell models. In addition, carotenoids can inhibit the expression of enzymes/proteins that play a role in inflammation, partly by suppressing the activation of the transcription factor NF-κB. Other flavonoids such as lycopene, quercetin, tyrosol, epigallocatechin, gallate, genistein, and myricetin also have a protective effect on intestinal-barrier function. On the other hand, fatty acids can act via cell-surface and intracellular receptors/sensors that control inflammatory cell signaling and gene expression patterns. Although eicosanoids produced from ω-6 fatty acids (such as arachidonic acid) have a pro-inflammatory role, eicosanoids from ω-3 fatty acids (such as eicosapentaenoic acid) have anti-inflammatory properties. It has been presented that the release of arachidonic acid from intra-epithelial lymphocytes after incubation with gliadin leads to the activation of cytosolic phospholipase A2 cPLA2, which results in the lymphocyte cytolysis and immune response of celiac disease. Furthermore, it has been shown that docosahexaenoic acid, as a long chain ω-3 polyunsaturated fatty acid, can disturb the pro-inflammatory effects of arachidonic acid.[ 93 , 101 ]

Celiac patients usually need to adhere to a strictly GFD for the rest of their lives. Different GF cereals and additives have been used in GF products; the additives contribute structure-building and water-binding properties to GF-baked goods. The comparison between previous studies showed that pseudocereals and legumes are appropriate choices for making GF products because of their significantly higher levels of protein, fat, fiber, and minerals. From an economic perspective, pseudocereals offer a cheaper alternative to wheat that can help increase dietary compliance by reducing the economic pressure of a GFD. Each method for the production of GF food suffers from limitations, such as nutrition deficiency or deterioration of functional properties. As a result, the unpalatability and weak functional properties must overcome while maintaining nutritional value and safety.

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