Cracker F5 was rated as the hardest (mean score 8.6), significantly harder than crackers F3 and F4 (*p* < 0.05) (mean scores 6.1 and 6.3), but not significantly harder than cracker F8 (mean score 7.4) (*p* > 0.05). The fat content in cracker F5 was lower than the fat content in the other three types of crackers, which most likely caused their higher hardness (Table 5). In addition to physical and chemical properties, fat has a positive effect on product viability by delaying the absorption of water from starch [32]. Therefore, the texture stability of cracker F5 could be the lowest.

**Table 5.** Energy and nutritive value of the different types of crackers (g/100 g of sample, *n* = 2, expressed on sample dry matter as mean ± standard deviation).

Samples within the same row marked with different letters are statistically significantly different (*p* < 0.05)

The results of the hedonic sensory analysis of the individual properties of the four bestrated crackers are shown in Figure 4. Cracker F4 was best-liked in all examined properties (appearance, odour, taste and flavour, and texture, as well as overall), with average scores for each property above 8 ("like very much"), followed by cracker F3 with slightly lower average scores, but not significantly different from F4 (*p* > 0.05). The differences in the bestrated cracker F4 as compared with cracker F3 were the addition of flax seeds in the recipe and the use of chives, while in the cracker F3, chia seeds and Mediterranean seasoning mix was used.

Previous studies on the addition of flax seeds in biscuit production have shown that partial replacement of wheat flour with ground flax seeds (up to 12%) does not affect the physical and sensory properties of the product [33]. However, Cukelj et al. [ ˇ 6] achieved sensory acceptability of a multigrain flax seed biscuit similar to that of white wheat biscuits by using a suitable combination of cereal flour with ground flax seeds.

If we compare the results of the hedonic and descriptive sensory analyses, crackers F3 and F4 differed significantly only in intensity of overall odour, which was higher for cracker F4, suggesting that the better acceptance of cracker F4 was influenced by the spices used.

If we compare the results of the hedonic sensory analysis conducted on nine crackers with the sensory analysis conducted on four crackers in terms of overall impression, the best-rated crackers in both analyses were cracker F4, followed by cracker F3. However, in the second sensory analysis, crackers F5 were rated better than F8, while in first analysis it was vice versa.

Crackers F5 and F8 differed significantly from the better-rated crackers F3 and F4 (*p* < 0.05). Appearance, taste, and flavour, as well as texture in the mouth were rated better for cracker F5 than for cracker F8. Odour is the only sensory property that was rated better for cracker F8 than for cracker F5.

There are several factors that might influenced the better acceptance of cracker F5 as compared with cracker F8. First, several types of flour (maize, millet and buckwheat) were combined in cracker F5, while only buckwheat flour was used in cracker F8. In the research of Šimurina et al. [34], a wholegrain buckwheat cracker was rated better in terms of taste as compared with control wheat cracker, while in terms of odour, the control wheat cracker was rated better. In addition, chia seeds and pumpkin seed meal were included in our cracker F5, while flax seeds and sweet potato puree were used our cracker F8. We can conclude that combining buckwheat flour with other flours leads to better acceptability of crackers.

#### *3.3. Nutritive Value, Fructan Content, and Labelling of Crackers*

The moisture content in the final products was below 5 g in 100 g of sample, except for cracker F8 in which the moisture content was around 8% and could cause crackers to have a lower shelf life as compared with the other three crackers. Significantly higher content (*p* < 0.001) of minerals was found in crackers F5 and F8 as compared with crackers F3 and F4. The addition of pumpkin seed meal and buckwheat flour (Table 5) contributed to higher mineral content in crackers F5 and F8.

Crackers made according to the F8 formulation had the highest fat content, significantly differing from crackers F4 and F5 (*p* = 0.048 and 0.004, respectively), but not from cracker F3 (*p* = 0.183). In addition, all the crackers were low in saturated fat. In the F3, F4, and F5 formulations, 31–35% of the energy value originated from fats rich in unsaturated fatty acids.

Although significantly lower protein content was found in crackers F3 and F8 than in crackers F4 and F5, the differences were small. Pumpkin seed meal, which contains up to 65% protein [35], contributed to the higher protein content of cracker F5. Proteins were a source of 22–24% of the total energy value of crackers. Given that more than 20% of the energy value of crackers in four different recipes comes from protein, it could be labelled as "high in protein" [25]. Nutritional or health claims in the labelling, presentation and advertising of products while ensuring the effective functioning of the market also aim to ensure a high level of consumer protection.

In crackers F3, F4, and F5, the proportion of calories derived from carbohydrates was around 44%, while in cracker F8 it was slightly lower, around 40%. Thus, the stated calorie proportions derived from high-value proteins and carbohydrates were slightly lower than the recommended values of ~25–32% and 45–55%, respectively (Academy of Nutrition and Dietetics, Chicago, IL, USA). To achieve the target values for carbohydrates and proteins, the amount of fat should be reduced and the amount of proteins increased in our formulations. All the types of crackers were "high in fibre", since they contained more than 15 g of fibre per 1000 kcal [25]. One serving of the crackers would contribute around 10% of the recommended daily intake (25 g). Nevertheless, crackers F4, F5, and F8 had higher proportions of soluble fibre (23–24%) as compared with cracker F3 with 17% soluble fibre (Table 5). Although insoluble fibre promotes regularity in a healthy digestive system, in patients with IBS, it can increase bloating, gas, cramping, etc., whereas soluble fibre helps improve IBS symptoms and slows the digestion of food [36]. Moreover, fructan levels in all four of the analysed crackers were below the cut-off value (Table 5). Thus, our crackers could be labelled as "low-FODMAP", "naturally rich in fibre", and "gluten-free".

Considering the content of soluble fibre, crackers F4, F5, and F8 would be suitable for IBS patients, while all of the four crackers would be suitable for patients who suffer from NCWS and celiac disease. Hence, it can be observed that higher total dietary fibre content contrasts important descriptive sensory properties of crackers such as taste and flavour, while soluble fibre content contrasts undesirable descriptive texture attributes and bitter taste (Table 5 and Figure 4).

To the best of our knowledge, there are only two certified low-FODMAP crackers on the market, and they can be found in Australia (FODMAP everyday. Available online: https: //www.fodmapeveryday.com/product-category/collections/low-fodmap-certified-brands/ fodmap-friendly-low-fodmap-certified/?paged=8 (accessed on 14 August 2022)). The crackers contain about 21 g of fats, 55–60 g of carbohydrates, 4–7 g of fibre, 10–14 g of proteins, and approximately 450 kcal per 100 g of product. If we compare our crackers with those from the market, it is evident that they have less fat, much more proteins and fibre, and still have low-FODMAP content. The reason for this is that our crackers had more complex compositions and more nutritionally valuable ingredients such as wholemeal flours and oilseed flours. Furthermore, our crackers were prepared with sourdough which contributed to their flavour and taste, but also assured low-FODMAPs [9]. In particular, we observed about a 2.5-fold reduction in expected fructan level (almost 0.3 g/100 g) in cracker R5 containing pumpkin seed cake. In conclusion, oilseeds in a moderate amount can be safely used in the formulation of low-FODMAP crackers with sourdough.

#### **4. Conclusions**

The development of high-fibre low-FODMAP crackers requires the selection of a larger number of raw materials, as well as chemical and sensory evaluations. Wholemeal millet is a suitable raw material for such crackers because it is high in fibre, but more importantly, high in soluble fibre while low in FODMAPs. Furthermore, oilseed flours, as well as flours from by-products (pumpkin seed meal) contribute to a higher nutritional value of crackers in terms of a higher content of protein, unsaturated fatty acids, and minerals. Moreover, crackers from wholemeal flours with a milder taste (maize and millet) are better accepted than those from buckwheat flour, which is characterised by a specific taste. At the same time, sweet potato and spices have a significant influence on the acceptance of the product, as does the addition of sourdough, which has the potential to improve the taste and appearance of the crackers. Unlike total dietary fibre content that obstructs important descriptive sensory properties of crackers such as taste and flavour, soluble fibre content confines undesirable descriptive texture attributes and bitter taste. Nevertheless, soluble fibre and fat content contrast crackers' hedonic qualities. In the development of products with a high fibre content for IBS sufferers, special attention should be given to the proportion of soluble fibre in total fibre, not only for consumer acceptance, but also for health reasons. Future studies should investigate novel processing techniques in the production of snacks for IBS and NCWS patients, as well as their shelf life.

**Author Contributions:** Conceptualisation, N.C.M., D.N.; methodology, K.R., K.V., S.D., and B.V.; ˇ validation, N.C.M., S.D., D. ˇ C., and D.N.; formal analysis, K.R., K.V., and S.D.; investigation, K.R., ´ N.C.M., and D.N.; resources, D.N.; data curation, K.R. and K.V.; writing—original draft preparation, ˇ K.R. and N.C.M.; writing—review and editing, S.D., N. ˇ C.M., D. ˇ C., and D.N.; visualisation, K.R. and ´ N.C.M.; supervision, D.N.; project administration, B.V.; funding acquisition, D.N. All authors have ˇ read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Croatian Science Foundation (IP-2020-3829 and DOK-2021-02). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the Croatian Science Foundation.

**Institutional Review Board Statement:** This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the University of Zagreb, Faculty of Food Technology and Biotechnology.

**Informed Consent Statement:** All subjects gave their informed consent for inclusion before they participated in the study.

**Data Availability Statement:** The data used to support the findings of this study are included in the article.

**Acknowledgments:** This work is based upon the work from COST Action 18101 SOURDOMICS— Sourdough biotechnology network towards novel, healthier and sustainable food and bioprocesses (SOURDOMICS. Available online: https://sourdomics.com/; https://www.cost.eu/actions/CA1 8101/, accessed on 14 August 2022), where the author D.N. is a Management Committee member and the author N.C.M. is a member of the Working Group 7. SOURDOMICS are supported by ˇ COST (European Cooperation in Science and Technology). COST is a funding agency for research and innovation networks. COST Actions help connect research initiatives across Europe and enable

scientist to grow their ideas by sharing them with their peers, thus, boosting their research, career, and innovation. We thank Lesaffre Adriatic Inc. for donating LivendoTM LV1 starter and SME Osijek for donating flax seeds.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Effects of Teff-Based Sourdoughs on Dough Rheology and Gluten-Free Bread Quality**

**Rosen Chochkov 1 , Daniela Savova-Stoyanova 1 , Maria Papageorgiou 2 , João Miguel Rocha 3,4 , Velitchka Gotcheva 5 and Angel Angelov 5,\***

**Abstract:** Production of gluten-free bread (GFB) with good quality characteristics represents a technological challenge. Our study aimed to obtain nongluten bread from cereals and pseudocereals with applying single cultures of *Pediococcus acidilactici*, *Pediococcus pentosaceus* and *Enteroccocus durans* as sourdoughs. The effect of sourdoughs on the quality traits of gluten-free (GF) dough and GFB was explored. The structural and baking properties of GF dough composed of teff, rice, corn, and sorghum flours were improved by adding xanthan gum (0.6%), guar gum (1.0%) and carboxymethyl cellulose (1.0%). The tested strains reached 108 cfu/g in teff flour and produced sourdoughs with a pleasant lactic aroma. The sourdough-fermented doughs were softer and more elastic compared to control dough and yielded reduced baking loss. Strain *Enterococcus durans* ensured the best baking characteristics of GF dough and the highest softness of the GFB during storage. Strain *Pediococcus pentosaceus* had the most pronounced positive effect on aroma, taste and aftertaste. Pan baking was found to be more appropriate to obtain stable shape and good-looking products. A careful starter culture selection is necessary for GFB development since a significant effect of strain specificity on dough rheology and baking characteristics was observed.

**Keywords:** teff; sorghum; sourdough; lactic acid bacteria; starter cultures; gluten-free bread; celiac disease; cereals; pseudocereals

#### **1. Introduction**

The incidence of gluten intolerance, or so-called 'celiac disease' (CD), has increased significantly over the last 50 years, affecting approximately 1% of the world's population. If untreated, it can cause a number of serious health complications. The only way to control celiac condition is to maintain a strict gluten-free diet throughout the affected person's life [1–3].

In the literature, various strategies for improving the quality of nongluten dough are suggested. However, they are mostly focused on the composition of flour mixes and food additives that aim to reproduce the viscoelastic properties of the gluten network and hence increase the development of bread loaf volume [4–7]. Dairy, soy and egg ingredients have also been used to improve the quality of gluten-free bread (GFB), but with limited success [8–11]. The need to improve the nutritional profile, appearance, taste and aroma of

Savova-Stoyanova, D.; Papageorgiou, M.; Rocha, J.M.; Gotcheva, V.; Angelov, A. Effects of Teff-Based Sourdoughs on Dough Rheology and Gluten-Free Bread Quality. *Foods*

**Citation:** Chochkov, R.;

Academic Editor: Alessandra Marti

**2022**, *11*, 1012. https://doi.org/ 10.3390/foods11071012

Received: 10 February 2022 Accepted: 25 March 2022 Published: 30 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

gluten-free breads still represents a challenge for researchers [12–14]. The latest innovative technological approaches include the application of high hydrostatic pressure, new baking methods (Ohmic heating) and sourdough technology [15,16].

The use of sourdough is the oldest biotechnological process to leaven bakery goods and to improve bread texture, aroma, and shelf life [17–19]. A number of studies have shown that sourdough can be used successfully to improve the processing characteristics of gluten-free doughs [20–23]. In some cases, this effect may be attributed to the potential of lactic acid bacteria to secrete extracellular polysaccharides, which could be a beneficial alternative to conventional thickeners used to modify the viscosity, structure and stability of a wide range of gluten-free foods [24]. These extracellular polysaccharides may act as a substitute for hydrocolloids used as food additives and, therefore, the application of lactic acid bacteria could represent a cost-efficient approach to improving the rheology of gluten-free doughs. In addition, these extracellular polysaccharides have a beneficial effect on the intestinal microbiome by selectively stimulating the growth of bifidobacteria and other beneficial microorganisms [25–32].

Cereals such as oats, sorghum and teff and various pseudocereals (buckwheat, amaranth, quinoa), tubers (cassava, potatoes), legumes (chickpea, soy, pea, lupin), nuts (acorns) and oil seeds (rapeseed, sunflower, canola) are used for the production of non-traditional flour types [6,10,11,33–39]. In most cases, they are superior to traditional cereal-based flours in terms of protein, essential amino acids, dietary fiber and vitamins.

One of the most widely used gluten-free cereals is rice (*Oryza sativa*). It is rich in carbohydrates (75–80%) and minerals, but protein content is only 6–7%. Potassium, magnesium, phosphorus, calcium, manganese and zinc are commonly found in rice flour. However, it is poor in trace elements due to the specific process of grain milling. Compared to corn, wheat and potatoes, rice is a relatively good source of thiamine (pantothenic acid, folic acid and vitamin E [40]. Corn flour (*Zea mays*) is combined in most cases with other types of flour to make gluten-free bread. In bakery products, it contributes to obtaining a dense and moist bread crumb. Corn flour is rich in essential nutrients, such as choline (21.6 mg/100 g), folate (48 µg/100 g), vitamins B1 (0.25 mg/100 g), B5 (0.66 mg/100 g), B6 (0.37 mg/100 g) and especially in minerals—potassium (315 mg/100 g), phosphorus (272 mg/100 g) and magnesium (93 mg/100 g) [41–45].

Teff flour (*Eragrostis tef*) is found to have a number of valuable nutritional characteristics compared to flours from more common crops. Its starch is slowly digestible and, therefore, has a low glycemic index (GI). It has a favorable amino acid composition and does not contain gluten [46,47]. It is a good source of unsaturated fatty acids (1.66 g/100 g) and is high in minerals, especially iron (7.63 mg/100 g) and calcium (180 mg/100 g) [48]. In addition, the high content of protein (13.3 g/100 g) and dietary fiber (8 g/100 g) makes teff flour a desirable raw material for various functional foods, some of which may bear nutritional claims such as "rich in protein" and "source of fiber" [49,50]. Teff starch has a slow retrogradation tendency, which could have a potentially positive impact on the shelf-life of baked products [51]. In addition, some authors report that the addition of teff to cereal-based sourdoughs (rice and buckwheat) modified the aroma profile of the breads, increasing the fruity, toasty and cereal notes [52–57], which contributes to developing more diverse baking products with positive consumer acceptance.

Sorghum (*Sorghum bicolor* L. Moench) is the fifth largest crop in the world. Sorghum grain has a high protein content varying from 4.4 to 21.1% with an average value of 11.4% [33,58]. Sorghum grain is gluten free, high in resistant starch, a rich source of nutrients, and most importantly, contains a diverse range of bioactive phenolic compounds [59,60]. It has been proven safe for people with celiac disease, but literature on its use in gluten-free foods is relatively scarce compared to that for corn and rice.

Since none of these gluten-free crops has satisfactory bakery performance when used alone, appropriate combinations of raw materials and processing techniques should be sought to develop successful gluten-free bakery products. Therefore, the aim of the present study was to explore the application of teff-based sourdoughs in the development of gluten-free bread with improved sensory and quality characteristics.

#### **2. Materials and Methods**

#### *2.1. Raw Materials*

The flours used in this study were teff flour (Adan Village Ltd., Sungurlare, Bulgaria), rice flour (Bodpie Food Ltd., Varna, Bulgaria), corn flour (Lubeks Ltd., Asenovgrad, Bulgaria) and sorghum flour obtained by laboratory grain milling at the University of Food Technologies, Bulgaria. Compressed yeast was supplied by Lesaffre Ltd. (Sofia, Bulgaria). Guar gum (E412) and xanthan gum (E415) were supplied by Cargill (Minneapolis, MN, USA), and carboxymethyl cellulose (E466) was supplied by Dow Wolff Cellulosics GmbH (Bomlitz, Germany).

#### *2.2. Flour Characterization*

Crude fat, protein, moisture, fiber and starch contents in the flours were determined according to the American Association of Cereal Chemists (AACC) methods 30–10, 46–12, 44–15, 32–05 and 76–13, respectively [61]. Protein content was calculated with a protein factor of 6.25.

#### *2.3. Lactic Acid Bacteria (LAB)*

Three lactic acid bacteria (LAB) strains were used in this study: *Pediococcus acidilactici* 02P108 (PA), *Pediococcus pentosaceus* 12R2187 (PP) and *Enteroccocus durans* 09B374 (ED) from the culture collection of the department of biotechnology, University of Food Technologies, Plovdiv, Bulgaria. The strains originate from typical Bulgarian sourdoughs [62,63]. Starter cultures for the sourdoughs were prepared from stock cultures of each strain stored in Microbank™ (Pro Lab Diagnostics Inc., Richmond Hill, ON, CA) by cultivation in MRS broth (de Man–Rogosa–Sharpe, Merck KGaA, Darmstadt, Germany) at 37 ◦C for 48 h.

#### *2.4. Sourdough Preparation*

Teff flour was used to prepare a separate sourdough with each LAB strain. Equal weights of flour and sterile water were mixed to obtain a dough yield (DY) of 280. Each starter culture was added to a batch of teff dough at an inoculum amount of 5 log colonyforming units (CFU)/g of dough. The sourdough variants were then fermented in an incubator at 37 ◦C for 24 h.

#### *2.5. Analyses of Sourdoughs*

Active acidity (pH) was determined by a pH meter (FiveEasy FE20, Mettler-Toledo GmbH, Greifensee, Switzerland). Total titratable acidity (TTA) was determined by titration with 0.1 N NaOH to pH 8.4 and expressed as mL of NaOH/10 g of sourdough, as described in AACC 02-31 [61]. The measurements were performed in triplicate. LAB viable cell counts were determined on MRS agar plates at the beginning and the end of the sourdough fermentation. The identity of the starter cultures in each sourdough batch was confirmed by colony morphology and microscopic observations.

#### *2.6. Bread Preparation*

In order to select the most appropriate formulation of a nongluten dough, gluten-free flours were mixed with an equal amount of baker's yeast and different combinations and ratios of xanthan, guar gum and carboxymethyl cellulose. The formulations of the three tested dough variants (A, B and C) are presented in Table 1.

After selecting the most appropriate dough variant, test gluten-free breads with sourdoughs and control bread with baker's yeast were prepared. Their formulations are presented in Table 2.

#### **Table 1.** Composition of gluten-free bread formulations.

**Table 2.** Composition of gluten-free breads with sourdoughs and control bread.

Kneading was performed by a single-phase process of dough preparation to obtain a dough with a homogeneous mass and an initial temperature of 25–26 ◦C. The dough was left to rest for 20 min and was then divided into pieces of 230 g—for floor bread and 440 g—for pan bread. After shaping, the dough was subjected to a final fermentation at 33 ◦C for 60 min in a fermenting chamber (Tecnopast CRN 45-12, Novacel Rovimpex Novaledo, Italy). The dough was then baked in an electric floor oven Salva E-25 (Salva Industrial S.L.U., Lezo, Spain), preheated to a temperature of 220–230 ◦C, for 17–18 min for floor bread (plain bread, baked by placing the loaf directly on the floor of the oven) and 22–25 min for pan bread (the loaf is baked in a pan). After baking, the breads were allowed to cool for 3 h at room temperature [64].

#### *2.7. Degree of Immersion of the Doughs*

The degree of immersion of the prepared doughs was measured by an automatic penetrometer AP-4/2 (Steinmeyer Mechatronik GmbH, Dresden, Germany). Each dough was divided into pieces of 13 g and placed in the sleeve of the penetrometer, which was then placed in a thermostat at 35 °C for 60 min. The immersion of the calibrated body in the dough for 5 s was automatically measured and expressed in penetrometer units (PU).

#### *2.8. Rheological Properties of the Doughs*

The following dough characteristics were determined by a farinograph (Brabender GmbH&Co. KG, Duisburg, Germany): water absorption (%), development time (min), stability (min), degree of softening (farinograph units (FU)) and consistency (FU), with modification of AACC Method 54-21.02 [61]. Farinographic analysis is only applied for wheat flour doughs where the reference value is 500 FU. In the present study it was used for nongluten flour combinations, with which the highest values achieved for FU were much lower (Table 3). Flour samples of 300 g were analyzed using the ICC standard method 115/1 [65]. Water absorption (WA) of the formulations was first adjusted to 65%, and then the other parameters of the doughs were measured.

**Table 3.** Effect of sourdoughs on the rheological characteristics of nongluten dough.

Mean values with different letter in superscript within the same column differ significantly (*p* < 0.05). Note: *Pediococcus acidilactici* 02P108 (PA); *Pediococcus pentosaceus* 12R2187 (PP); *Enteroccocus durans* 09B374 (ED). DDT, dough development time.

#### *2.9. Bread Quality*

#### 2.9.1. Physical Properties

The quality of the prepared breads was assessed by the following characteristics. Bread loaf volume was determined after baking and cooling the breads for 3 h at room temperature by a rapeseed displacement method [61]. The specific volume was calculated by the ratio between volume (cm3 ) and mass (g) of each sample. Bread height and diameter were measured by a caliper, and the shape stability (height/diameter) was calculated [66]. Bake loss (%) was determined following weighing each loaf before and after baking [67]. The bread loaves were wrapped in plastic bags and stored at room temperature (27 ± 2 ◦C) to determine the storage time (in days) until mold growth became visible [68,69].

#### 2.9.2. Crumb Elasticity

Total and plastic deformation were measured by an automatic penetrometer and expressed as penetrometric units (PU) [69]. A 4-mm thick crumb sample was cut from the bread and placed on the flat surface of the lifting table, which was raised until the upper surface of the sample lightly touched the lower end of the immersion body. The value of penetration of the immersion body in the sample after 5 s represented total deformation. The steel disk was removed and the immersion system unloaded. Then the measurement was repeated for 10 s, and the recorded value in PU represented plastic deformation. Elastic deformation was calculated as the difference between total and plastic deformation.

#### *2.10. Sensory Analysis*

Sensory analyses of the obtained breads were performed by a descriptive panel consisting of 25 panelists (52% women and 48% men) aged 22–60 years, who were familiar with sensory analysis of foods but not specifically trained in the evaluation of sourdough breads. The analyses were carried out according to ISO 6658:2017 [70]. The panelists were asked to score eight parameters, namely shape, crust color, crumb color, porosity, aroma, chewability, taste and aftertaste. They expressed the intensity of each attribute on a 9-point hedonic scale (9—extremely good; 1—extremely bad).

#### *2.11. Statistical Analyses*

All analyses were conducted in triplicate. The obtained data were subject to one-way ANOVA using XLSTAT version 2019.1.2 (AddinSoft Inc., New York, NY, USA). Comparison among least-squares means were performed by Tukey's test; differences were considered significant at *p*-value < 0.05 [71].

#### **3. Results and Discussion**

In recent years, there has been an increased interest toward using nonconventional raw materials for sourdough preparation, including both fermenting matrices and starter cultures [72–77]. In this line, the benefits of nongluten flours and lactic acid bacteria as well as applied sourdough technology were studied with the goal of developing gluten-free breads with improved nutritional and quality characteristics.

#### *3.1. Flour Composition*

Four types of nongluten flours were selected as raw materials for gluten-free bread preparation: teff, rice, sorghum and corn. The raw materials selection was based on literature data for the cultivars' chemical composition. Table 4 shows the detailed chemical composition (content of moisture, protein, fiber, starch and lipids, in dry-matter (DM) basis) of the nongluten flours used in this study.

**Table 4.** Chemical composition of the nongluten flours.

Mean values with different letter in superscript within the same column differ significantly (*p* < 0.05).

The obtained data shows that protein and fiber content of teff and sorghum flour are much higher compared to the values of these parameters for rice and corn. Protein content of sorghum was 12.49%, and in teff it was 10.20%. Furthermore, fiber content was 12.39 and 9.56%, while the values for corn and rice flour were very low—3.74 and 1.47%, respectively. These results confirm that teff and sorghum are excellent sources of protein and dietary fiber as reported by other authors [48,78–81], and could, therefore, be preferred raw materials for the development of nongluten breads with additional functionalities (rich in protein and fiber). Other authors reported approximately 11–13% of protein, approximately 80% of complex carbohydrates, and 2.4–3.0% of fat for teff [34,82]. Sorghum and teff are also generally characterized by good technological properties [83]. Apart from fiber, teff is also an excellent source of iron and contains far more calcium, potassium and other essential minerals than other grains [34,84–89].  It is important to point out that teff contains fat that is not easily oxidized—which results in a longer shelf-life of teff flour compared to other nongluten flours [72]. Our analyses showed that starch content was of similar levels for teff, sorghum and corn—viz., 74.5, 72.2 and 73.4%, respectively, while starch in rice flour amounted to 84.7%, which was more than 10% higher compared to the other nongluten flours. Other authors report similar results for starch content of the analyzed raw materials [72,73,81,90,91].

#### *3.2. Formulation of the Control Gluten-Free Dough Matrix*

To develop a gluten-free bread with good nutritional and quality characteristics, the initial step of the study was to formulate a flour blend based on the functional chemical composition as well as the technological characteristics of nongluten cereals and pseudocereals. Based on the available literature data and preliminary trials, four types of flour were selected to formulate the bread matrix—teff, rice, sorghum and corn flour.

A technological challenge with developing nongluten bakery products is that the doughs do not have an adequate structure consistency (elasticity) to retain the gas formed during fermentation. Therefore, it is necessary to use other ingredients/food additives such as proteins, starches, gums and hydrocolloids with water-binding and structure-building properties, which are able to compensate for the lack of gluten in the dough [92].

The appropriate combination of structure-forming additives and their amounts depends on the type of the nongluten flour or the specific flour combination, since these have a significant effect on the quality characteristics of the baked products. Curic et al. ´ (2007) [93] supplemented a nongluten flour mix (rice flour, corn starch) with 1, 2 and 3% of xanthan, guar gum, pectin or cellulose as stabilizing additives toward the improvement of the structure of the obtained gluten-free bread and found that 3% guar gum had the best structure-forming effect.

In the present study, different combinations of xanthan, guar gum and carboxymethyl cellulose at different ratios to the nongluten flour base were tested aiming to find the most efficient gluten-replacement strategy (Materials and Methods, Section 2.6). Dough and baking characteristics of the three formulations are presented in Tables 5 and 6.

**Table 5.** Fermentation characteristics of nongluten dough formulations.

Variant A—xanthan 1.0% and guar gum 1.0%; Variant B—xanthan 0.6%, guar gum 1.0% and CMC 1.0%; Variant C—xanthan 0.6%, guar gum 1.0% and CMC 3.0%. Mean values with different letter in superscript within the same column differ significantly (*p* < 0.05).

**Table 6.** Baking characteristics of nongluten dough formulations.

Variant A—xanthan 1.0% and guar gum 1.0%; Variant B—xanthan 0.6%, guar gum 1.0% and CMC 1.0%; Variant C—xanthan 0.6%, guar gum 1.0% and CMC 3.0%. Mean values with different letter in superscript within the same column differ significantly (*p* < 0.05).

The obtained results show that Variant B yielded a significantly higher specific volume of both floor bread (1.40 cm3/g) and pan bread (1.85 cm3/g) compared to Variants A and C (Table 6). These values were positively correlated with the highest values of the degree of immersion before and after fermentation of Variant B (Table 6) and with low bake loss—that is, 20.87% for floor bread and 17.72% for pan bread of dough variant B (Table 6). Variant C had a significantly lower shape stability (height/diameter = 0.36) compared to Variants A and B, which did not differ in this parameter. These results clearly show that the higher hydration of the nongluten dough enables a more active fermentation, but the appropriate combination of structure-forming additives is of key importance for the baking characteristics of the nongluten breads. Based on the obtained results, Variant B was selected as the control nongluten dough formulation for the next steps of the study. In terms of baking method, pan breads showed better baking characteristics, and to commercialize such nongluten formulations, pan baking would be more appropriate to obtain goodlooking products.

#### *3.3. Sourdough Fermentation*

The sourdoughs applied for leavening of the main gluten-free flour mix were prepared only from teff flour with the addition of the three different cultures of lactic acid bacteria. The kinetics of sourdough fermentation was monitored by the changes in pH, TTA and the total viable counts of the respective starter culture. Results from these analyses are presented in Table 7.

**Table 7.** Fermentation kinetics of teff sourdoughs.

Mean values of the same parameters with different letter within the same row differ significantly (*p* < 0.05).

The obtained results clearly indicate a good capacity of the three tested strains to ferment teff flour, while increasing their biomass and producing organic acids, which resulted in sourdoughs with a pleasant lactic acid aroma. Indeed, the genera *Pediococcus* and *Enterococcus* are homofermentative or homolactic bacteria, thus releasing solely lactic acid from fermentation—and known as hexose fermentation pathway or Embden–Meyerholf– Parnas (EMP) pathway. Data analysis showed no statistical differences in the yielded biomass concentration at the end of the fermentations among the three tested LAB strains (8.72–8.83 log CFU/g). The lowest pH value (3.88) was reached in the sourdough with strain *Pediococcus pentosaceus* 12R2187, but it was not significantly different compared to the other two sourdoughs. One of the strains used to prepare sourdough was of *Enteroccocus durans* species, which is not commonly found in sourdoughs. It was interesting to observe that this strain yielded a final TTA value of 18.4, which was significantly higher compared to the other two LAB strains and indicated the highest capacity of strain *Enteroccocus durans* 09B374 to produce organic acids.

#### *3.4. Development of Gluten-Free Bread with Sourdough*

#### 3.4.1. Degree of Immersion in the Nongluten Sourdoughs

Three nongluten doughs were prepared by using as a matrix the preselected dough variant B (Section 3.2) without yeast and by adding equal amounts of teff sourdoughs prepared with the three selected lactic acid bacteria strains—*Pediococcus acidilactici* 02P108, *Pediococcus pentosaceus* 12R2187 and *Enteroccocus durans* 09B374. Dough variant B with yeast was used as the control. All nongluten doughs were kneaded according to the established methodology (Section 2.6). The results from analyzing the degree of immersion in the nongluten doughs are presented in Figure 1.

The initial degree of immersion of the control dough was significantly higher (195 PU) (*p* < 0.01) compared to each of the three samples with added sourdoughs (160–164 PU). However, the difference between these three samples was not significant. After 20 min of fermentation, the degree of immersion of the control sample increased by 87.7%, and for all sourdough-fermented doughs, the values more than doubled (335–345 PU). However, for all three sourdough-fermented samples, the degree of immersion was significantly lower than that of the control sample (366 PU). These results indicate that the sourdoughfermented doughs are more resistant and less elastic compared to the control dough fermented by baker's yeast (*Saccharomyces cerevisiae*). These observations differ from the results reported by Wolter et al. (2014) [94], where sourdough addition led to decreased dough strength resulting in softer dough. Other authors also confirm that sourdough

fermentation increased the elasticity and reduced the stiffness of doughs [95]. However, these teams studied wheat-based doughs, while with nongluten doughs this effect is the opposite, as observed in our study. The reduced elasticity could be attributed to the nongluten flour composition and the specific fermentation capacity of the tested strains. Other studies on the rheological properties of gluten-free sourdoughs prepared with lactic acid bacteria also found that the addition of sourdough reduced the elasticity of the dough and improved the dough strength [69,96,97]. *Foods* **2022**, *11*, x FOR PEER REVIEW 9 of 19

**Figure 1.** Degree of immersion in nongluten doughs prepared with the addition of sourdoughs with single cultures lactic acid bacteria. Note: PU—penetrometric units; PA—sample with *Pediococcus acidilactici* 02P108; PP—sample with *Pediococcus pentosaceus* 12R2187; ED—sample with *Enteroccocus durans* 09B374. Different small letters for each time measurement indicate significant difference between mean values (*p* < 0.01). **Figure 1.** Degree of immersion in nongluten doughs prepared with the addition of sourdoughs with single cultures lactic acid bacteria. Note: PU—penetrometric units; PA—sample with *Pediococcus acidilactici* 02P108; PP—sample with *Pediococcus pentosaceus* 12R2187; ED—sample with *Enteroccocus durans* 09B374. Different small letters for each time measurement indicate significant difference between mean values (*p* < 0.01).

The initial degree of immersion of the control dough was significantly higher (195 PU) (*p* < 0.01) compared to each of the three samples with added sourdoughs (160–164 PU). However, the difference between these three samples was not significant. After 20 min of fermentation, the degree of immersion of the control sample increased by 87.7%, and for all sourdough-fermented doughs, the values more than doubled (335–345 PU). In our study, after 20 min of fermentation, strain PP produced a significantly softer dough compared to the other two strains. Such findings confirmed that the reduced elasticity was attributed to the LAB fermentation and that strain specificity has an effect on nongluten dough stability.

#### However, for all three sourdough-fermented samples, the degree of immersion was sig-3.4.2. Effect of Sourdoughs on the Rheological Characteristics of Nongluten Dough

nificantly lower than that of the control sample (366 PU). These results indicate that the sourdough-fermented doughs are more resistant and less elastic compared to the control The results from the rheological analysis of the nongluten doughs obtained with the addition of sourdoughs with different lactic acid bacteria are depicted in Table 3.

dough fermented by baker's yeast (*Saccharomyces cerevisiae*). These observations differ from the results reported by Wolter et al. (2014) [94], where sourdough addition led to decreased dough strength resulting in softer dough. Other authors also confirm that sourdough fermentation increased the elasticity and reduced the stiffness of doughs [95]. However, these teams studied wheat-based doughs, while with nongluten doughs this effect is the opposite, as observed in our study. The reduced elasticity could be attributed to the nongluten flour composition and the specific fermentation capacity of the tested strains. Other studies on the rheological properties of gluten-free sourdoughs prepared with lactic acid bacteria also found that the addition of sourdough reduced the elasticity of the dough and improved the dough strength [69,96,97]. In our study, after 20 min of fermentation, strain PP produced a significantly softer A farinograph is a useful tool for the determination of the optimal water content for dough preparation. In addition, it provides information about dough stability and dough development time [98]. In general, the water-absorbing capacity of flour depends on the protein content, the particle size, the amount of starchy grains with impaired integrity and some other factors. In the present study, the addition of hydrocolloids also contributed to the water absorption and to the improved and rheological properties. Our preliminary experiment with different WA values (55%, 65% and 70%) of nongluten flour basis showed that 65% provided best dough consistency as well as best specific volume and porosity of the bread (data not shown). Based on these findings, the WA of the formulations in the current study was first adjusted to 65%, and then the other parameters of the doughs were measured.

dough compared to the other two strains. Such findings confirmed that the reduced elasticity was attributed to the LAB fermentation and that strain specificity has an effect on nongluten dough stability. 3.4.2. Effect of Sourdoughs on the Rheological Characteristics of Nongluten Dough The results from the rheological analysis of the nongluten doughs obtained with the addition of sourdoughs with different lactic acid bacteria are depicted in Table 3. A farinograph is a useful tool for the determination of the optimal water content for dough preparation. In addition, it provides information about dough stability and dough The consistency of the control sample was 20.69% higher than that of the experimental samples. No significant differences were observed in the consistency (290 FU) of the samples with added sourdoughs. Significant differences between the control and the sourdough-leavened doughs were also observed for the other tested parameters: dough development time (DDT), stability and relaxation of the dough. The DDT of the sourdoughadded samples was reduced by 3 times (2 min) for sample PP and by 4 times (1.5 min) for samples PA and ED. These results are in contrast to the observations of Tafti et al. (2013) [99] who found no effect of spray-dried sourdough addition on wheat dough development time. Again, the difference might be attributed to the different kind of dough matrix.

development time [98]. In general, the water-absorbing capacity of flour depends on the protein content, the particle size, the amount of starchy grains with impaired integrity

uted to the water absorption and to the improved and rheological properties. Our preliminary experiment with different WA values (55%, 65% and 70%) of nongluten flour basis showed that 65% provided best dough consistency as well as best specific volume and porosity of the bread (data not shown). Based on these findings, the WA of the for-

In our study, the control sample had significantly higher stability (10 min) than the sourdough-fermented samples. However, the values of 6.5–7.5 min also indicated good stability of the doughs obtained with sourdough addition and with dough samples PP and PA showing significantly better effect compared to the sample ED. The degree of dough softening of the control sample was 10 FU, while the sourdough-fermented doughs showed a significantly higher value (30 FU), which is still not too high for this parameter and was the same for the three test samples. Tafti et al. (2013) [99] also observed that the degree of softening significantly increased with an increase in the sourdough level, whereas dough stability was significantly reduced. The results obtained in our study indicate that the use of sourdoughs requires some additional matrix optimization to achieve the same dough rheology as the yeast-fermented dough.

#### 3.4.3. Quality Assessment of Gluten-Free Breads Leavened with Sourdough

To assess the baking characteristics of the gluten-free breads with added sourdough, two types of bread—floor and pan bread—were prepared from each sourdough-leavened bread variety. Fermentation and baking of all dough samples were carried out under equal conditions, according to the adopted technology. Results from the quality assessment of their baking characteristics are presented in Table 8.

**Table 8.** Baking characteristics of nongluten breads with sourdough.

Mean values with different letter in superscript within the same column differ significantly (*p* < 0.05). Note: *Pediococcus acidilactici* 02P108 (PA); *Pediococcus pentosaceus* 12R2187 (PP); *Enteroccocus durans* 09B374 (ED).

The specific volumes of the floor bread samples prepared with strains PA and ED were significantly higher compared to the control and sample PP, with strain *Enteroccocus durans* yielding the highest value (1.70 cm3/g). Comparison of pan bread samples showed that samples PP and PA had significantly lower (20% for sample PP) specific volumes than the control sample and sample ED. These results indicate that strain specificity is important in terms of the generated specific volume of nongluten breads, and in our study, strain *Enteroccocus durans* gave the best performance in leavening the nongluten dough.

Literature data on the effect of LAB on the specific volume of gluten-free breads are diverse. According to some authors, the addition of sourdough to GF breads does not have a significant influence on the specific volume [100,101]. Cappa et al. (2016) [17] reported that sourdoughs have been effective in improving bread volume and softness, which confirms the positive effects observed in our experiments. Other studies also showed that sourdough gluten-free bread had a higher specific volume and was less firm than GF bread fermented with baker's yeast alone [102,103].

In terms of shape stability (height/diameter ratio, H/D), strain ED gave a significantly higher value of 0.48 compared to the other three tested doughs. Similar to this result, Falade et al. (2014) [104] reported that the addition of lactic acid bacteria increased the bread height after baking.

It is interesting to note that all three LAB-leavened samples had significantly lower baking loss compared to the yeast-leavened control sample, with the lowest value (15.65%) among floor breads observed for sample ED, and the lowest among pan breads (12.25%) observed for sample PA. This positive effect could be attributed to the organic acids produced by the bacteria, which strengthen the structure of the gels in the gluten-free doughs. Therefore, the gas retention in the bread is greater. Our observations confirm the findings of Wolter et al. (2014) [94] and indicate that the application of sourdoughs

does affect the baking characteristics of the gluten-free bread, mostly by a significantly pronounced reduction in baking loss. A strain-specific effect on the analyzed parameters was also observed, and this effect differed with respect to the type of bread—i.e., floor or pan. sourdoughs does affect the baking characteristics of the gluten-free bread, mostly by a significantly pronounced reduction in baking loss. A strain-specific effect on the analyzed parameters was also observed, and this effect differed with respect to the type of bread—i.e., floor or pan.

which confirms the positive effects observed in our experiments. Other studies also showed that sourdough gluten-free bread had a higher specific volume and was less firm

In terms of shape stability (height/diameter ratio, H/D), strain ED gave a significantly higher value of 0.48 compared to the other three tested doughs. Similar to this result, Falade et al. (2014) [104] reported that the addition of lactic acid bacteria increased

It is interesting to note that all three LAB-leavened samples had significantly lower baking loss compared to the yeast-leavened control sample, with the lowest value (15.65%) among floor breads observed for sample ED, and the lowest among pan breads (12.25%) observed for sample PA. This positive effect could be attributed to the organic acids produced by the bacteria, which strengthen the structure of the gels in the gluten-free doughs. Therefore, the gas retention in the bread is greater. Our observations confirm the findings of Wolter et al. (2014) [94] and indicate that the application of

#### 3.4.4. Sensory Profile of Nongluten Breads with Sourdough 3.4.4. Sensory Profile of Nongluten Breads with Sourdough

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than GF bread fermented with baker's yeast alone [102,103].

the bread height after baking.

In general, the use of LAB-inoculated sourdough can improve the quality of bread with regards to various characteristics such as taste, staleness, odor, chewability, softness, moisture content, pH, acidity and texture [105]. In the present study, the gluten-free breads leavened with sourdoughs differed in appearance from the control mainly by a better color of the crust (Figure 2), especially of sample ED. The crust of the three samples was thin, smooth and soft. In general, the use of LAB-inoculated sourdough can improve the quality of bread with regards to various characteristics such as taste, staleness, odor, chewability, softness, moisture content, pH, acidity and texture [105]. In the present study, the gluten-free breads leavened with sourdoughs differed in appearance from the control mainly by a better color of the crust (Figure 2), especially of sample ED. The crust of the three samples was thin, smooth and soft.

**Figure 2.** Sensory profile of nongluten pan breads with sourdough. Note: *Pediococcus acidilactici* 02P108 (PA); *Pediococcus pentosaceus* 12R2187 (PP); *Enteroccocus durans* 09B374 (ED). **Figure 2.** Sensory profile of nongluten pan breads with sourdough. Note: *Pediococcus acidilactici* 02P108 (PA); *Pediococcus pentosaceus* 12R2187 (PP); *Enteroccocus durans* 09B374 (ED).

Generally, all three strains reached biomass content at the level of 108 CFU/g, but the differences in the fermenting capacity showed variations in acid production as well as the composition of the produced organic acids. These differences resulted in variations in the sensory characteristics of the obtained nongluten breads (Section 3.4.4). Generally, all three strains reached biomass content at the level of 108 CFU/g, but the differences in the fermenting capacity showed variations in acid production as well as the composition of the produced organic acids. These differences resulted in variations in the sensory characteristics of the obtained nongluten breads (Section 3.4.4).

The ratio between lactic and acetic acids s an important factor affecting the aroma of bread [106], and its influenced by the fermenting microorganisms, the fermentation temperature and the type of flour or flours [107]. In the present study, the aroma, taste The ratio between lactic and acetic acids s an important factor affecting the aroma of bread [106], and its influenced by the fermenting microorganisms, the fermentation temperature and the type of flour or flours [107]. In the present study, the aroma, taste and aftertaste were found pleasant for all samples prepared from the nongluten flour mix. However, these characteristics were most pronounced in the sample with strain *Pediococcus pentosaceus* 12R2187 (PP), with a significant difference compared to the other samples.

According to Moore et al. (2008) [100], acidification during sourdough fermentation increases polysaccharide swelling that can partially replace gluten and improve the structure of gluten-free bread. In our study, the development of bread porosity in the sourdoughfermented bread samples did not differ from the control sample. The middle of all samples was soft and slightly moist, and slight sticking to the teeth was observed while chewing. Indeed, only sample PA gave a significant difference compared to control regarding this parameter. Data from the sensory analysis showed positive acceptance of the prepared nongluten breads. Some sensory characteristics were significantly influenced by the LAB strain specificity, with the most pronounced positive effect shown by strain *Pediococcus pentosaceus* 12R2187 (PP).

#### 3.4.5. Shelf-Life Estimation of Nongluten Bread with Sourdough 3.4.5. Shelf-Life Estimation of Nongluten Bread with Sourdough

strain *Pediococcus pentosaceus* 12R2187 (PP).

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samples.

Bourne (1978) [108] described the use of instrumental texture profile analysis extensively, using force, deformation, and work measurement to determine the texture parameters for hardness, fracturability, cohesiveness, adhesiveness, springiness, gumminess and chewiness. In the present research, estimation of the shelf life of the prepared gluten-free pan breads with sourdoughs was based on the time of occurrence of mold growth, and the analysis of the deformation characteristics—viz., total, elastic and plastic deformation—were measured by an automatic penetrometer of the bread crumb. Pan breads were selected to explore deformation characteristics since this method of baking showed better baking characteristics of the nongluten formulations. The total deformation of the breads leavened with lactic acid bacteria (33–35 PU) was more than two times higher than that of the yeast-leavened control sample (15 PU) (Figure 3). This trend was generally preserved until the end of the experiment (72 h), while total deformation gradually decreased for all samples. Bourne (1978) [108] described the use of instrumental texture profile analysis extensively, using force, deformation, and work measurement to determine the texture parameters for hardness, fracturability, cohesiveness, adhesiveness, springiness, gumminess and chewiness. In the present research, estimation of the shelf life of the prepared gluten-free pan breads with sourdoughs was based on the time of occurrence of mold growth, and the analysis of the deformation characteristics—viz., total, elastic and plastic deformation—were measured by an automatic penetrometer of the bread crumb. Pan breads were selected to explore deformation characteristics since this method of baking showed better baking characteristics of the nongluten formulations. The total deformation of the breads leavened with lactic acid bacteria (33–35 PU) was more than two times higher than that of the yeast-leavened control sample (15 PU) (Figure 3). This trend was generally preserved until the end of the experiment (72 h), while total deformation gradually decreased for all samples.

and aftertaste were found pleasant for all samples prepared from the nongluten flour mix. However, these characteristics were most pronounced in the sample with strain *Pediococcus pentosaceus* 12R2187 (PP), with a significant difference compared to the other

According to Moore et al. (2008) [100], acidification during sourdough fermentation increases polysaccharide swelling that can partially replace gluten and improve the structure of gluten-free bread. In our study, the development of bread porosity in the sourdough-fermented bread samples did not differ from the control sample. The middle of all samples was soft and slightly moist, and slight sticking to the teeth was observed while chewing. Indeed, only sample PA gave a significant difference compared to control regarding this parameter. Data from the sensory analysis showed positive acceptance of the prepared nongluten breads. Some sensory characteristics were significantly influenced by the LAB strain specificity, with the most pronounced positive effect shown by

**Figure 3.** Effect of the different LAB strains on the total deformation of nongluten bread. Note: PU—penetrometric units; PA—sample with *Pediococcus acidilactici* 02P108; PP—sample with *Pediococcus pentosaceus* 12R2187; ED—sample with *Enteroccocus durans* 09B374. Different small letters for each time measurement indicate significant difference between mean values (*p* < 0.01). **Figure 3.** Effect of the different LAB strains on the total deformation of nongluten bread. Note: PU—penetrometric units; PA—sample with *Pediococcus acidilactici* 02P108; PP—sample with *Pediococcus pentosaceus* 12R2187; ED—sample with *Enteroccocus durans* 09B374. Different small letters for each time measurement indicate significant difference between mean values (*p* < 0.01).

It is interesting to note that the total deformation differences between the three samples with sourdoughs were not significant during the 48 h of fermentation, and only at the end of the process did samples PP and PA have a significantly lower total deformation (21 and 20 PU, respectively) compared to sample ED (26 PU).

The total deformation of the control sample was reduced by 53% for 72 h, while the average reduction for the breads with sourdough addition was 25%, with the largest decrease observed for sample PP (40%), and the lowest for sample ED (26%). These results clearly show that the application of LAB improves the softness of bread throughout storage time, with the strongest effect observed for strain *Enteroccocus durans* 09B374 (ED). In a study on the application of sourdough *Lactobacillus* strains to obtain gluten-free bread, Di Cagno et al. (2008) [103] also found that the addition of lactic acid bacteria resulted in lower hardness of gluten-free bread crumb during storage.

Results from analyzing the plastic deformation of sourdough-leavened nongluten breads are displayed in Figure 4.

It is interesting to note that the total deformation differences between the three samples with sourdoughs were not significant during the 48 h of fermentation, and only at the end of the process did samples PP and PA have a significantly lower total defor-

The total deformation of the control sample was reduced by 53% for 72 h, while the average reduction for the breads with sourdough addition was 25%, with the largest decrease observed for sample PP (40%), and the lowest for sample ED (26%). These results clearly show that the application of LAB improves the softness of bread throughout storage time, with the strongest effect observed for strain *Enteroccocus durans* 09B374 (ED). In a study on the application of sourdough *Lactobacillus* strains to obtain gluten-free bread, Di Cagno et al. (2008) [103] also found that the addition of lactic acid bacteria re-

Results from analyzing the plastic deformation of sourdough-leavened nongluten

mation (21 and 20 PU, respectively) compared to sample ED (26 PU).

sulted in lower hardness of gluten-free bread crumb during storage.

breads are displayed in Figure 4.

**Figure 4.** Effect of lactic acid bacteria on the plastic deformation of nongluten bread. Note: PU—penetrometric units; PA—sample with *Pediococcus acidilactici* 02P108; PP—sample with *Pediococcus pentosaceus* 12R2187; ED—sample with *Enteroccocus durans* 09B374. Different small letters for each time measurement indicate significant difference between mean values (*p* < 0.01). **Figure 4.** Effect of lactic acid bacteria on the plastic deformation of nongluten bread. Note: PU—penetrometric units; PA—sample with *Pediococcus acidilactici* 02P108; PP—sample with *Pediococcus pentosaceus* 12R2187; ED—sample with *Enteroccocus durans* 09B374. Different small letters for each time measurement indicate significant difference between mean values (*p* < 0.01).

The plastic deformation of the sourdough-leavened breads was considerably greater (around threefold more) compared to the control, and it gradually decreased until the end of the experiment. The crumb of sample ED retained its plasticity to the greatest extent, which was most pronounced at the end of the 72 h test. Plasticity of this sample decreased by 25% between the third and the 72nd hour compared to a 60% reduction observed in the control. For the other two LAB-leavened samples, plasticity decreased by an average of 44%. After 24 h storage, the plastic deformation of sample ED became significantly higher compared to sample PA, and this trend was maintained until the end of the experiment, when plastic deformation of sample ED was significantly higher than sample PP as well. The difference between samples PP and PA was not significant during the course of the entire duration of the experiment. These results indicate that the microbial strain specificity affects the plastic deformation of nongluten breads during storage, and in this study strain *Enteroccocus durans* 09B374 (ED) provided the highest softness of the nongluten bread in terms of total and plastic deformation. The plastic deformation of the sourdough-leavened breads was considerably greater (around threefold more) compared to the control, and it gradually decreased until the end of the experiment. The crumb of sample ED retained its plasticity to the greatest extent, which was most pronounced at the end of the 72 h test. Plasticity of this sample decreased by 25% between the third and the 72nd hour compared to a 60% reduction observed in the control. For the other two LAB-leavened samples, plasticity decreased by an average of 44%. After 24 h storage, the plastic deformation of sample ED became significantly higher compared to sample PA, and this trend was maintained until the end of the experiment, when plastic deformation of sample ED was significantly higher than sample PP as well. The difference between samples PP and PA was not significant during the course of the entire duration of the experiment. These results indicate that the microbial strain specificity affects the plastic deformation of nongluten breads during storage, and in this study strain *Enteroccocus durans* 09B374 (ED) provided the highest softness of the nongluten bread in terms of total and plastic deformation.

The third analyzed shelf-life parameter for the nongluten breads prepared with the additions of sourdoughs was elastic deformation (Figure 5). The third analyzed shelf-life parameter for the nongluten breads prepared with the additions of sourdoughs was elastic deformation (Figure 5).

Elastic deformation measurement at 3 h of storage showed the same average values (5 PU) for all tested samples. The relaxation capacity of the gluten-free bread samples PP and PA did not change after 24 h and remained at 4 PU at 48 and 72 h, while sample ED behaved similar to the control sample and also did not change between 48 and 72 h. Results for samples PP and PA were significantly different (*p* < 0.01) compared to control and sample ED, which indicates the effect of strain specificity on the elastic properties during storage.

Many other studies also demonstrated that sourdough gluten-free bread was less firm than gluten-free bread leavened with baker's yeast alone [101,103,109,110]. Shelf-life rheological tests made by Moore et al. (2008) [101], however, showed that the addition of sourdough to a gluten-free mix led to increased firmness and elasticity overtime, which indicated that a LAB strain could be used to produce gluten-free bread with increased quality and shelf life. A study of Moroni et al. (2009) [24] also showed that the use of sourdough in nongluten bread development had positive effects on the crumb structure. Staling was delayed and longer shelf-life was achieved. These effects are mostly associated

with the production of lactic and acetic acids, as well as exopolysaccharides during fermentation with lactic acid bacteria. In addition, the use of LAB-inoculated sourdoughs in bread preparation may contribute antifungal properties, thereby increasing the shelf-life of the bread even at a reduced salt content [111]. *Foods* **2022**, *11*, x FOR PEER REVIEW 14 of 19

**Figure 5.** Effect of lactic acid bacteria on the elastic deformation of nongluten bread. Note: PU—penetrometric units; PA—sample with *Pediococcus acidilactici* 02P108; PP—sample with *Pediococcus pentosaceus* 12R2187; ED—sample with *Enteroccocus durans* 09B374. Different small letters for each time measurement indicate significant difference between mean values (*p* < 0.01). **Figure 5.** Effect of lactic acid bacteria on the elastic deformation of nongluten bread. Note: PU—penetrometric units; PA—sample with *Pediococcus acidilactici* 02P108; PP—sample with *Pediococcus pentosaceus* 12R2187; ED—sample with *Enteroccocus durans* 09B374. Different small letters for each time measurement indicate significant difference between mean values (*p* < 0.01).

#### Elastic deformation measurement at 3 h of storage showed the same average values **4. Conclusions**

(5 PU) for all tested samples. The relaxation capacity of the gluten-free bread samples PP and PA did not change after 24 h and remained at 4 PU at 48 and 72 h, while sample ED behaved similar to the control sample and also did not change between 48 and 72 h. Results for samples PP and PA were significantly different (*p* < 0.01) compared to control and sample ED, which indicates the effect of strain specificity on the elastic properties during storage. Many other studies also demonstrated that sourdough gluten-free bread was less firm than gluten-free bread leavened with baker's yeast alone [101,103,109,110]. Shelf-life rheological tests made by Moore et al. (2008) [101], however, showed that the addition of sourdough to a gluten-free mix led to increased firmness and elasticity overtime, which indicated that a LAB strain could be used to produce gluten-free bread with increased The selected combination of nongluten flours and additives was adequate for obtaining nongluten bread with good quality characteristics. The three tested LAB strains demonstrated good capacity to ferment teff flour into sourdough, reaching different levels of acidification. The application of teff-based sourdoughs had a positive effect on various technological and sensory characteristics of the nongluten doughs and breads. The sourdough-fermented doughs were softer and more elastic compared to the control dough fermented by baker's yeast. The application of sourdoughs resulted in a significantly pronounced reduction of baking loss. A strain-specific effect on the analyzed quality parameters was observed, and this effect also differed with respect to the bread type—floor or pan bread. Pan baking resulted in better bread characteristics and proved to be more appropriate for commercialization of nongluten bread formulations.

quality and shelf life. A study of Moroni et al. (2009) [24] also showed that the use of sourdough in nongluten bread development had positive effects on the crumb structure. Staling was delayed and longer shelf-life was achieved. These effects are mostly associ-The obtained nongluten breads had positive sensory acceptance. Strain specificity had a significant effect on some sensory characteristics of the products and on bread softness during storage.

ated with the production of lactic and acetic acids, as well as exopolysaccharides during fermentation with lactic acid bacteria. In addition, the use of LAB-inoculated sourdoughs in bread preparation may contribute antifungal properties, thereby increasing the shelf-life of the bread even at a reduced salt content [111]. The study demonstrated that the application of sourdoughs in the nongluten flour matrix is a successful approach for gluten-free bread development. Strain specificity is significant for dough rheology and the baking characteristics, and it is, therefore, important to perform a careful starter culture selection.

**4. Conclusions** The selected combination of nongluten flours and additives was adequate for obtaining nongluten bread with good quality characteristics. The three tested LAB strains demonstrated good capacity to ferment teff flour into sourdough, reaching different lev-**Author Contributions:** Conceptualization, A.A., V.G., R.C.; methodology, A.A., R.C., M.P.; experimental work, R.C., D.S.-S.; data analysis, A.A., V.G.; resources, A.A., M.P.; writing, R.C., V.G., A.A., J.M.R., M.P.; supervision, A.A. All authors have read and agreed to the published version of the manuscript.

els of acidification. The application of teff-based sourdoughs had a positive effect on various technological and sensory characteristics of the nongluten doughs and breads. **Funding:** This research was funded by the Bulgarian National Science Fund, grant number KP-06- PN-36/11 and by the Bulgarian Ministry of Education and Science under the National Research

The sourdough-fermented doughs were softer and more elastic compared to the control dough fermented by baker's yeast. The application of sourdoughs resulted in a significantly pronounced reduction of baking loss. A strain-specific effect on the analyzed Programme "Healthy Foods for a Strong Bio-Economy and Quality of Life" approved by DCM # 577/17.08.2018.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The present study is part of the work of COST Action 18101 SOURDOMICS— Sourdough biotechnology network towards novel, healthier and sustainable food and bioprocesses (https://sourdomics.com/ (accessed on 10 March 2022); https://www.cost.eu/actions/CA18101/ (accessed on 10 March 2022) supported by COST (European Cooperation in Science and Technology) (https://www.cost.eu/ (accessed on 10 March 2022). COST is a funding agency for research and innovation networks. The author J.M.R is the Chair and Grant Holder Scientific Representative, the author A.A. is a Management Committee Member and member of Working Groups "Screening and characterization of cereal flours and sourdough microbiota" and "Design and development of sourdough starter cultures for breadmaking and other agri-food products", the author M.P. is the viceleader and leader of the working group "Recovery, characterization and selection of autochthonous conventional and nonconventional (pseudo)cereal seeds", and the author V.G. is a member of Working Group "Screening and characterization of cereal flours and sourdough microbiota".

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

AACC, American Association of Cereal Chemists; CD, celiac disease; CFU, colonyforming units; C-GFB, control gluten-free bread; CMC, carboxymethyl cellulose; DDT, dough development time; DM, dry-matter; DY, dough yield; ED, *Enteroccocus durans* 09B374; EMP, Embden-Meyerholf-Parnas; FU, farinograph units; GFB, gluten-free bread; GI, glycemic index; H/D, Height/Diameter ratio; LAB, lactic acid bacteria; MRS, de Man-Rogosa-Sharpe; PA, *Pediococcus acidilactici* 02P108; PP, *Pediococcus pentosaceus* 12R2187; PU, penetrometer units; TTA, total titratable acidity.

#### **References**

### *Review* **Gluten-Free Bread and Bakery Products Technology**

**Zuzana Šmídová \* and Jana Rysová**

Food Research Institute Prague, Radiová 7, 102 00 Prague, Czech Republic<EMAIL_ADDRESS>**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Gluten, a protein fraction from wheat, rye, barley, oats, their hybrids and derivatives, is very important in baking technology. The number of people suffering from gluten intolerance is growing worldwide, and at the same time, the need for foods suitable for a gluten-free diet is increasing. Bread and bakery products are an essential part of the daily diet. Therefore, new naturally gluten-free baking ingredients and new methods of processing traditional ingredients are sought. The study discusses the use of additives to replace gluten and ensure the stability and elasticity of the dough, to improve the nutritional quality and sensory properties of gluten-free bread. The current task is to extend the shelf life of gluten-free bread and bakery products and thus extend the possibility of its distribution in a fresh state. This work is also focused on various technological possibilities of gluten-free bread and the preparation of bakery products.

**Keywords:** gluten-free products; bread; bakery products; cereals; enzymes; sourdough

#### **1. Introduction**

A gluten-free diet is the only treatment for people suffering from gluten intolerance. Gluten consumption leads to a range of gluten-related disorders, such as coeliac disease, dermatitis herpetiformis (cutaneous manifestation of coeliac disease), gluten ataxia and non-coeliac gluten sensitivity [1]. A gluten-free diet requires the use of gluten-free cereals—corn, rice, sorghum, millet, teff—and pseudo-cereals—buckwheat, quinoa, amaranth, canihua—but also other foods that are naturally gluten-free—potatoes, tapioca, nuts, oilseeds, legumes, fruits and vegetables [2]. The main challenges for food technologists are bread, bakery products, pastry and pasta. Because of the absence of gluten, other substances needed to maintain the texture, volume, satisfactory crumb, shelf life and sensory quality must be used. These include the use of hydrocolloids, sourdough or enzyme preparations. The use of them is intended to change the recipe and the production technology.

Gluten-free bread and other gluten-free bakery products are very unusual for a consumer accustomed to classic wheat or wheat–rye bread. Toth et al. (2020) have shown that 70.8% of the asked consumers were dissatisfied with gluten-free breads due their texture and taste [3]. Gluten-free breads usually have a less flexible crumb, which hardens faster, and which is easy to crumble. The taste of these products is also different, depending, of course, on the ingredients used. Gluten-free products are easily accepted by people who have been suffering from gluten intolerance since childhood. The acceptance of a glutenfree diet, and at the same time, the acceptance of gluten-free bread in adults diagnosed with gluten intolerance later in life, is more difficult.

According to Codex Standard 118-1979 [4], gluten represents a protein fraction from wheat, rye, barley, oats or their hybrids and derivatives that some people are intolerant to and that is insoluble in water and 0.5 M sodium chloride solution. Water-insoluble prolamins and glutelins (collectively referred to as gluten) usually make up 70–80% of cereal grain proteins. They are the most important cereal proteins from a technological point of view. In this sense, gluten is a specific structure, a viscoelastic gel that gives wheat dough and bakery products its unique properties. This gel is formed after the addition of water and kneading, when the wheat proteins gliadins and glutenins swell, and with

**Citation:** Šmídová, Z.; Rysová, J. Gluten-Free Bread and Bakery Products Technology. *Foods* **2022**, *11*, 480. https://doi.org/10.3390/ foods11030480

Academic Editors: Maria Papageorgiou and Theodoros Varzakas

Received: 10 January 2022 Accepted: 2 February 2022 Published: 7 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the simultaneous access of oxygen as a complex, a three-dimensional viscoelastic system (gluten in the original technological sense) is created which ensures the required viscoelastic properties of the dough. The result is a three-dimensional sufficiently strong and flexible continuous network capable of maintaining a large volume of gas, and thus ensuring the sufficient volume, shape and texture of the products. erties of the dough. The result is a three-dimensional sufficiently strong and flexible continuous network capable of maintaining a large volume of gas, and thus ensuring the sufficient volume, shape and texture of the products. Prolamins and glutelins are represented by a number of related proteins with somewhat different amino acid composition and structure (e.g., gliadin proteins are usually up

and bakery products its unique properties. This gel is formed after the addition of water and kneading, when the wheat proteins gliadins and glutenins swell, and with the simultaneous access of oxygen as a complex, a three-dimensional viscoelastic system (gluten in the original technological sense) is created which ensures the required viscoelastic prop-

*Foods* **2022**, *11*, x FOR PEER REVIEW 2 of 18

Prolamins and glutelins are represented by a number of related proteins with somewhat different amino acid composition and structure (e.g., gliadin proteins are usually up to several dozen for each wheat variety). In particular, the prolamins of wheat (as well as spelt, Khorasan, einkorn and emmer), rye and barley and their hybrids (Triticale, Tritordeum) cause a disease in predisposed individuals, which is called celiac disease. The relationship between oat prolamins and celiac disease is still the subject of debate. Wheat gliadin fractions are divided into four subfractions: α-, β-, γ- and ω-gliadins (Figures 1 and 2). All subfractions of α-/β-/γ-/ω—gliadins can cause celiac disease in predisposed individuals, and ω-5 gliadins allergic reaction. Celiac disease is caused by two amino acid sequences: ProSer-Gln-Gln (PSQQ) and Gln-Gln-Gln-Pro (QQQP) [5]. to several dozen for each wheat variety). In particular, the prolamins of wheat (as well as spelt, Khorasan, einkorn and emmer), rye and barley and their hybrids (Triticale, Tritordeum) cause a disease in predisposed individuals, which is called celiac disease. The relationship between oat prolamins and celiac disease is still the subject of debate. Wheat gliadin fractions are divided into four subfractions: α-, β-, γ- and ω-gliadins (Figures 1 and 2). All subfractions of α-/β-/γ-/ω—gliadins can cause celiac disease in predisposed individuals, and ω-5 gliadins allergic reaction. Celiac disease is caused by two amino acid sequences: ProSer-Gln-Gln (PSQQ) and Gln-Gln-Gln-Pro (QQQP) [5].

**Figure 1.** Gluten structure from the UNIPROT database https://www.uniprot.org/ (accessed date 31 January 2022) [6]. The picture shows (**a**) glutenin LMW subunit; (**b**) α,β-gliadin; (**c**) γ-gliadin; (**d**) glutenin HMW subunit. AlphaFold produces a per-residue confidence score (pLDDT) between 0 and 100. Some regions with low pLDDT may be unstructured in isolation. The colours represent the model confidence: dark blue–Very high (pLDDT > 90); light blue–Confident (90 > pLDDT > 70); yellow –Low (70 > pLDDT > 50); red–Very low (pLDDT < 50). **Figure 1.** Gluten structure from the UNIPROT database https://www.uniprot.org/ (accessed date 31 January 2022) [6]. The picture shows (**a**) glutenin LMW subunit; (**b**) α,β-gliadin; (**c**) γ-gliadin; (**d**) glutenin HMW subunit. AlphaFold produces a per-residue confidence score (pLDDT) between 0 and 100. Some regions with low pLDDT may be unstructured in isolation. The colours represent the model confidence: dark blue–Very high (pLDDT > 90); light blue–Confident (90 > pLDDT > 70); yellow –Low (70 > pLDDT > 50); red–Very low (pLDDT < 50).

**Figure 2.** Scanning electron micrograph of the gluten dough prepared in Brabender Farinograph from a wheat flour after 8 min. of mixing (FI-8, magnification: 2600×). Adapted from [7]. **Figure 2.** Scanning electron micrograph of the gluten dough prepared in Brabender Farinograph from a wheat flour after 8 min. of mixing (FI-8, magnification: 2600×). Adapted from [7].

#### **2. Raw Materials for Gluten-Free Bread and Bakery Products**

**2. Raw Materials for Gluten-Free Bread and Bakery Products** The specific technological properties of typical gluten cereals in the production of gluten-free bread and bakery products need to be replaced. Rice, corn, or sorghum and other gluten-free cereals are the basis of the diet in many countries around the world. For the preparation of bread and bakery products from gluten-free raw materials, it is necessary to ensure the volume and cohesion of the dough. Rapid staling of these products is a big problem. The nutritional value of wheat and gluten-free bread can be different. Table 1 shows the comparison of nutritional values between wheat flour and gluten-free bread The specific technological properties of typical gluten cereals in the production of gluten-free bread and bakery products need to be replaced. Rice, corn, or sorghum and other gluten-free cereals are the basis of the diet in many countries around the world. For the preparation of bread and bakery products from gluten-free raw materials, it is necessary to ensure the volume and cohesion of the dough. Rapid staling of these products is a big problem. The nutritional value of wheat and gluten-free bread can be different. Table 1 shows the comparison of nutritional values between wheat flour and gluten-free bread baking mixtures (or flour, respectively) and Table 2 the composition of fresh glutencontaining and gluten-free buns. The nutritional values strong depend on the raw material composition of these products and are not uniform.

baking mixtures (or flour, respectively) and Table 2 the composition of fresh gluten-containing and gluten-free buns. The nutritional values strong depend on the raw material **Table 1.** Nutritional values of wheat flour and gluten-free bread baking mixtures. The values were obtained from the product packages or available on https://itesco.cz/ (accessed on 24 January 2022).

Fats (g) 1 1.9 4.4 0.7 5.6 0.9

**Table 2.** Nutritional values of wheat buns and gluten-free buns. These values were obtained from

**Nutritional Values per 100 g per 100 g** Energy (kJ) 1352 1144 Energy (kcal) 320 272 Fats (g) 5.4 8.9

of which saturates (g) 1.6 1.8 Carbohydrates (g) 55.8 42

**Fresh Bun Conventional Gluten-Free**

the product packages or available on https://itesco.cz/ (accessed of 24 January 2022).

of which saturates (g) 0.2 0.5 1.9 0.1 0.6 0.2 Carbohydrates (g) 69 81.9 42 84 66 80 of which sugars (g) 2 3.8 < 0.5 < 0.5 0.8 1.4 Proteins (g) 12 3.2 2.3 2.4 7.2 2.7 Fiber (g) 2 - 1.1 - 6.0 4.4 Salt (g) <0.005 0.2 1.4 1.5 2.5 0.83

**Table 2.** Nutritional values of wheat buns and gluten-free buns. These values were obtained from the product packages or available on https://itesco.cz/ (accessed on 24 January 2022).

In addition to basic gluten-free ingredients such as gluten-free flours and starches, technologically and nutritionally functional ingredients such as hydrocolloids of cereal and non-cereal origin [8], fruit or vegetable fiber [9,10], flax and chia seeds [11,12], psyllium [13], modified starches (e.g., [14]) and proteins [15] from many sources need to be added to achieve sufficient bread volume, crumb softness and shelf life. The addition of fiber, through its hydration, affects the quality of the bread. Besides the beneficial health effects, fiber improves texture, specific volume, apparent viscosity, consistency, texture, sensory quality and shelf life. This is due to the ability to bind water, form a gel and thicken [8]. The key parameters are fiber length, polymerization degree, soluble/insoluble fiber ratio and fiber interactions with other ingredients [16–19]. Soluble fiber improves dough; coarse fiber reduces gas retention [13,20]. The use of enzyme preparations improves the colour of crumbs, supports the production of flavors, increases specific volume and prevents starch retrogradation [21]. Krishna et al. (2019) [11] evaluated the effect of 1% (total base) addition of flax seed powders from flax (*Linum usitatissimum*) and acacia seed powder on the pasting properties, texture and volume of gluten-free bread. The addition of all seed powders reduced crumb hardness by 30–65% and increased specific loaf volume by 50%. These textural improvements were caused by water absorption capacity and emulsifying ability. A darker bread crumb was observed after flax addition, whereas after acacia addition, dark particles were visible. Scanning electron microscopy of these breads showed the absence of holes in the pore surface and viscoelastic starch–protein network. Steffolani et al. (2004) [12] observed a reduction in specific volume and an increase in bread hardness after the addition of chia seed or flour into rice breads, when the effect was more evident with the flour than with seeds. Chia addition minimized weight loss during baking. Chia flour addition led to a darker crust and crumb. No significant differences between the different breads in acceptability were noted; however, chia seed breads presented better texture than controls. Additionally, Sandri et al. (2017) [22] prepared chia-containing rice breads with acceptable sensory properties when the best formulations were prepared from rice flour blends with 5, 10 and 14% whole chia flour. The overall acceptability scores were 8.7, 8.1 and 7.9 out of the 10-point scale, and were very similar to their white gluten-free bread and wheat bread counterparts. The addition of chia flour was acceptable up to 14%. The use of 5–14% whole chia flour increased the levels of lipids, proteins and dietary fiber compared with the white gluten-free breads.

Moreover, there is a trend to use fermentation processes in bread baking so that the products resemble sensory properties of sourdough bread, e.g., the use of sourdough made from gluten-free flour (e.g., [23]). In addition to organic acids, the lactic bacteria also produce free amino acids as precursors of flavours, contemporary degrade phytates and starch and change the fiber solubility. The resulting organic acids and antimicrobial substances extend the shelf life of the bread. Some lactic acid bacteria produce exopolysaccharides, which can affect the rheological properties of the dough [24].

Another possibility of raw material modification is the use of controlled germination. Germinated seeds are characterized by improved taste and nutritional properties. Germination activates seed enzymes, the partial degradation of storage substances into simpler sugars or peptides occurs and the availability of minerals increases. Similar to fermentation, germination also produces a number of secondary metabolites and leads to the degradation of antinutritive substances [25–27].

An integral part of gluten-free dough is emulsifiers, which enable easier processing of the dough and soften the crumb. Lecithin, mono- and diglycerides of fatty acids and esters of fatty acids with lactic acid are used for this purpose. Milk, egg yolk (except for bread), soy protein, sunflower and lupine flour can also serve as emulsifiers [28,29].

The basic raw materials for gluten-free bread and bakery production are gluten-free flours or native starches to which additional ingredients must be added that "substitute gluten" and ensure optimal dough properties. Flours represent more complex materials in comparison with starches, which also include proteins and a low amount of lipids, as well as some minor components such as fiber, vitamins and minerals. Therefore, they are more convenient. For gluten-free products, there is only scarce information concerning flour requirements. These flours differ in starch characteristic (e.g., amylose and amylopectin ratio), in protein amount, and in particle size and their distribution. As far as gluten-free wheat starch present on the market is concerned, it does not have harmful effects on most celiacs; however, celiac people are still unwilling to consume products with wheat-based ingredients [30].

#### **3. Gluten-Free Dough Specifications**

Gluten-free dough is a very complex semi-liquid system that contains polysaccharides and other structure-forming components, viscosity-increasing substances and doughstabilizing substances. It is characterized by high density and low elasticity. Gluten-free dough contains more water than conventional wheat dough. The amount of water depends on the nature of the basic raw materials, their ability to absorb water and the granulation of the raw materials. Additionally, the kneading, its length and its speed are very important. Prolonged kneading increases the specific volume of bread [31].

When baking, the proteins are denatured with increasing temperature, and the starch gelatinization occurs. A sufficiently strong and flexible spatial structure should be created to maintain the expanding gas bubbles and not collapse during baking or cooling of the product. However, gluten-free flours and starches alone do not create such a structure. Therefore, hydrocolloid addition is necessary because of their swelling and water binding capacity [8,32]. Proper hydration affects the conformation of polymer molecules and the rheological properties of the dough. It also determines the texture and softness of the crumb and the crunchiness of the crust. The less hydrated dough provides a small volume of bread, the more hydrated dough can be processed better and the fermentation takes place better in it. Without the addition of other raw materials, the product is irregular in shape, not very cohesive and the crumb is not sufficiently supple and flexible. If the gluten-free recipe contains less protein, the product has usually light crust. There are not enough amino acids available to enter the Maillard reaction [33,34].

After baking, the crust on the surface is firm and crunchy, whereas the crumb retains moisture. After a certain storage time, the moisture in both parts of the bakery product begins to equalize. The water retained in the crumb diffuses to the surface of the product; the crust softens and may deform. On the contrary, through this redistribution process the crumb loses water, thus reducing the flexibility and suppleness of the bread (Table 3). Starch tends to return from an amorphous state to a crystalline form. Starch is recrystallized (retrograded) and bread is staling. The crumb is now brittle and incoherent or hard [35]. Rapid staling and thus limited shelf life are a big problem with gluten-free products. Short shelf life limits the possibility of the sale of fresh gluten-free bread and is one of the reasons for using dry mixtures for home baking of gluten-free bread and bakery products [33].

**Table 3.** The table shows the generalized differences between wheat dough and bread and between gluten-free dough and bread. Adapted and modified according to [34,36–40].

The viscoelastic properties of different doughs significantly influence the volume, and the crumb texture of gluten-free baked goods [41]. One of the most important factors affecting the quality of bread and bakery products is the flour particle size. For bread production, bigger particle size is more suitable and particles below 80–100 µm should not be used if gluten-free bread with high volume and soft crumb is desired. Flours with very large particles may result in breads with sandy texture; therefore, 200 µm is the maximum particle size. Flours with larger particles have been proven to reduce the dough gas retention capacity as well as the final bread volume [30].

#### *3.1. Proteins in Gluten-Free Dough and Products*

Proteins improve the nutritional value of gluten-free products. The choice of flour and possibly another source of protein affects the rheological properties of the dough and the water binding in the dough. Proteins interact with starch and lipids and together contribute to the stability of the dough and the structure of the product. They also give the impression of full product flavour. Proteins can be of plant origin (legumes, soya, gluten-free cereals, rapeseed, canola, sunflower, potato), animal origin (whey, egg, casein, caseinate) or microorganism-, algae-, seaweed- and insect-based [15].

Conventional proteins represent egg and milk proteins. Eggs are very useful in forming the structural network, but they are not usually used in bread. Milk proteins, including caseinates and whey protein concentrates, are sources of calcium and can bind moisture satisfactorily. They have a positive effect on the colour and volume of bread and bakery products. For example, protein-rich gluten-free bread with the addition of 15% whey protein concentrate and 3% of HPMC were prepared by Rustagi et al. (2018) [42]. However, many celiacs do not tolerate lactose and must omit milk from their diets [43]. *Foods* **2022**, *11*, x FOR PEER REVIEW 7 of 18

The source of proteins are naturally gluten-free cereals—rice, corn, teff, sorghum, Job's tears (Figure 3). These cereals contain prolamins too (an ethanol-soluble protein fraction), but the molecules of these proteins do not contain amino acid sequences that are toxic to people with celiac disease. Rice is often used as the basis for gluten-free bread formulations. Gluten-free oats have also very good properties [44]. The zein protein, a prolamin from corn, behaves similar to gluten when heated to 35–40 ◦C. Both corn zein and sorghum kafirin increase the plasticity of the dough [31,45]. The source of proteins are naturally gluten-free cereals—rice, corn, teff, sorghum, Job's tears (Figure 3). These cereals contain prolamins too (an ethanol-soluble protein fraction), but the molecules of these proteins do not contain amino acid sequences that are toxic to people with celiac disease. Rice is often used as the basis for gluten-free bread formulations. Gluten-free oats have also very good properties [44]. The zein protein, a prolamin from corn, behaves similar to gluten when heated to 35–40 °C. Both corn zein and sorghum kafirin increase the plasticity of the dough [31,45].

**Figure 3.** Images of dough samples containing starch with zein, thermally treated-zein (zein heated in vacuum at 160 °C, Z-V-160) and gluten. Adapted from [46]. The white arrows in the image represent the scale - they indicate the distance of 20 m. **Figure 3.** Images of dough samples containing starch with zein, thermally treated-zein (zein heated in vacuum at 160 ◦C, Z-V-160) and gluten. Adapted from [46]. The white arrows in the image represent the scale-they indicate the distance of 20 µm.

Cereal flours are combined in gluten-free bread and bakery products formulations with flour from other crops and with starches. A combination of cereals with legumes is nutritionally advantageous. Pseudocereals also contain proteins with a preferred amino acid composition [8]. This category includes buckwheat, amaranth and quinoa. For example, Föste et al. (2013) [47] used various buckwheat milling fractions, rice and corn flour and fermented buckwheat brans for gluten-free bread preparation. The specific bread volume, porosity and crumb texture can be improved by using buckwheat flour. Soya is a traditional gluten-free ingredient. Soy protein has a very advantageous amino acid composition, participates in interactions with other substances, binds water well and slows down the staling of bread. Legume flours from pea, chickpea, lupine, lentil and bean are rich in proteins with a high lysine content. They significantly affect the dough quality. Lupine and soya flour show emulsifying properties because of the lecithin content. Legumes reduce the glycemic index of food products. The disadvantage of legumes is their typical taste [35,48–50]. Recently, other flours, such as from nuts and seeds, have appeared in the range of bakery products. Typically, walnut flour and peanut flour are relatively expensive, but very suitable for some formulations. Fat in walnut flour or other nuts flours (except for coconut) contains polyunsaturated fatty acids, and nut proteins are also of very high quality due to their composition. Coconut flour binds water very well, too [51,52]. Cereal flours are combined in gluten-free bread and bakery products formulations with flour from other crops and with starches. A combination of cereals with legumes is nutritionally advantageous. Pseudocereals also contain proteins with a preferred amino acid composition [8]. This category includes buckwheat, amaranth and quinoa. For example, Föste et al. (2013) [47] used various buckwheat milling fractions, rice and corn flour and fermented buckwheat brans for gluten-free bread preparation. The specific bread volume, porosity and crumb texture can be improved by using buckwheat flour. Soya is a traditional gluten-free ingredient. Soy protein has a very advantageous amino acid composition, participates in interactions with other substances, binds water well and slows down the staling of bread. Legume flours from pea, chickpea, lupine, lentil and bean are rich in proteins with a high lysine content. They significantly affect the dough quality. Lupine and soya flour show emulsifying properties because of the lecithin content. Legumes reduce the glycemic index of food products. The disadvantage of legumes is their typical taste [35,48–50]. Recently, other flours, such as from nuts and seeds, have appeared in the range of bakery products. Typically, walnut flour and peanut flour are relatively expensive, but very suitable for some formulations. Fat in walnut flour or other nuts flours (except for coconut) contains polyunsaturated fatty acids, and nut proteins are also of very high quality due to their composition. Coconut flour binds water very well, too [51,52].

An alternative solution is the use of insect proteins, e.g., cricket flour which improves the texture of gluten-free bread [31]. An alternative solution is the use of insect proteins, e.g., cricket flour which improves the texture of gluten-free bread [31].

The use of proteins in the form of protein concentrates or isolates from different sources in gluten-free baked goods leads to the quality and nutritional profile improvement [15]. Through the comparison of plant- and animal-based proteins, Gorissen et al. [53] reported lower content of essential amino acids in plant-based protein isolates than in animal-based proteins. Differences in the composition of the amino acid spectrum of gluten-free raw materials are known, especially in the composition of essential amino ac-The use of proteins in the form of protein concentrates or isolates from different sources in gluten-free baked goods leads to the quality and nutritional profile improvement [15]. Through the comparison of plant- and animal-based proteins, Gorissen et al. [53] reported lower content of essential amino acids in plant-based protein isolates than in animal-based proteins. Differences in the composition of the amino acid spectrum of gluten-free raw materials are known, especially in the composition of essential amino acids. Cereal proteins

ids. Cereal proteins are deficient in lysine, and some cereals also in threonine and tryptophan. Therefore, from this point of view, it is recommended to combine different types of

ingredients makes easier to ensure the presence of other nutrients, such as vitamins or

minerals.

are deficient in lysine, and some cereals also in threonine and tryptophan. Therefore, from this point of view, it is recommended to combine different types of plant proteins and thus optimize the ratio of amino acids. The combination of different ingredients makes easier to ensure the presence of other nutrients, such as vitamins or minerals.

#### *3.2. Starch in Gluten-Free Dough and Products*

Starch, together with flours from gluten-free crops, is one of basic ingredients in glutenfree bread and bakery products. It is involved in the formation of the crumb structure, responsible for the volume and colour of the product. It is also used as a thickening, gelling, stabilizing, moisture retention and anti-staling agent [54]. According to Abdel-Aal (2009) [55], starch influences gluten-free products in three ways: it enhances crumb softness, ensures dough consistency and affects starch gelatinization. Starch is stored in starch grains of various sizes and shapes according to its plant source. Individual starches differ in their composition, size and shape depending on the plant species and the interactions between genes and environment [54]. The amylose starch fraction forms single chains, whereas amylopectin is branched with a significantly larger molecule. When heated in suspension/dough, the starch grains swell, are partially solubilized [56], and gradually lose their cohesiveness. Starch gelatinization occurs at a temperature of 50–70 ◦ C, when their chains are released, and a viscous solution is formed from the suspension. Upon cooling, the viscosity increases, new bonds are formed between the molecules and a gel is formed. During storage, the gel further changes, loses water and eventually retrogrades. Amylose retrogradation proceeds faster than the same process for amylopectin. It follows that by choosing the type of starch, it is possible to partially influence the staling of the bread [54]. Starch behavior may be affected by bound lipids.

Native starches are the most commonly used in gluten-free products, e.g., potato, corn, rice and tapioca, and pea starch has also appeared. Specially prepared gluten-free wheat starch is also used for its properties ensuring an optimal bread texture [15,57]. Modified starches are produced for food purposes have a wide range of physical properties according to the purpose they are used. Starches can be modified by heating of the starch solution or by heating in the dry state; the heating can be performed by drying or extrusion. Chemical modification of starch is also possible [58]. For gluten-free products, starches with good water absorption and slow retrogradation are selected. Specially modified starches are suitable for frozen products. A new modification is the so-called superheated starch, prepared by heating the starch suspension to high temperatures until dissolved and then cooling to form a spreadable gel with a creamy consistency [59,60]. Additionally, various types of banana flour can be applied as well as the direct use of bananas in the dough [61].

There are significant differences in granular structure among various types of starches, which affect their ability to produce high quality gluten-free baked goods. When the baked goods are based on starch, they show higher volume, lower hardness and a lighter crust since Maillard reactions are reduced. The starch addition results in softer, and resilient crumbs. The type of starch also influences the quality of the baked goods. For the specific gluten-free formulations different mixtures of flours and starches must be optimized [30,41].

Preventing the retrogradation of starch and thus prolonging the shelf life of gluten-free bread and bakery products can be achieved in several ways:

#### 3.2.1. Use of Enzyme Preparations

The most common enzyme preparations use amylases, which improve the colour of crumbs and support the production of flavors. Amylases partially degrade amylopectin and thus modify the starch recrystallization process [62–64]. Transglutaminase improves the dough viscoelasticity and decrease crumb hardness, and cyclodextrinase also enhances dough viscoelasticity, leading to improvement in shape index and crumb firmness [65].

Cyclodextrin glycosyltransferases form cyclic structures from starch with different affinities for water outside and inside the ring. Lactase and tyrosinase create crosslinks of non-starch polysaccharides with proteins with the use of phenolic substances [25]. Oxidases such as lipoxygenase, sulfhydryl oxidase, glucose oxidase and peroxidase stabilize the dough; for example, glucose oxidase added to rice bread improved volume and reduced stiffness. Proteases and peptidases improve the interaction between protein molecules and starch and reduce the viscosity of the dough [31,66]. Microbial transglutaminase, which forms covalent bonds between the free epsilon-amino group of lysine and the amide group of glutamine, is used to promote the formation of a spatial network of gas bubble trapping molecules. Transglutaminase supports the rheological and viscoelastic properties of the dough [67–71]. Silva et al. (2020) [72] tested gluten-free bread from red rice flour and cassava flour, with the addition of transglutaminase and chitosan at concentrations of 0%, 1% and 2%. Bread with chitosan and transglutaminase showed lighter brown coloration because of incomplete Maillard reaction and low specific volumes, probably related to chitosan interference with yeast fermentation. With the use of chitosan, viscosity increased. Bread containing chitosan had a lower rate of staling due to water retention.

The use of enzymes influences the quality of the gluten-free baked goods, and the effect depends on the type of the flour used. Some enzymes have positive effects on product volume and delay staling [30].

#### 3.2.2. Use of Hydrocolloids

The application of hydrocolloids is crucial for the quality of gluten-free bread. Hydrocolloids swell and form a gel. This heated gel thickens the mass of dough forming the walls of gas bubbles, preventing the loss of gas released during whipping, leavening or from raising agents. After baking, hydrocolloids stabilize the crumb structure, bind water, and prevent rapid starch retrogradation. They stabilize the product during freezing. Due to the higher water binding, the recipes with the addition of hydrocolloids contain higher doses of water [32,73].

Vegetable gums (guar gum, locust bean gum, arabic gum, tara gum, carob, konjac gum), beta-glucans, pentosans and arabinoxylans, cellulose derivatives (methyl cellulose MC, carboxymethyl cellulose CMC, hydroxypropylmethyl cellulose HPMC), microbial exopolysaccharides (xanthan, dextrins) and seaweed polysaccharides (agar, carrageenans) are used to produce gluten-free bread and bakery products [42,74–76]. Flax flour or ground chia seeds are sometimes used to increase viscosity.

Cellulose derivatives, especially CMC and HPMC, are among the most used hydrocolloids in gluten-free dough. They can interact with other raw materials in the matrix. They are most often combined with other types of thickeners, proteins, and emulsifiers [66]. For example, Liu et al. (2018) [77] compared the effect of HPMC, CMC, xanthan gum and pectin on the behavior of steamed potato dough. The addition of 2% HPMC increased mostly the specific volume of bread and porosity and reduced the stiffness of the crumb by almost 29%. The addition of hydrocolloids significantly reduced the content of both readily available and slowly available starch and, conversely, increased the content of resistant starch. Model experiments with HPMC and rice flour on Mixolab examined the effect of HPMC dose (1–3%) and water dose (90–110%) on the rheological properties of gluten-free dough and crumb quality. The optimal dose is 2.2% HPMC and 110% water) [78]. Lazaridou et al. (2007) [79] showed that 1% CMC and 2% pectin led to breads with improved breads volume, porosity and crumb elasticity. With the use of HPMC, Hager et al. (2013) [80] observed an increased volume in corn and teff breads, a decreased size of rice breads and a positive effect on the crumb hardness of each bread.

Salehi (2019) [41] dealt with the application of HPMC, CMC and other hydrocolloids in rice flour dough. HPMC in combination with carrageenan forms a softer crumb. To slow down the aging of bread, the addition of CMC or HPMC 0.1–0.5% is recommended.

Kaur and Chopra (2018) [61] tested 74% corn starch bread with tapioca, rice flour and 2.2% HPMC. Belorio and Gómez (2020) [81] tested the use of different types of hydrocolloids (HPMC, xanthan and psyllium) in rice and corn bread and the effect of water levels. The water dose has been optimized to form a thermoreversible gel that provides a sufficient volume of bread. For corn bread with added HPMC, the optimal hydration was 80%; for rice bread with HPMC, the hydration was higher—100%.

Fruit and vegetable pomace containing fiber and antioxidants can also be used [9,41,82]. Djeghim et al. (2021) [9] observed the addition of various by-products with gluten-free bread formulations based on corn and chickpea flours (2/1 *w*/*w*)—orange and apple pomace, tomato peel, pepper peel, prickly pear peel and prickly pear seed peel on the dough rheology and properties of gluten-free breads. They found out that the addition of the above-mentioned by-products significantly improved the specific volume of gluten-free bread, with values increasing from 1.48 to 2.50 cm3/g, and increased the maximum dough height, the total CO2 production and CO2 retention coefficient.

The effect of apple, orange and carrot pomace powders, on dough rheology and quality characteristics of the rice sweet bakery were studied by Kirbas et al. (2019) [10]. With an increase in the content of pomace powders, the dough elasticity, specific gravity and apparent viscosity increased. The addition of pomace powder increased crumb hardness and decreased the specific volume of the rice-based sweet bakery products. The addition of 5% of orange pomace powder had the highest acceptance scores with respect to the colour, flavour, texture, appearance and acceptability of the products.

The comparison of breads and bakery products prepared from gluten-free flour alone and with various hydrocolloids (gums) showed that the incorporation of hydrocolloids led to a significant improvement in the texture, volume, color, appearance, flavor and overall acceptability. Different hydrocolloids have slightly different effects on rheology, texture and other properties, thus affecting the resulting quality of various types of gluten-free breads and bakery products. For example, xanthan gum is able to maintain unchanged texture parameters during storage; the addition of xanthan, carrageenan and guar gums decrease dough extensibility, whereas arabic gum and HPMC lead to increased extensibility [41]. HPMC is preferred to other hydrocolloids since it provides gluten-free products with appropriate physical characteristics, higher specific volumes of products and better sensory properties [30]. Moreover, the use of hydrocolloids is the easiest way to raise the content of dietary fiber in gluten-free baked goods [8].

#### 3.2.3. Microbial Fermentation in Gluten-Free Bread Production

The use of sourdough is a traditional procedure in conventional baking technology. In the preparation of gluten-free bread, starter cultures began to be applied later, because gluten-free raw materials have a specific composition different from rye flour; therefore, the classical culture of rye sourdough bacteria and yeasts may not grow sufficiently in gluten-free substrates. Although it is possible to gradually "dilute" rye flour with glutenfree raw material during repeated fermentation so that the proportion of rye is reduced to a minimum value, such a process would take a long time, and there would still be the danger of the presence of gluten traces [83]. Therefore, suitable strains of microorganisms capable of fermenting rice, buckwheat, sorghum or corn flour are sought. The choice of a suitable starting culture will significantly affect the resulting properties of the dough and the product. Additionally, the oilseed, chia and flaxseed sourdoughs can be used [84]. During fermentation, the dough is acidified. At the same time, the enzymes naturally contained in the flour are also activated and break down high molecular weight substances, and thus make them more accessible. The activity of the cereal grain's own enzymes is combined with the action of microbial enzymes. Substances affecting the taste and smell of the products are formed. Fermentation increases the swelling of carbohydrates and improves the viscoelastic properties of the dough. The fermentation products include organic acids with a predominance of lactic and acetic acids, but some strains also produce propionic acid [85–89]. These acids significantly increase the shelf life of the bakery

products. For example, Kaur and Chopra (2018) [61] deal with the use of teff flour and rice sourdough as a possible combination. Bacterial strains of sourdough microflora, for example *Lactobacillus reuterii* or *Weisella cibaria*, are able to produce the exopolysaccharides fructan, levan, dextran or reuteran. These polysaccharides naturally increase the viscosity of the dough and thus contribute to the formation of the product texture. The presence of these polysaccharides reduces the hardness of the crumb, improves its porosity, and slows down the staling of the bread [25,31]. Additions of dried sourdough are also being applied. The advantage of dried sourdough is its standardized quality, the disadvantage is the possible inactivation of living microbial strains during the drying process [90]. A nontraditional sourdough using *Lactobacillus sanfranciscensis* for fermentation of chia, quinoa and hemp flour to produce gluten-free corn/rice bread was tested by Jagelaviciute and Cizeikiene (2021) [87]. This sourdough showed a decreased pH, specific volume and rate of bread staling and, on the other hand, increased bread porosity compared with bread made only with chia, quinoa or hemp seed flour. The use of non-fermented chia and hemp flour increased the firmness and the rate of bread staling, whereas use of non-traditional hemp and quinoa sourdough reduced the rate of bread staling.

The use of sourdough in gluten-free baked goods leads to products with improved technological and nutritional properties [23], which are softer, tend to stale more slowly and have a delayed mould spoilage rate and thus a prolonged shelf life. Sourdough also brings nutritional benefits because it makes minerals more available and its presence leads to the production of exopolysaccharides, which function as hydrocolloids [30].

#### *3.3. Gluten-Free Bread and Bakery Products Spoilage*

Because gluten-free bread contains more water, it has a higher water activity. It is not usually baked using sourdough; therefore, the possibility of infestation by mould and other microorganisms is a significant problem. Mould species involved in bread and bakery products spoilage have been identified; they are represented by fungi of the genus *Penicillium*, *Cladosporiun*, *Neurospora or Rhizopus*, *Aspergillus*, *Fusarium*, *Mucor*, *Endomyces*, *Chrysonilia*, etc. [91,92]. Mould contamination leads to off-flavour generation and mycotoxins production, endangers human health and causes economic losses and consumer dissatisfaction [93]. Baked goods can also be attacked by yeasts, e.g., of the genera *Pichia*, *Candida* or *Torulaspora* and bacteria of the genus *Bacillus* (e.g., *B. subtilis*, *B. amyloliquefaciens*, *B. licheniformis*, *B. cereus*), with *B. amyloliquefaciens* as the main species causing rope spoilage [91,94]. The same microorganisms cause spoilage of gluten-free products [91].

To ensure the shelf life of bread and bakery products, various physical methods can be applied. Ultraviolet light, infrared treatment, microwave heating and ultra-high-pressure treatments can be used for bakery products preservation [95]. The disadvantages include low penetration ability of ultraviolet light, higher cost of infrared treatment or condensation problems asssociated with microwave heating [96]. The application of modified atmosphere or gamma irradiation alone does not give advantageous outputs, in practice, more types of protective factors will have to be combined. The use of antimicrobial compounds extracted from plants—biopreservation—provides very promising results and is considerably efficient in slowing down the growth of fungi [93].

For the distribution of finished bread, buns and other bakery products, it is necessary to choose packaging material with good barrier properties. It is possible to directly use packaging with antimicrobial and antioxidant properties, ethanol emitting or carbon dioxide emitting packaging, moisture absorbing packaging or packaging ensuring chemical preservation of the product, for example by potassium acetate, calcium propionate or potassium sorbate (Figure 4) [96–98]. Barrier packaging using oxygen absorbers and the use of modified active packaging (MAP) is a way to extend the shelf life of products. Modified atmosphere packaging [99] is now commonly used for food packaging. The use of natural essential oils is also tested for wheat bread and other bakery products' shelf life extension; the use of this treatment can be proposed in gluten-free bakery products, too [91,100–102].

**Figure 4.** Innovative food packaging systems adapted and modified from [96,101]. Synergies between particular packaging types are marked with brackets.

The use of gluten-free sourdough improves the microbial stability of bread and bakery products as well as their taste [103,104] and represents another tool of biopreservation. In addition to lactic acid, sourdough bacteria also produce acetic and propionic acids with antimicrobial properties. Axel et al. (2016) [103] found out that the addition of *Lactobacillus reuteri* R29 containing sourdough extended the shelf life by 2 days for rice and quinoa bread compared with controls. Similar results were achieved with *Lactobacillus amylovorus* DSM19280-inoculated quinoa sourdough bread [104]. Some lactic acid bacteria in sourdough also form bacteriocins directed against competing microorganisms, and thus improve the shelf life of bakery products [105–107].

Many possibilities exist as to how to avoid spoilage in gluten-free baked goods. To prevent spoilage of these products and prolong their shelf life, it is necessary to combine several types of protective measures and thus use their synergistic effect.

#### *3.4. New Technologies in Gluten-Free Dough and Bread Preparation*

Recently, several technological processes have been tested to influence the properties of gluten-free dough and improve baking. Treatment of the dough with a high pressure (pascalization) of 100–1000 MPa reduces the temperature of starch gelatinization and change the properties of proteins, including crosslinking. Starch swells and gelatinizes without granules degradation; the extent of swelling depends on the intensity and length of pascalization. This changes the viscoelastic properties of the dough, increases its flexibility, but sometimes also its viscosity [25,31]. The experiments were also performed using ultrasound and micromilling to reduce flour particles. However, no positive effects on bread volume and porosity have been found [31,45].

The properties of gluten-free dough can also be influenced by heating of dry ingredients before dough preparation. Protein denaturation and partial gelatinization of the starch occurs, which increases the flexibility of the dough and the ability to retain gas. The dough viscosity, resistance and stiffness increase, as well as the dough volume [31,108]. Microwave heating was used to heat the rice flour with a moisture content of 20–30%. Proteins denatured after opening their three-dimensional structure. The specific volume and elasticity of bread has significantly improved, and the staling of bread has slowed down [109]. The "Instant controlled pressure drop" technology based on the heating of gluten-free flour for a short time under reduced pressure was tested on a mixture of rice and bean flour. The temperature was in the range of 100–165 ◦C, and the pressure of

5 kPa and the heating time of 20–60 s were used. The appropriate heating conditions of gluten-free raw materials were determined so that the bread baked from the heat-treated mixture resembled the control wheat bread [110].

Microwave and infrared technologies are also tested in the gluten-free bread baking process. Microwave heating would be cost effective and fast, but the resulting product had low volume, a solid crumb and was rapidly subject to the staling process. The poorer energy penetration into the bread mass is the disadvantage of infrared heating technology, but the resulting product was better sensory evaluated. The so-called "jet-impingement" using hot air convection heating on the surface of the bread was also tested. Homogeneous heat transfer occurred, but the process was energy consuming. The disadvantage was the formation of a solid crumb and dense texture, loss of water and aroma. The starch gelatinization was not complete and starch digestibility was thus decreased. The combination of both methods was recommended for this reason [111,112].

Ohmic heating was the other tested technology. The food material was heated up by its resistance during the electric current passage. The advantage of this process would be the homogeneity of the heating [66]. Another possibility of heating is the partial baking under the reduced pressure. In the first stage, bread was baked at a normal pressure at 180 ◦C, and after the formation of a solid crust preventing the bread collapsing, it was baked at a pressure reduced to 60 kPa. No changes in bread volume or stiffness were observed, but product moisture was lost and the crust colour was affected. After vacuum baking, other types of starch crystals were formed in the bread and the bread tended to grow stale more slowly [113].

The new technologies improving the gluten-free dough and products quality and shelf life have been still evolving. Some of them have proven to be appropriate. It has been shown that some promising methods do not give completely satisfactory results. Other technologies need to be combined. Research in this area will certainly continue. In practice, however, the economic side of the process would be crucial. Gluten-free bakery products should be not only of high quality, but also be affordable for customers.

#### **4. Clean Label vs. Gluten-Free Products**

Green label means that the products do not contain additives. New consumer demands are moving in this direction [114]. However, in gluten-free products, this is very difficult because of the poor baking properties of gluten-free raw materials, missing texturing properties of wheat gluten, etc. Only some of the used additives are appropriate for clean label products. A solution can be achieved using reformulations [115]. To mimic the gluten function hydrocolloids are used which are all classified as food additives and have their E numbers in accordance with EU regulation No. 1333/2008 [116]. Psyllium and beta-D-glucan can be used as gluten replacements in clean label formulations [117]. As far as enzymes are concerned, there are some exceptions they do not have E numbers.

Chia seeds, buckwheat flour and flax flour absorb water very well; lupine flour or soy flour can serve as natural emulsifiers, thus they do not have E numbers. An appropriately chosen starting culture for the preparation of sourdough improves the shelf life and taste of the bakery products, but can also modify the rheology properties of the dough due to the production of exopolysaccharides [118]. The sourdough can also be used in clean label formulations. Flours modified by heating or extrusion are tested instead of E numbers labeled thickeners [119].

#### **5. Conclusions**

Gluten intolerance is becoming more common in the population, and patients with this intolerance must follow a gluten-free diet. Bread and other bakery products are staple foods and pose a problem in a gluten-free diet due to their short shelf life and the need to replace gluten. Naturally gluten-free cereals and pseudocereals, but also milled legumes, seeds and nuts, are being increasingly used for the preparation of gluten-free baked goods. The additions of hydrocolloids are traditionally used in gluten-free product formulations. Recently, amylase, transglutaminase and other enzymatic preparations have been applied to bakery gluten-free mixtures. The use of fermentation for native sourdough preparation or the addition of dried sourdough to dry baking mixes improves the taste and shelf life of gluten-free bread. The quality of gluten-free dough and bread is improved by the addition of modified starches and protein isolates or concentrates. Rheological properties of gluten-free dough, the texture and sensory quality of gluten-free bread also improve the utilization of sourdough with specific microbial strains selected for the gluten-free raw materials. Newly tested baking technologies could improve the texture and slow down the staling of these products. New packaging materials and packaging methods can affect the shelf life of gluten-free bread and pastries. In the future, it is possible to anticipate the use of other non-traditionally processed gluten-free raw materials as well as new technologies for the sourdough preparation and bread baking.

**Author Contributions:** Both authors contributed to the article equally, for 50% and 50% in data searching, writing—original draft preparation, writing—review and editing, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Agriculture of the Czech Republic, institutional support RO0318, METROFOOD-CZ project MEYS, grant number LM2018100 and METROFOOD-PP (EU Horizon 2020 Grant agreement No. 871083).

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

## *Review* **The Role of Hydrocolloids in Gluten-Free Bread and Pasta; Rheology, Characteristics, Staling and Glycemic Index**

**Alina Culetu 1 , Denisa Eglantina Duta 1 , Maria Papageorgiou 2 and Theodoros Varzakas 3,\***