gluten free bread research paper

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• Published: 01 May 2019

Recent practical researches in the development of gluten-free breads

• Hiroyuki Yano   ORCID: orcid.org/0000-0002-0910-854X 1  

npj Science of Food volume  3 , Article number:  7 ( 2019 ) Cite this article

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

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Introduction.

The aroma emanating from a bread bakery is unmistakably alluring. The flavor and crunchy texture of wheat breads sharpen our appetite and satisfy our basic human cravings for comfort as well as nutrition. Indeed, human beings are so enchanted by bread that it is much more than a “staple food”; it has been called “the staff of life”. Breadmaking has a long and fascinating story. 1 , 2 , 3 , 4 It is generally accepted that breadmaking dates back to the New Stone Age, from 8000 to 10,000 BC, and originated around the Fertile Crescent and consisted of emmer and einkorn wheat grains. 1 At first the grains were consumed as porridge. Then, grains that had been hand-crushed using knocking stones were mixed with water and baked on a heated stone with a cover of hot ash, resulting in an unfermented, flat bread. Later, around 6000 BC, people in southern Mesopotamia started using sourdough, 5 speculated to have been developed accidently in an abandoned mixture of flour and water. This first leavened bread dough, which contained fermentation gas, swelled up in the baking process. In ~3000 BC, the Egyptians improved bread by adding yeast, developing what would become the prototype of modern bread. They dehulled and milled wheat grains using saddle querns, the most ancient type of quern stones, 6 which were later replaced by rotary querns and are used even today. Breadmaking and beer production in Egypt are closely related and are considered evidence of a high degree of civilization. 7 Bread was made not only with flour prepared from raw grains, but sometimes also with malt (germinated grains). Moreover, water with a blend of cooked and uncooked malt was used in brewing. The mixture was strained free of husk before inoculation with yeast.

The precise origin of bread has still not been determined. Recent reports show archaeobotanical evidence that the origins of bread date back to 14,400 years ago. 8 Progress in archaeology will eventually clarify the origin of bread, along with some sense of how bread fits into the larger culture of ancient civilizations. Wheat bread is now one of the most representative food in the world. A unique property of wheat gluten realizes bread with high quality. However, some genetically predisposed people cannot eat wheat bread, because gluten causes harmful reactions to them. In this short review, we will summarize the gluten-dependent swelling mechanism of wheat bread and the recent scientific effort to make bread without gluten.

Modern wheat breadmaking

Simply stated, breadmaking is composed of three steps: mixing/sheeting, fermenting, and baking processes. 9 In the mixing process, wheat flour, water, yeast, sugar, salt, oil, and other components are mixed and kneaded. Here, the ingredients are blended homogeneously and hydrated, resulting in the development of the all-important gluten network. 10 Gluten is made from two major wheat proteins together comprising 85% of wheat endosperm protein: gliadin and glutenin. Kneading of wheat dough promotes the hydrogen bonding and disulfide cross-linking interactions of these proteins, eventually producing a viscoelastic and highly conformational protein network termed “gluten”. 11 Yeast grows fast in the dough, feeding on supplemental sugar, until it consumes all available oxygen. Then, it shifts metabolism from aerobic respiration to anaerobic fermentation. In the subsequent fermentation process, yeast generates fermentation gas, mainly composed of carbon dioxide and other components, such as ethanol:

In wheat dough, the gas is confined in the continuous “gluten matrix”, 12 which is composed of the viscoelastic gluten network and other components, such as starch granules and water (Fig. 1a ). Thus, in the beginning of the fermentation process, many small gas cells are produced throughout the dough, like so many small balloons. As the fermentation proceeds, each small gas cell grows bigger, and the dough rises. In the following baking process, the gas cell inflates further by heat, resulting in the expansion, namely, “oven spring” of the dough. 13 The starch molecules are gelatinized by heat, so that the gluten matrix forming the envelopes of the “balloons” become hardened, thus constructing the stable crumb framework. 14 Concurrently, the crust, or surface of the bread dough, is hardened as well as browned by the Maillard reaction between the sugars and amino acids. 15 Finally, the breadmaking is completed, emitting a fresh aroma. 16

Comparison of the swelling mechanism ( a ) and appearance ( b ) of fermenting wheat dough and additive-free, gluten-free (GF) rice batter

The preparation of ingredients, especially flour, is also a critical step. Wheat grain is composed mainly of three parts: the endosperm, germ, and bran. 17 In the endosperm, which is the major constituent of the polished grain, starch granules are embedded in a protein matrix. 18 Wheat flour is produced by grinding whole-wheat grains or polished ones mechanically. 19 Impact mills, such as hammer mills and pin mills, accomplish particle size reduction by exposing seeds to a set of rotating hammer or pins that fracture the seeds, while roller and stone mills compress the seeds between two hardened surfaces. 20 During the milling of wheat grains, a portion of the starch granules are mechanically damaged. 21 The extent of the damage depends on wheat variety (hard or soft type) as well as milling conditions. In the mixing and fermentation steps of breadmaking, damaged starch accelerates the absorption of water to the starch granules, resulting in the activation of local amylases, leading to the degradation of starch molecules into dextrin and maltose. 22 Consequently, yeast activity and the final bread volume is increased. However, excessive starch damage produces wet or sticky dough and bread with poor quality. Thus, control of flour quality in terms of the starch damage is critical in the milling industry. 23

In other words, intact and damaged starch granules each have their respective role in the making of wheat bread—and, as we will show, in rice-flour breads as well. In the case of wheat dough, intact starch granules constitute the gluten matrix, while damaged ones activate fermentation. Generally, the extent of starch damage in commercially available wheat flours is 10–15%. 19

Social demand for gluten-free food

Gluten intolerance.

While the unique viscoelastic property of gluten realizes wheat bread with high quality, some people choose to or must follow a gluten-free diet. Recent reviews well summarize the background and status quo of gluten-free diets, 24 , 25 so only the outline will be mentioned here. Gluten intolerance includes autoimmune celiac disease (CD), wheat allergy, and non-celiac gluten sensitivity (NCGS). Celiac disease is an autoimmune disorder caused by genetic as well as environmental factors. 26 In CD patients, ingestion of gluten leads to small intestinal damage, typically leading to malabsorption. Its prevalence in the United States and Europe is estimated to reach about 1%. Gluten protein has protease-resistant regions in its structure. 27 Digestion of gluten in the human gastrointestinal tract generates “pathogenic” peptides that occasionally reach the lamina propria, where the peptides are deamidated by local transglutaminase. 28 The modified gluten peptides have a higher affinity to human leukocyte antigen (HLA)–DQ2 as well as HLA–DQ8 molecules, 29 which are present only in the small percentage of people carrying the HLA–DQ2 or the DQ8 haplotype. 30 This bonding results in the presentation of the gluten peptides to T cells, thereby triggering further malignant immune response in those with CD. In addition, tissue transglutaminase cross-links covalently to gliadin molecules. The protein complexes with new epitopes are considered to trigger the primary immune response as well. Antibodies against tissue transglutaminase are characteristic of CD. 31

In contrast, food allergy to wheat is characterized by T helper type 2 (Th2) activation, which can result in immunoglobulin E (IgE) and non-IgE-mediated reactions. 32 The IgE-mediated wheat allergy reactions usually occur immediately after contact of wheat, and are characterized by the occurrence of wheat-specific IgE antibodies in serum. Ingestion of wheat causes food allergy, while inhalation of wheat causes respiratory allergy to genetically predisposed individuals. A food allergy to wheat may cause a life-threatening reaction, such as anaphylaxis and wheat-dependent, exercise-induced anaphylaxis. 33 In contrast, repetitive exposure to wheat flour may cause baker’s asthma or rhinitis, mostly characterized as occupational allergic diseases. 34 Non-IgE- mediated food allergy reactions to wheat usually occur hours or even days after ingestion of wheat products and are characterized by chronic eosinophilic inflammation of the gastrointestinal tract. 35 There is a variability among reports of wheat allergy prevalence due to the differences in the diagnostic criteria, methodology, age, and geography. 36 The prevalence of wheat allergy is estimated to be 0.9% in the United Kingdom (based on questionnaire response), 37 3.6% in the United States (based on measurement of anti-wheat-specific IgE antibodies), 38 and 0.2% in Japan (based on a combination of questionnaire-based examination, skin prick test, and serum omega-5 gliadin-specific IgE test). 39

Non-celiac gluten sensitivity (NCGS) is a recently proposed, increasingly recognized clinical condition in patients in whom celiac disease and wheat allergy have been ruled out. It is characterized by intestinal and extra-intestinal symptoms triggered by the ingestion of gluten-containing foods. 40 Due to the lack of a reliable biomarker, confirmation of an NCGS diagnosis relies only on a double-blind placebo-controlled (DBPC) gluten challenge. 41

So far, a gluten-free diet is the only safe and effective treatment for the above conditions of gluten intolerance. 32

Gluten-free “lifestylers”

Demand for gluten-free foods is not limited to the gluten-intolerant population. Although it is not clear whether a gluten-free diet is beneficial for one’s health, some gluten-tolerant consumers believe that gluten-free food products are simply healthier. 42 , 43 This can be partly explained by a kind of “health halo” effect, making consumers believe that products with “free-from” label are healthier overall. 44 Besides, some popular books by bestseller authors, athletes, and celebrities have encouraged a gluten-free diet. An online questionnaire survey demonstrated that 41% of non-celiac athletes, including Olympic medalists, follow a gluten-free diet 50–100% of the time, and that adoption of the diet in most cases was not based on a medical rationale and may have been driven by the perception that gluten removal provides health benefits and an ergogenic edge. 45 Approximately 13% of young adults are reported to value gluten-free food; this population is more likely to engage in other healthy dietary behaviors, such as eating breakfast daily and eating more fruits/vegetables while simultaneously pursuing questionable behaviors, such as using diet pills to control weight. 42

A double-blind randomized study found that the supposed health benefit of a gluten-free diet has no evidence base in individuals who do not have celiac disease or irritable bowel syndrome, demonstrating that gluten is unlikely to be the culprit for gastrointestinal symptoms or fatigue in otherwise healthy individuals. 43 Moreover, commercially available gluten-free food products tend to contain ingredients with less diversity and less nutritional quality compared with their gluten-containing counterparts. 46 , 47 Other studies claim that despite recent improvements in the formulation and availability of gluten-free foods, they still are less available and more expensive than gluten-containing versions. 48 They generally have adequate levels of fiber and sugar, but lower levels of protein and higher levels of fat compared with their gluten-containing counterparts. 48 Also, very few gluten-free foods are fortified with micronutrients. 48

The gluten-free products market was valued at USD 4.18 billion in 2017 and this is projected to reach USD 6.47 billion by 2023, at a compound average growth rate of 7.6% during the forecast period. 49 The gluten-free diet has become the mainstream rather than just supporting a niche market.

Developments of gluten-free breads

As mentioned in the previous sections, demand for the development of gluten-free foods is growing. 50 Much of the focus is on bread products, as bread is an important staple food. Rice is considered a suitable substitute for wheat, as it is available worldwide and is less allergenic. So, development of rice-based gluten-free breads is the main topic of this review. It is not easy to make bread without using wheat flour or gluten, as bread’s quality depends on the properties and functionality of gluten. 25 In a wheat flour dough, the gluten matrix, composed mainly of the protein network of gluten, starch granules, and water (Fig. 1a ), encloses the fermentation gas, making small “balloons”. Thus, the dough rises as the fermentation proceeds. On the other hand, hydration of flour from gluten-free cereals, such as rice, results in a runny “batter” rather than viscoelastic “dough” as their proteins do not possess the network-forming properties typically found in gluten. 51 Therefore, the fermentation gases rise to the surface while starch granules and yeast settle. 52 Generally, a gluten-free batter without a thickening agent, such as hydrocolloids, becomes foamy. 53 , 54

Several efforts have been made in the development of gluten-free breads. Typical gluten-free breads contain hydrocolloids (e.g., xanthan gum, guar gum, etc.) which increase the viscosity of the liquid phase, keeping the starch granules, yeast, and gas bubbles suspended in the fermentation process. 52 , 55 The subsequent baking process gelatinizes the starch and hardens around the hydrocolloid membrane surrounding the air bubbles to set the crumb structure. As a surface-active hydrocolloid, hydroxypropyl methylcellulose (HPMC) behaves somewhat differently. It has hydrophobic methyl ester/hydroxypropyl groups in addition to hydrophilic cellulose regions. Thus, HPMC stays at the gas/liquid interface, uniquely stabilizing the bubbles and preventing coalescence. 52 , 56 Moreover, as HPMC is thermoreversible, 57 it also helps harden the bubble membrane in the baking process. 58

Another recent approach includes enzymatic treatment of gluten-free batter. 51 Transglutaminase (EC 2.3.2.13) catalyzes the acyl-transfer reaction between primary amino groups on protein-bound lysine residues and γ-carboxyamide groups on protein-bound glutamine residues. 59 Thus, transglutaminase is capable of introducing covalent cross-links between proteins. 60 The protein cross-linking ability has been shown to transform weak gluten into a strong gluten, with measurable effects on rheological behavior. 61 The addition of transglutaminase, along with HPMC, to a gluten-free rice batter resulted in its improved elastic and viscous behavior, as well as a higher specific volume and softer crumbs in the resulting bread. 62 The improvement in the viscoelastic properties of the rice batter appeared to be associated with the enhanced capability of the rice flour to retain the carbon dioxide produced during proofing. The quantitative decrease of free amino groups of proteins suggested that this improvement was due to the cross-linking of protein, that is, the generation of a gluten substitute, supplementing the role of HPMC in the baking of rice bread. 62 Microstructure analyses of a rice-based bread fortified with skim milk or egg powder using confocal laser-scanning microscopy (CLSM) verified that addition of transglutaminase promoted the formation of a protein network in the gluten-free bread that mimicked the gluten network in wheat breads. 63 The networking efficiency of transglutaminase depends on both the correct protein substrates and the level of enzyme addition. Thus, formation of the appropriate protein network under the right conditions should improve the overall quality of gluten-free bread by enhancing loaf volume and crumb characteristics, as well as appearance.

Improvement of the gas-retaining capability of gluten-free batter using protease, a seemingly paradoxical strategy for cross-linking, is also in progress. Protease has actually been used to weaken wheat dough by cleaving a portion of the gluten network. 64 However, treatment of a brown rice batter with bacterial protease improved bread quality by significantly increasing the specific volume while decreasing crumb hardness and chewiness. 65 CLSM images of the bread crumbs suggested that the gelatinized starch phase was the main structure component in the protease-treated bread. Thus, protease may partially degrade the large macromolecular protein complex embedding starch granules, 66 , 67 resulting in improved continuity of the starch phase as well as better rheological properties of the batter. Treatment of rice batter with a protease from Aspergillus oryzae increased its viscosity and resulted in bread with a high specific volume. Optical microscopic observation of the batter suggested that partially degraded protein, possibly glutelin, and starch granules formed aggregations containing voids. 54 This fine network of interlinked protein‒starch aggregates resulted in gas cell stabilization. 54 Proteases are mainly categorized into four classes based on the catalytic mechanism: metallo, serine, cysteine, and aspartyl proteases. 68 Comparative analyses of the proteases 69 , 70 demonstrated that metallo, serine, and cysteine proteases, but not aspartyl protease, are effective additives for improving the quality of gluten-free rice breads.

Application of the redox regulation

Addition of glutathione, a ubiquitous natural peptide, facilitated the deformation of rice batter, thus increasing its elasticity in the early stages of bread baking and increasing the volume of the resulting bread. 53 , 71 Below, we would like to introduce briefly how glutathione can be used in making gluten-free rice bread. The disulfide bond is a cross-link between two cysteine residues and plays an important role in the structure/function of proteins. 72 Redox regulation, control of reduction/oxidation of the disulfide bonds, as well as phosphorylation are the two major post-translational modifications of proteins. 73 Thioredoxin (Trx), 74 a small 12 -kDa protein, and glutathione, 75 a natural tripeptide, play central roles in the redox-dependent regulatory mechanisms.

Trx reduces the disulfide bond of its target protein specifically. In the reactions below, oxidative status is abbreviated as “OX” and reduced status is abbreviated as “RED”:

Glutathione (GSH) is a tripeptide with a free SH group. Two molecules of glutathione occasionally cross-link with an intermolecular disulfide bond to make “oxidized” glutathione (GSSG). Glutathione’s reaction occasionally entails glutathionylation (GL): 76

Redox regulation has been a key target of Dr. Bob Buchanan’s laboratory, University of California, Berkeley, after he clarified the Trx-dependent regulatory mechanism in photosynthesis. 77 , 78 In the proteomic analyses of plant biochemistry mostly performed by the Berkeley group, 79 , 80 , 81 , 82 we have found that redox regulation occurs in many aspects of plant life and plays critical roles in plant biology: seed germination/maturation, photosynthesis, defense against oxidative stress/pathogens, and others. 83 Then, thinking in the opposite direction, modification of the disulfide bonds in biology, that is, artificial activation of the redox regulatory mechanism, might lead to the production of a new, useful plant. Following this hypothesis, overexpression of Trx in plants was first tried in the starchy endosperm of barley. 84 The transformant germinated earlier than the wild type. Also, enzymes in charge of starch mobilization appeared earlier. As fast germination of barley seeds reduces the production cost and improves the quality of beer, 85 the results suggest the practical utility of Trx transformants. Conversely, underexpression of Trx in white wheat seed has been tried. White wheat has received increasing attention, as it is naturally white and needs no bleaching for uses, such as breadmaking. However, white wheat grains tend to germinate on the spike before harvest. 86 The preharvest sprouting (PHS) reduces the crop yield as well as the quality of the seeds and the flour. Rainfall or high humidity in the grain-filling season leads to PHS, and causes farmers significant financial losses. 87 Suppression of Trx in the starchy endosperm led to improved PHS resistance in the transformants 88 without affecting the crop yield or flour quality. 89

These two findings reported by the Berkeley group are the first discovery that control of Trx expression, that is, artificial redox regulation, affects the physiological processes of plants. Although risk assessment of genetically modified organisms (GMOs) is a critical issue, 90 the characteristics of these and other trial model plants provide the possibility of the industrial application of redox regulation. 91

More recently, we have sought to use this strategy to enable rice batter to confine fermentation gas. Glutathione was added to rice batter in an attempt to transform the intramolecular disulfide bonds of rice proteins into intermolecular disulfide bonds and eventually form a gluten-like network. Both reduced glutathione (GSH) and oxidized glutathione (GSSG) were found to be successful in swelling gluten-free rice batter and bread. 53 , 71 However, contrary to our expectations, analysis of the proteins revealed that no gluten-like protein network was formed. In contrast, microstructure and biochemical analyses suggested that glutathione cleaved the disulfide-linked glutelin polymers embedding the starch granules. The glutelin polymer has been suggested to work as a hindrance to the absorption of water by starch molecules when water is added to a rice flour; 66 glutathione may fray this barrier to make the batter more consistent and viscous, thereby improving its gas-holding capability in the fermentation process, 53 as is the case with protease-treated rice batter. 65 Although the number of its applications in food processing has been limited so far, 91 glutathione appears to be a promising tool for developing food with new properties. Glutathione is usable as a food ingredient in the United States 92 and some east Asian countries. For example, glutathione-based oral dietary supplements have been accorded the status of a Generally Recognized as Safe (GRAS) constituent with Section 201(s) of the Federal Food, Drug, and Cosmetic Act of the US Food and Drug Administration (US-FDA). 93

On the other hand, usage of glutathione for food has some limitations. First, glutathione is not usable as a food in all countries. In Japan, for instance, it is recognized as medicine, and cannot be incorporated as a food additive. 94 Second, GSH-added rice batter has been shown to yield a slight amount of hydrogen sulfide (0.43 ppm) and methyl mercaptan (0.106 ppm) in the headspace gas of the bread. 71 Generation of hydrogen sulfide in heated meat or purified GSH is well known; 95 indeed, a slight amount of hydrogen sulfide contributes to the pleasant aroma of cooked meat 96 and rice. 97 Usage of GSSG in breadmaking instead of GSH significantly reduced the generation of these sulfur compounds, 71 and sensory evaluation demonstrated that the aroma of GSSG-added rice bread was almost equivalent to that of non-added bread. 98 However, we sought to develop rice bread without glutathione or any other additives.

In the process of developing glutathione-added rice bread, we found that the control sample, that is, “non-added bread”, occasionally swelled in fermentation. Although it collapsed mostly in the following baking process, we expected that if optimal conditions could be found, we could make an additive-free, gluten-free rice bread from solely the basic ingredients: rice flour, water, yeast, sugar, salt, and oil.

Additive-free, gluten-free rice bread

The development of additive-free, gluten-free rice bread has taken a trial-and-error rather than a strategic approach. 99 , 100 First, we tried several commercially available rice flours and found that flours with low-starch damage (<5%) were the most suitable. The physical property of the gluten-free rice batter appeared quite different from the familiar viscoelastic wheat dough. It had an appearance and texture of a slurry with low viscosity. So, lots of “cooking tips” have been discerned for the breadmaking process. For example, as rice batter tends to make lumps, we paid attention in the mixing procedure to avoid lumps. Also, the dried yeast needs to be dissolved completely. Generation of bubbles of different sizes due to heterogeneous distribution of dried yeast may result in their coalescence 101 and a sudden shrinkage of the batter in the fermentation process. The breadmaking processes, i.e., mixing of the batter, fermentation and baking, as well as tips for successful making in the respective processes, are mentioned in a later section.

To clarify how the gluten-free batter swells without additives, we sought to investigate the microstructure of the fermenting batter. The fermenting batter appeared like a meringue and was quite different from wheat dough, which is so viscoelastic that its full mass can be lifted with a scoop (Fig. 1b ). As it was not easy to freeze the fragile batter without destroying the delicate structure, a sectioned specimen for microscope observation could not be made. Instead, freshly made batter was sandwiched between a microscope slide and a coverslip and the batter was left at room temperature to ferment there. Optical microscopic observation revealed the microstructure: bubbles covered by starch granules (Fig. 2 ). The structure was entirely different from that of the typical wheat dough, in which gas cells are surrounded by the gluten matrix made by a network of gluten protein and starch granules. 102 In contrast, it had a similar structure to a “particle emulsion” 101 in which rice granules stabilize the interface between oil and water (Fig. 2 ). 103 Thus, it was suggested that the bubble observed in an additive-free, gluten-free rice batter had the structure of a “particle foam” (Figs. 1a , 2 ). 101

Explanatory figure of particle emulsion/foam. Adapted from refs. 99 , 100 . Scale bar: 30 µm. Copyright (2017), with permission from Elsevier

The hypothetical mechanism is illustrated in Fig. 2 . Generally, oil and water do not mix. However, when they are mixed well in the presence of a detergent, microscopic oil droplets covered by detergent molecules disperse throughout water. This is a classic emulsion. Likewise, aeration of water in the presence of detergent results in a foam. A small amount of air is surrounded by a thin film of water, in which detergent molecules stabilize the boundary.

At the beginning of the 20th century, solid particles were found able to adsorb onto the interface between oil and water, and play a similar role to that of detergent molecules. 104 , 105 This is called a “particle-stabilized emulsion” or “particle emulsion”. Starch granules of native rice, maize, wheat, 103 quinoa, 106 high-pressure treated corn starch granules, 107 chemically modified waxy maize and tapioca, 108 as well as rice starch granules 109 have been reported to form particle emulsions. A particle-stabilized foam occurs in the same manner. Particle emulsions/foams have received renewed attention during the past decade, as recent advancement in nanoparticle technology accelerates research trends. 110 , 111 Moreover, such foams have potential applications in a wide variety of industries, including foods, pharmaceuticals, and cosmetics. One of the key advantages of the mechanism for foodstuff applications is that microparticles of biological origin, such as starch granules, cellulose, or protein particles, work as stabilizers. 101 Our report showed for the first time that rice starch granules stabilize particle “foam” in an additive-free, gluten-free rice batter. 99

The breadmaking processes and tips for the successful gluten-free breadmaking from rice flour are summarized in Fig. 3 . In the early stage of fermentation, yeast produces fermentation gas, composed mainly of carbon dioxide and alcohol. Ordinarily, the batter cannot hold the gas and becomes foamy. 53 , 54 However, if rice flour with low-starch damage is used and breadmaking is performed with the right conditions, the fermentation gas is trapped in the batter. 99 Thus, small bubbles appear throughout the batter. The small bubbles are particle foams in which fermentation gas is surrounded by starch granules. As the fermentation proceeds, the fragile bubbles gradually grow bigger, making the whole batter rise. Here, it is critical to keep the temperature stable, as fragile bubbles tend to burst in fluctuating temperatures. In the late stage of fermentation, the swollen bubbles should be heated rapidly to make the starch granules gelatinize, that is, to solidify the bubble walls. The most swollen bubbles are the most fragile, so rapid heating is the key.

Summary of the procedures for making additive-free rice bread and “cooking tips” for each step. Adapted from ref., 100 with permission

The overall process resembles the synthesis of a polyacrylamide hydrogel, in which modified nanoparticles stabilize an air/water (acrylamide solution) emulsion, and the macroporous structure is fixed by thermal-induced polymerization. 112

We have investigated several commercially available rice flours and found that rice flours with less starch damage (<5%) make bread with a higher specific volume. 99 Higher starch damage tends to facilitate greater absorption of water by starch granules. 113 The hydrophobicity/hydrophilicity ratio determines the aptitude of starch granules to form particle foam. 114 Thus, to prevent destabilization of the fragile bubbles in the fermentation process, it is important to maintain the proper hydrophobicity/ hydrophilicity ratio. Our success in making bread using flour with less starch damage, that is, less water absorption, seems consistent with the hypothetical mechanism. In this context, reduction of surface tension by hydrophobic treatment of rice starch granules was successful in making a stable particle emulsion. 108 , 109

From another point of view, if rice starch granules are capable of constituting a particle foam, they should have the ability to mimic the function of detergents, that is, to reduce the surface tension of water. Starch granules with less starch damage (4.7 w/w%) effectively reduced the surface tension of water from 73 to 35 mN/m. In contrast, starch granules with higher starch damage (9.8 w/w%) were not as effective, reducing the surface tension to only 47 mN/m. 99

Starch granules show emulsion-forming ability by stabilizing the water/tetradecane interface. 108 So, similar experiments were conducted using starch granules with low- and high-starch damage (Fig. 4 ). Both starch granules made stable water/tetradecane emulsions (Fig. 4a ). However, the microstructures of the emulsions were somewhat different (Fig. 4b ). Optical microscopic analyses of the emulsions showed that starch granules with less starch damage (LD) covered the oil droplets densely. In contrast, in the case of rice granules with higher starch damage (HD), swollen granules were occasionally seen, and the oil droplets were not covered completely. Thus, rice granules with low-starch damage demonstrated better particle-emulsion-forming ability compared with the high-starch-damage counterparts. This was consistent with the observation that rice starch granules with low-starch damage were suitable for constructing particle foam, that is, to make additive-free rice bread.

a Water/tetradecane emulsions formed by starch granules at different rice flour concentrations. From left to right: control (no flour), addition of rice flour with low-starch damage (20% w/w, 50% w/w), as well as high-starch damage (20% w/w, 50% w/w). b Optical microscopic analyses of the emulsion. Rice flour with low- (LD) and high- (HD) starch damage was compared. Adapted from ref. 99 Scale bar: 100 µm for ×100, and 30 µm for ×400, respectively. Copyright (2017), with permission from Elsevier

All these three observations support the hypothetical particle foam theory. Verification studies are in progress in our lab.

Several approaches in the development of gluten-free bread by our own laboratory and others have been introduced in this review, together with the social and scientific context of these efforts. The research is aimed to improve the quality of life of celiac disease or wheat allergy patients. Better bread quality (flavor, texture, and volume), reduced production cost, and wider availability are all important issues. 115 For example, so far, rice bread lacks the mouth-watering aroma of freshly baked wheat bread. It is not clear whether this is inevitable or whether a better selection of ingredients or an improved breadmaking procedure could lead to improvement of the aroma and flavor of rice bread, such that it becomes comparable with that of wheat bread. Besides, rice breads tend to be sticky compared with wheat bread. Also, gelatinized rice starch tends to retrograde faster, 116 so the bread is prone to become stale and hardened faster, 117 resulting in a shorter shelf life. 118 Using rice varieties with intermediate amylose content and a low water absorption index may give superior crumb properties. 119

Recent wide availability of household breadmaking countertop appliances has prompted our laboratory and others to develop gluten-free bread recipes suitable for these machines. Providing specific ingredients, such as fitted rice flour sold along with the breadmaker, may help consumers experience success in making custom gluten-free bread at home. Improving the machines by incorporating an induction-heating (IH) system may be suitable for making “particle-foam” type rice bread, as an IH system guarantees stable temperature control in fermentation as well as rapid heating in the baking process. 120 Addition of micronutrients and functional food ingredients is also an important theme. Further studies may thus improve the bread quality to be comparable to that of wheat bread and to improve the quality of wheat-sensitive patients’ life through providing a satisfactory diet.

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Acknowledgements

We appreciate Dr. Bob Buchanan and Dr. Peggy Lemaux, University of California, and Dr. Wallace Yokoyama and Dr. James Pan, USDA, for useful discussions. Dr. Shigeru Kuroda is also appreciated for his encouragement throughout this work.

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Texture profile analysis and sensory evaluation of commercially available gluten-free bread samples

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The need for better quality gluten-free (GF) bread is constantly growing. This can be ascribed to the rising incidence of celiac disease or other gluten-associated allergies and the widespread incorrect public belief, that GF diet is healthier. Although there is a remarkable scientific interest shown to this topic, among the numerous studies only a few deals with commercially available products. The gap between research and commercial reality is already identified and communicated from a nutritional point of view, but up to date texture studies of commercial GF breads are underrepresented. In this study, 9 commercially available GF bread were compared to their wheat-based counterparts from texture and sensory viewpoints. Results showed that among GF loaves products, some performed significantly better at hardness and springiness attributes during the 4-day-long storage test compared to the wheat-based products. Two of GF cob breads performed significantly better or on the same level as the wheat-based cob regarding to hardness and cohesiveness during 3 days. Among sensorial properties mouth-feel, softness and smell were evaluated as significantly better or similarly for some GF versus wheat-based products. Two GF bread had more salty taste which reduced the flavor experience. Both the texture and sensory data of the storage test indicate that the quality of some GF bread products has significantly improved in the recent years; they stayed comparable with their wheat-based counterparts even for a 4-day-long storage period.

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Introduction

However, Celiac disease (CD) was already mentioned by Aretaeus of Cappadocia probably in the second century (AD) [ 1 ], it became an emphasized scientific and commercially important topic in the last decades. The consumption of gluten-free (GF) products is significantly increasing, just as the demand for good-quality GF products [ 2 ]. The underlying reason for the expanded interest can be attributed to better diagnostical methods of CD, wheat allergy, non-celiac gluten sensitivity and dermatitis herpetiformis [ 3 , 4 ], and to the widespread incorrect public belief that GFD is healthier [ 5 ].

Gluten—as a term used to encompass prolamin proteins—can be found in wheat, barley and rye, including all their subtypes and genus [ 6 ]. It is the key structure-forming protein, which is the most common and important protein ingredient in the bakery industry. Absence of gluten in the GF formulation ends up with much weaker gas-holding properties; therefore, it causes low loaf volume [ 7 ], crumbling texture, poor color [ 8 ], choky dry mouth-feel and shorter shelf life [ 9 , 10 , 11 ].

Consumer survey studies revealed that the consumers are satisfied with the quality of GF sweets, biscuits and pasta, but still significant improvement is needed in GF bread and cakes to meet the consumers’ expectations [ 12 , 13 , 14 ]. The constantly growing number of published articles shows that several approaches were studied mostly using different modified starches, pseudocereals, enzymes, protein supplementation and/or hydrocolloids to improve the quality and nutritional properties of GF flours and breads [ 6 , 15 ]. Among these numerous studies, only a few deals with commercially available products, the majority rather focuses on self-made prototypes from different raw materials. The publications that are based on commercial products concentrate on composition, nutrition values and/or prices. Based on their detailed and thorough study by examining 228 commercial products Roman et al. [ 12 ] declared a gap between commercial reality and research. Studying the ingredient list of breads they noticed that the commercial breads do not seek to use one single starch or gluten replacer, but a combination of several ingredients to optimize bread quality (hydrocolloids, acidifiers, emulsifiers, leavening agents, preservatives, and aromas or flavorings). They observed that some ingredients which have momentous attention and focus in the scientific world (e.g., pseudocereals) are hardly used in commercial products. On the UK market, GF products are 159% more expensive than their regular version, most GF bread and flour products contain higher amount of salt, fat and sugar, while some GF products are lower in fiber and protein content [ 16 ]. Similar differences were found on the Italian market [ 17 , 18 ]. Spanish market sample study revealed that sodium, fat and cholesterol content were significantly higher in 20 commercial GF bread samples due to having egg, different oils like coconut, olive, sunflower, palm [ 19 ]. Although it is true that dietary fiber and sugar levels are more adequate than in the past, the GF diet might lead to CD patients’ inadequate intake of fats, proteins, sodium and vitamins [ 20 ].

In general, it can be declared that GF products are significantly more expensive compared to their wheat-based counterparts, and their on-shelf availability can be limited [ 21 , 22 , 23 ].

The studies mentioned above, give important and valuable information for the scientific community and draw attention to the gap between research and commercial reality. Despite the fact that this gap is already identified and communicated from a nutritional point of view and regarding ingredients, up to date rheological studies are hardly available dealing with commercial GF breads (Table 1 ).

Considering the rapid and constant development and changes in the GF bakery industry (ingredients, technologies, consumer needs), more and more GF bakeries are appearing on the market and selling freshly baked, preservative-free bread products. These products are based on different ingredients and recipes, but trying to be comparable with the gluten containing products in terms of lookalike, size, taste and shelf life. Therefore, it would be essential to continuously examine the textural and sensory properties of the GF freshly baked and sold bread products available on the different local markets. Following this approach, the current study aims to compare these GF commercially available, preservative-free bread products with their gluten containing wheat flour-based counterparts, focusing on their texture and sensory properties.

Materials and methods

Bread samples.

The studied 9 different GF commercial bread samples were purchased from different specialized GF bakeries, while the wheat-based products from a supermarket. All the samples were selected with the aim to compare them regarding the product’s name, appearance and packaging. Special attention was taken to ensure that the products did not contain preservatives and gas or modified atmosphere in the packaging. In this study, three types of bread were selected: cob (artisan, round shaped bread), white and wholegrain loaf (baked in loaf tin). From each bread type, GF and wheat-based products were selected and compared (Table 2 ).

All the samples considered in this study were sliced and ready to eat, without prior heating requirement. Ingredients and nutrition values of the samples which were noted on the product’s packaging are presented in Table 3 .

Texture measurement

Texture profile analysis (TPA) was performed at room temperature using Stable Micro Systems TA.XT2. Samples were taken and measured from the first, middle and last third of the sliced bread products, doing 7 different measurements on different slices of the same bread sample. The measurement was placed on the middle of the bread slices, avoiding region near to the crust. Each bread slice had 12 mm thickness. The applied settings were 35 mm diametric acryl cylindrical probe, 50% strain, 5 mm/sec crosshead speed and 5 s of waiting time between the two measurements. Firmness, cohesiveness and springiness were the main representative parameters of the sample texture. Results obtained from the GF and wheat bread samples were compared and followed up.

Sensory evaluation

During the sensory evaluation group of 15 people (13 females and 2 males, aged between 22 and 47 years) tested the bread samples. The ethical statement for the study was applied from the Hungarian University of Agriculture and Life Sciences and informed consent was obtained from each subject prior to their participation in the study. Subjects confirmed not having any known gluten, rye, milk protein, egg or lactose consumption-related disorder. All participants were recognized as regular bread consumers, consuming bread at least once per day.

The assessors received 1 full slice of the sample without any spreading, and were asked to appraise the intensity of 17 sensory attributes, which were described as relevant ones for GF bread by Pagliarini et al. [ 34 ] to cover appearance, color, taste and texture. For evaluation purpose a continuous, unstructured 10 cm long line scale with extremes at the ends (absolutely not intense and immensely intense) was used for every attribute. Samples were served with 3-digit codes on white plastic plates under white light at room temperature.

Data analysis

Received data were analyzed with IBM SPSS Statistics 25.0.2.2 software. Significant difference between the measured groups was determined by one-way analysis of variance (ANOVA) with 95% confidence level. Tukey HSD test was used after normality and standard deviation homogeneity test. Linear discriminant analysis (LDA) was performed to examine the separability of each bread type. Sensorial test data were analyzed by ANOVA. When there is significant difference, Tukey test was applied using a level of 5% of significance.

Results and discussion

Nutritional values of the bread samples.

In line with the previously published data, the examined GF bread samples contained different starches, hydrocolloids, fibers and protein supplements all at the same time. The type of starches (corn, tapioca, potato, and rice) and hydrocolloids (HPMC, guar gum, xanthan gum) were the most commonly used ones among various GF breads on different markets [ 12 , 17 ]. The fiber and salt content of C1, WL1 and WG1 samples were higher while the protein content was lower than in their wheat-based counterparts. Lower protein level was also detected for GF breads previously [ 12 ], but in this case of C1, WL1, WG1 samples according to the statement on the manufacturer’s website keeping the protein level low was a conscious decision, so their products can be used for people diagnosed with phenylketonuria (PKU) as well. People with PKU have to follow a low protein and phenylalanine containing diet [ 35 ]; therefore, these products are suitable not just for celiac people. Following gluten-free option as dietary practice is known and should be followed [ 36 ]. The energy and carbohydrate values were similar between the GF and wheat-based samples expect for WL2, which had the lowest level of energy and carbohydrate level among all the samples.

Texture profile changes

Results of the TPA measurements during the shelf-life test are presented in Table 4 . Overall, it can be seen that C2 sample had significantly ( p  < 0.05) higher hardness but lower cohesiveness during the whole study. C1 was significantly softer on day 1, but not different from CW on the following days. C3 showed non-significant difference in hardness from CW during the whole study. Among the GF white loaf samples compared to WLW, WL1 was significantly lower in hardness except on day 3, while WL2 was also significantly softer versus WLW except for day 2. WL3 after day 1 was not significantly different from WLW. In case of whole grain loaves, on day 1 all the GF samples were significantly softer than WGW. On the following days, there were no significant difference detected among them, except for day 3, when WG1 was significantly softer versus WGW.

High cohesiveness leads to no disintegration during mastication, in case of low cohesiveness the bread crumbles [ 37 ]. Crumbling texture of GF bread during storage test was reported in the last decades, raising awareness as a general quality issue of these products [ 38 ]. Moore et al. [ 25 ] experienced decrease in cohesiveness ( p  < 0.01) in GF bread samples after a two-day storage. In this study, all the GF white loaf samples had significantly higher cohesiveness during the storage test versus the wheat-based white loaf sample. In case of whole grain samples, WG1 was not significantly different in cohesiveness from WGW. WG2 and WG3 samples showed significantly higher values compared to WGW until day 4, when only WG2 was different. Among cob samples C2 and C3 were significantly different from CW, and in general C2 was different from the other cob samples during the whole study.

In bread, springiness is associated with freshness, and products with low values are linked with crumb brittleness [ 27 ]; therefore, having high springiness values during the shelf life is desired. In this study WL2 sample showed significantly ( p  < 0.05) lower springiness during the 4-day-long storage test compared to all other bread samples. Despite the level of springiness grew day by day, but on the 4th day, it could barely reach 80%, still being more rigid. During the storage test, WL3 had significantly higher springiness values versus WLW sample, while WL1 was significantly better or comparable with WLW. Among the whole grain samples, WG2 showed higher springiness values every day compared to WGW, while WG1 and WG3 were better or comparable with WGW. Within the cob samples only on day 1 C1 showed significantly lower springiness value, but on the other days all the GF cob samples were comparable with the wheat-based cob.

Resilience characterizes the beginning of a sample’s elasticity and calculated from the ratio of the area under curve of the second half of the first cycle to the first half of the cycle. Reduction in springiness and resilience reflects alteration of the crumb elasticity [ 39 ]. The GF white loaves and the GF whole grain samples showed higher ( p  < 0.05) resilience values compared to their wheat-based counterparts. This is in line with the springiness values, where the GF samples had higher or comparable values. In case of the cob samples, C3 always had higher ( p  < 0.05) values than CW, C1 on days 1 and 4, while C2 was all the way consentaneous with CW.

According to the results, C1, C3, WL1, WL3, WG1, WG2 and WG3 bread samples in general can be described as soft and spongy [ 33 ] as they had comparable or lower hardness, higher springiness and resilience values than their wheat-based counterparts. From cohesiveness point of view the mentioned samples performed better or comparable to their wheat-based counterparts.

LDA results (Fig.  1 ) showed that WLW and WGW samples were classified as different groups from the others during the whole storage test. The significant difference in cohesiveness and resilience for both group, the springiness of WLW and the hardness of WGW attributes led together to show these samples as different product groups from the others.

LDA results of the storage test ( a with all samples; b without C2 sample; c without C2 and WL2 samples)

Due to its hardness results C2 was also classified as a separate group (Fig.  1 a), and WL2 because of its springiness and resilience attribute (Fig.  1 b). LDA result without these two groups (Fig.  1 c) showed an overlap between C1 and CW samples (79.3% of cross-validated grouped cases were correctly classified). This result clearly showed that the quality and texture profile attribute changes of C1 during a 4-day-long storage test were as good as the highest quality wheat-based product’s considered to be artisan.

Mean ratings (given in cm) for the 17 sensory descriptors of the 12 bread samples are presented in Table 5 . Less homogeneous crumb porosity for GF bread samples were previously reported [ 40 , 41 ], which was linked to high starch and low protein content, impacting the dough interfacial properties and rheological attributes. Pagliarini et al. [ 34 ] found commercial GF bread product with uniform crumb porosity but with higher protein value. The commercial GF samples included in the study had significantly lower protein content versus the wheat-based ones, but received as high or even significantly higher values for crumb homogeneity perception. The reason for that could be linked to more effective protein supplements and/or better understanding of starch–protein–hydrocolloid interactions.

From crumb color behavior point of view, participants found this attribute at same or more intense level than their wheat-based counterparts, except for WL3 and WG3. This result showed that it was achievable with the combination of GF ingredients like starches (corn, tapioca, rice), pseudocereals (amaranth, buckwheat) and fibers (apple, potato, psyllium) to create crumb color for GF breads, which was typical for the wheat-based counterparts, and preferable even for non-celiac consumers. However, the exact ratio just based on the ingredient list information could not be determined. The improvement of crumb and crust color intensity indicating that appearance, as one of the most important factors at bread purchasing had significantly improved in the last decade in the case of fresh baked GF breads.

One of the biggest struggle with GF bread formulations had been their flavor. GF products were often described as having dry, tasteless or unpleasantly strong corny taste [ 15 , 17 , 27 , 34 ]. In this study the GF samples did not have type-unusual corny and cheesy flavor and/or odor. From taste point of view, the two most dominant difference were detected by the saltiness of C1 and the sweetness of WGW. Latter can be explained with the highest level of added sugar (3.8 g/100 g).

WLW was characterized by the most intense fermented taste and smell, which was probably due to the presence of sourdough.

Concerning texture properties, sensory results were in line with the instrumental measurements. The link between hardness and springiness measurement and softness scores was confirmed, they strengthened each other. C2 sample was the hardest during all days, which was reflected in the sensory test as well with the least intense softness value. WL2 sample is not just hard, but also rubbery. However, the exact level of ingredients was not mentioned on the labels, which can be linked to a higher level of hydrocolloids.

In general, checking all the texture properties together, C1, WL1 and WG1 samples were performing at the same level or better ( p  < 0.0.5) compared to their wheat-based counterparts.

This study aims to provide up to date data regarding the so far neglected topic of texture and sensory aspects of commercially available, freshly baked, preservative-free GF bread products designed for celiac consumers. Results show that the market has the ability to produce preservative-free, ready-to-eat bread products with comparable texture properties and attributes to their wheat-based counterparts during storage at room temperature. The higher fiber and the comparable or even lower energy and carbohydrates values decrease the gap in the nutrition area between GF and wheat-based bread products. In the future, it would be important that shelf-life studies aiming to evaluate the texture and sensory qualities of GF bread samples would concentrate on the commercially available GF products and in that case, these results and parameters could be used as reference. If the focus would shift more to the commercially available GF products, it would become more apparent that these products are not as low quality anymore. The hardness, springiness and cohesiveness data of the storage test prove the very opposite, the quality of these products has significantly improved during the last few years.

Availability of data and material

Data which support the outcome of the study are available from the corresponding author upon request.

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Acknowledgements

The authors appreciated Barbara Pém and Nikolett Lázár for their high-level assistance in proofreading and editing. The authors acknowledge the Hungarian University of Agriculture and Life Sciences’ Doctoral School of Food Science for the support in this study.

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Tóth, M., Kaszab, T. & Meretei, A. Texture profile analysis and sensory evaluation of commercially available gluten-free bread samples. Eur Food Res Technol 248 , 1447–1455 (2022). https://doi.org/10.1007/s00217-021-03944-2

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How Welsh scientists aim to boost white bread’s nutritional value

07-May-2024 - Last updated on 07-May-2024 at 13:28 GMT

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White bread is made from refined flour, which means the bran and germ were removed during processing, resulting in a product that's lower in fiber. Bran and germ are rich in vitamins and minerals; however, these are stripped out during the processing of white flour.

It’s important to note that not all white bread is created equal and many sold in the UK are fortified with vitamins and minerals to improve their nutritional content.

White bread ​ ​ has a higher glycemic index (GI) compared to its whole grain counterpart, meaning its causes spikes in blood sugar levels, which may contribute to weight gain, insulin resistance and an increased risk of type 2 diabetes.

And finally, some varieties of white bread contain additives and preservatives to improve shelf life and texture, which some experts claim could be detrimental to health in the long run.

Despite this raft of negative elements, white bread remains a consumer favorite, ​ ​ thanks to its soft and fluffy texture and its mild flavor.

It’s been a staple in many households for generations, being affordable and convenient. People also typically stick with what they were brought up with, especially if the household shied away from the ‘less palatable’ whole grain varieties.

This has resulted in the ‘fiber gap’ – the disparity between the recommended intake of dietary fiber and the actual consumption of fiber by a population.

The consequences of not meeting the recommended fiber intake can include digestive issues, such as constipation, as well as an increased risk of chronic diseases like heart disease, type 2 diabetes, and certain types of cancer.

Bridging the fiber gap

To help bridge this gap, ​ ​researchers from Aberystwyth University are investigating the addition of nutritionally dense ingredients like peas, oats and beans to enhance the nutritional profile of white bread flour.

The project has won funding from Innovate UK’s ‘Better Food For All’ initiative, one of 47 projects to receive a share of £17.4m. These projects are focused on improving food quality, creating functional foods, boosting nutrition, developing new proteins and extending the shelf life of healthy and fresh foods.

“These projects showcase the extensive range and quality of innovation within the agri-food sector of the UK,” said Dr Stella Peace, executive director for the Healthy Living and Agriculture Domain at Innovate UK.

“With global challenges like food security, sustainability, and nutrition, creative solutions are needed to make a tangible impact.

“At Innovate UK, we are committed to driving transformational change in food production and manufacture to shape the future economy and society as a whole.”

Aberystwyth University is recognized as a leading center for the development of new oat, bean and pea varieties, with 65% of all oats in the UK grown from varieties developed there. The research project will make use of the state-of-the-art facilities and resources at the Aberysthwyth’s innovation campus, AberInnovation.

“This is a very exciting opportunity to improve people’s diets, especially those who favor the look and sensory attributes of white bread,” said Dr Catherine Howarth, principal investigator from the Institute of Biological, Environmental and Rural Sciences (IBERS), a department of Aberystwyth within its Faculty of Earth and Life Sciences.

“The project underlines how our leading plant research here in Wales can make a difference to people’s lives. We hope this will be another chance to put our work – especially on beans, peas and oats – to very good use.”

A positive impact on both people and the planet

The team at Aberystwyth in Ceredigion have also enlisted the help of organic millers Shipton Mill to drill down into the time-honored milling and blending techniques used to produce white flour.

Regenerative agriculture ​ ​ is a topic widely covered by Bakery&Snacks. To learn more, search regenerative agriculture ​ ​ or link here ​ ​ and here. ​ ​

The Gloucester-based mill was founded in 1979 with the aim to produce flour from grains farmed by like-minded farmers who promote biodiversity and who value soil – an increasingly popular farming method called regenerative agriculture.

History, too, is inbred in the mills’ foundation. According to its website, it’s built on the site of an ancient mill recorded in the Doomsday book of 1086  and equipped with second-hand machinery that has proved its worth, such as the roller stands that date back to 1920.

“In milling, our craft is to provide bakers with excellent and reliable results that work with nature and what the climate and seasonality can offer,” said Chris Holister, head of Product Development and Artisan Support at Shipton Mill.

“This project builds on our belief that variety and nature-friendliness is the way to measure the success of a crop, not speed and growth. We hope that this work can help make for a healthier and happier diet for very many people.

“With projects like this, we in the UK food industry have a chance to make a positive impact: creating innovative products and solutions that could both improve people’s health and create jobs in the sector.”

Significant social and economic benefits

Addressing the fiber gap, Dr Amanda Lloyd, senior researcher from the Department of Life Sciences at Aberystwyth University said poor diet plays a major role in ill-health, chronic diseases and a significant portion of cancer cases.

“Obesity rates are very high in the UK, with projected costs for the NHS at £9.7 billion by 2050 and society at nearly £50 billion annually. Using our expertise at the University, we hope that this project can play a role in tackling this growing issue of diet-related poor health and wellbeing.

“The project will also bring significant social and economic benefits to the UK and will further establish the UK as a leader in the flour and flour-based foods markets.”

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Development, Analysis, and Sensory Evaluation of Improved Bread Fortified with a Plant-Based Fermented Food Product

Miriam cabello-olmo.

1 Biochemistry Area, Department of Health Science, Public University of Navarre, 31008 Pamplona, Spain

Padmanaban G. Krishnan

2 Dairy and Food Science Department, South Dakota State University, Brookings, SD 57007, USA

Miriam Araña

Maria oneca, jesús v. díaz.

3 Pentabiol S.L., Polígono Noain-Esquiroz s/n, 31191 Pamplona, Spain

Miguel Barajas

Maristela rovai, associated data.

The data are available from the corresponding authors.

In response to the demand for healthier foods in the current market, this study aimed to develop a new bread product using a fermented food product (FFP), a plant-based product composed of soya flour, alfalfa meal, barley sprouts, and viable microorganisms that showed beneficial effects in previous studies. White bread products prepared with three different substitution levels (5, 10, and 15%) of FFP were evaluated for physical characteristics (loaf peak height, length, width), color indices (lightness, redness/greenness, yellowness/blueness), quality properties (loaf mass, volume, specific volume), protein content, crumb digital image analysis, and sensory characteristics. The results revealed that FFP significantly affected all studied parameters, and in most cases, there was a dose–response effect. FFP supplementation affected the nutritional profile and increased the protein content ( p < 0.001). The sensory test indicated that consumer acceptance of the studied sensory attributes differed significantly between groups, and bread with high levels of FFP (10 and 15% FFP) was generally more poorly rated than the control (0%) and 5% FFP for most of the variables studied. Despite this, all groups received acceptable scores (overall liking score ≥ 5) from consumers. The sensory analysis concluded that there is a possible niche in the market for these improved versions of bread products.

1. Introduction

Consumer behaviors have changed dramatically in recent decades, probably because of increased concerns about the impact of food quality on overall health. This situation encourages healthy eating habits by reducing the consumption of unhealthy products and promoting the selection of more nutritious foods [ 1 ]. Among these, functional foods (FF) have gained increasing acceptance in recent years worldwide [ 2 ]. They refer to food products that confer a beneficial effect beyond basic nutrition, and when consumed regularly, can promote health and protect against disease, even though they are not medication [ 3 , 4 ]. They render an invaluable health benefit to the consumer, driven mainly by a complex of biologically active components, including probiotic and prebiotic compounds, bioactive peptides, metabolites, and antioxidant molecules [ 5 , 6 ]. To illustrate, FFs have been proven to be helpful in many noncommunicable diseases, such as type 2 diabetes, cardiovascular disease, and cancer [ 4 ], as well as in improving intestinal barrier function [ 7 ] and in alleviating food intolerance [ 8 ].

Due to food producers’ increasing interest in healthier food products, considerable efforts have been made to develop improved foods. A promising approach involves enhancing the nutritional quality of the marketed products. Bread, traditionally made of wheat flour, is one of the oldest foods and is widely consumed in most cultures [ 9 , 10 ]. It is an important part of the Mediterranean diet and can take part in a healthy diet [ 11 ]; however, regular bread often presents nutritional deficiencies (i.e., micronutrients, fiber, antioxidants) [ 12 ]. There are some factors affecting the nutritional value of bread products, including the type of starter culture (sourdough vs. yeast fermentation) [ 13 , 14 ], degree of refinement (i.e., whole-grain flour vs. refined flour) [ 15 , 16 ], and type of fermentation (backslopping vs. Chorleywood breadmaking process) [ 17 , 18 ].

In the last few years, multiple investigations have been conducted to improve bread and bakery products’ quality. Several strategies can be implemented, such as decreasing salt [ 19 , 20 ] or carbohydrate content [ 21 ], or increasing protein content [ 22 , 23 ] in bakery products. Other strategies rely on formulating new bakery products by partially replacing wheat flour with alternative flours of greater nutritional value, commonly known as composite flours [ 10 , 24 , 25 ]. Some examples describe the enrichment of bread with composite flours made from cereals and pseudocereals (wheat, oat, spelt, rye, quinoa) [ 26 , 27 ], grains (amaranth) [ 24 ], fibers (fructans) [ 28 ], oilseeds (chia, sunflower, flaxseed, and pumpkin) [ 29 ], fruits and vegetables (blueberry and grapefruit) [ 30 ], tubers (tiger nuts and sweet potato) [ 31 , 32 ], and by-products from the food industry [ 33 , 34 ]. Few studies have attempted to include fermented ingredients in bread formulations, using yoghurt [ 35 ], kefir [ 36 ], and fermented quinoa flour [ 27 ]. Curiously, even traditional bakery products, such as Iranian barbari bread [ 37 ] or Chinese steamed bread [ 38 ], have been fortified with different ingredients that increase their nutritional properties.

Frequently, changes in food formulation can cause unexpected changes in the physicochemical and sensory properties of the final product [ 39 , 40 ], and substantial modifications in traditionally marketed products could be rejected by regular consumers [ 41 ]. For example, the incorporation of some ingredients can lead to significant changes in the final products’ odor [ 42 ] or color [ 43 ], and thus condition consumer choice. In this line, some authors have described food neophobia, the reluctance to try new and unfamiliar food, as strongly influencing consumers´ acceptance of new FF products and thus consumer choices [ 44 , 45 ].

The present study examined bread fortified with different concentrations (0, 5, 10, and 15%) of fermented food product (FFP). FFP primarily comprises legumes and cereals, and also includes live microorganisms and fermentable compounds, as well as bioactive ingredients generated during the production process, collectively making it a health-promoting product. FFP was initially intended for livestock supplementation, and previous studies have shown beneficial effects in animals, including improved health status in rabbits [ 46 ] and dairy calves [ 47 ], and a similar product by the same producers provoked a greater energy efficiency for milk production and fiber digestibility in lactating goats [ 48 ]. Additionally, preclinical studies in type 2 diabetes rats fed FFP showed that the product protected against the development and progression of type 2 diabetes [ 49 ], and two other studies with a probiotic bacteria isolated from FFP also showed beneficial effects in type II diabetes mellitus management [ 50 , 51 ].

In this context, we attempted to develop a novel food product from bread by using FFP as a high-nutritional-value component, to serve as an alternative vehicle for providing the product to humans. The objectives of the present study were (1) to develop bread with different enrichment levels of FFP; (2) to evaluate the influence of different FFP replacement levels on the physical characteristics, color indices, quality properties, protein content and crumb structure of the final bread products; and (3) to explore whether the incorporation of FFP in the studied proportions led to products with acceptable sensory properties.

2. Materials and Methods

2.1. product description.

FFP is derived from a plant-based food product manufactured and distributed by a Spanish company (Pentabiol S.L, Noáin, Spain) and is commercialized for livestock supplementation (FDA Registered). Its main components are soya flour, alfalfa meal, and barley sprouts, along with a combination of lactic acid bacteria (LAB) and nonbitter beer yeast (marketed under industrial property protection) that goes through two fermentative processes. Its microbiological composition includes 2.0 × 10 5 colony-forming units (CFU)/g total bacteria, 4.6 × 10 7 CFU/g Lactobacilli , and 1 × 10 5 CFU/g yeast and fungi. The nutritional and physicochemical characteristics of FFP are summarized in Supplementary Table S1 . FFP harbors numerous microbial metabolites generated during industrial processing and partially preserves its microbial community during storage [ 52 ]. Thus, it can be defined as fermented food with hitherto undefined microbial content [ 53 ].

2.2. Experimental Design

We studied three bread formulations with different replacement levels of bread flour by FFP (5, 10, and 15% of bread flour substitution equivalent to Bread-5, Bread-10, and Bread-15 groups, respectively), and bread without FFP (Bread-0) was included as a control. The flour blends were prepared and tested in triplicate, for a total of 12 bread loaves. For the sensory analysis (See Section 2.9 ), 12 extra loaves were prepared ( n = 4 each group).

The outcome measures included loaf dimensions (maximum height, length, and width), mass and volume, specific volume (SV), crumb and crust color, protein content in the bread products, crumb digital image analysis, and consumer panel evaluation.

2.3. Ingredients and Preparation

Bread wheat flour (Gold Medal Premium Quality, General Mills, Minneapolis, MN, USA), salt, sugar (pure granulated cane sugar, C&H, New York, NY, USA), active dry yeast (Red Star, Wisconsin, USA), and water were used in each experiment. Extravirgin olive oil (EVOO) (La Española Olive oil, Vilches, Jaén, Spain) was used as liquid shortening. All ingredients were purchased from a local grocery store and stored at room temperature (RT) until use. FFP provided by the manufacturer (Pentabiol S.L, Noáin, Spain) was obtained from the same production batch. FFP was processed as follows before carrying out the experiments: (1) it was milled to a fine flour using a 0.5 mm sieve; (2) the resulting flour was autoclaved at 121 °C for 15 min to sterilize the product and improve preservation; (3) flour was stored in a sterile glass container at 4 °C until use. Supplementary Figure S1 shows pictures of FFP at each stage and Supplementary Table S1 compares the nutritional profiles of FFP and the bread flour.

2.4. Bread Production

A bread machine (SKG Automatic Bread Baker model 3920, Hong Kong, China) was used in the baking process to obtain a consistent baking method. This model included 19 automatic cooking programs, three loaf sizes (1, 1.5, and 2 lb), and three toast colors (light, medium, and dark).

Based on this experimental design, 12 bread loaves were prepared using different bread flour replacement levels. The ingredients and proportions of each blend are listed in Table 1 . All liquid ingredients (water and EVOO) were weighed and carefully placed in a bread machine pan before dry ingredients (flour mixture, sugar, and salt) were incorporated. For Bread-5, Bread-10, and Bread-15 groups, corresponding amounts of FFP were added to the dry ingredients and adequately mixed. Activated dry yeast was then added to the mixture. Each mixture was cooked using the “Basic bread” cooking program selecting a 1.5 lb loaf and medium crust color. The baking program lasted 3 h and consisted of a 10 min kneading step, 3 min rest, 5 min kneading followed by 10 min rest, and a final kneading phase for 20 min. Subsequently, there were two rising phases of 42 and 40 min each. Finally, baking was performed at 100 °C for 50 min.

Bread formulations.

Ingredients based on a flour weight percentage. FFP: Fermented food product.

When the cooking program was completed, the loaves were removed from the pan and cooled at RT. The loaf mass and volume were then determined, and the loaves were packaged in Ziploc ® bags, labeled, and stored at 4 °C or −20 °C until further analysis.

2.5. Bread Physical Properties

The maximum height (to the top of the mound), length (to the greatest extent), and width of each bread loaf were measured using a regular ruler. The color attributes of bread were determined using a chromameter (Konika Minolta CR 414, Tokyo, Japan). For this purpose, the lightness ( L *), redness/greenness ( a *), and yellowness/blueness ( b *) of the bread crust and crumb were determined in triplicate for each bread loaf.

2.6. Protein Content

With the purpose to checking nutritional changes following bread fortification with FFP, protein content (%) was determined in each group according to the Dumas method with a Rapid N Max Exceed (Elementar, New York, NY, USA). Nitrogen (N) was determined following the AOAC Method of Analysis 935.36, and the protein content (%) in the sample was estimated as N × 6.25. Protein values were adjusted for dry matter.

2.7. Bread Quality Attributes

Once the loaves were cooked and cooled at RT, the mass, volume, and SV were determined. Loaf volume (mL) was determined using the mustard seed displacement method, according to the AACC 10-05.01 approved method. The SV was calculated by dividing the loaf volume by the load mass (cm 3 /g) as previously described [ 25 , 42 ].

2.8. Digital Analysis of Bread Crumb Structure

For deeper insight into the structural changes with different percentages of FFP, digital image analysis was carried out in all the groups using the C-Cell Image Analysis System (Calibre Control Intl. Ltd., Warrington, UK, version 2.0). Each bread loaf was cut transversally into 1 cm thick slices using a food slicer (Chef´s Choice 610, Avondale, PA, USA). The first two slices were discarded, and the third to fifth slices were used for analysis. To assess the differences between the cell structures of the different groups, we studied the following parameters: slice brightness, slice area (mm 2 ), cells area (%), number of cells; main length (mm), cell volume (mm 3 ), cell diameter (mm), and cell wall thickness (mm). Measurements were recorded in triplicate for each bread loaf.

2.9. Sensory Evaluation by Consumer Panelists

Sensorial analysis was performed to study consumer acceptance of experimental bread groups (Bread-0, Bread-5, Bread-10, and Bread-15). To evaluate the sensory attributes of bread, panelists and potential consumers at South Dakota State University (SDSU, Brookings, SD, US) were recruited via email. A total of 42 panelists (64% female) aged 20–54 from 16 different nationalities (American, Bangladeshi, Brazilian, Cameroonian, Chinese, Colombian, Ethiopian, Honduran, Indian, Iranian, Mexican, Native American, Nepalese, Sri Lankan, Turkish, and Venezuelan) were recruited from among the university students and staff. All participants were declared regular bread consumers. The subjects received written information about the test and all the participants provided their informed consent to participate in the study. The study was exempt from ethical committee review.

Twelve bread loaves were baked on the previous two days: four for the appearance evaluation and eight for the sensory evaluation. The samples for the appearance evaluation were frozen (−20 °C) and the samples for the sensory evaluation were refrigerated (4 °C) and kept in a plastic Ziploc ® bag for freshness. On the day of the sensory test, frozen and refrigerated samples were left at RT. Prior to the sensory test, the bread loaves were cut transversally into 1 cm slices. Slices were cut in half longitudinally, and the test sample consisted of one half-piece of each bread group, including both crumb and crust. The samples were coded with random three-digit codes, and the sequences were randomized. All groups were presented at the same time to the panelists, along with water and McIntosh apples to cleanse the palate between samples. The panelists were asked to evaluate the appearance, crumb and crust color, odor, hardness, taste, aftertaste, and overall liking of the codified samples using a questionnaire ( Supplementary Figure S3 ). For the evaluation, a 9-point hedonic scale was used, being 1 “terrible,” 5 “maybe good, maybe bad,” and 9 “great” ( Supplementary Figure S4 ). Values between 1–4 indicated that panelists rejected the product, whereas values equal to or greater than 5 indicated that panelists accepted the product. The questionnaire included an example of the scale to assist the volunteers. Liking of aftertaste was graded with either “yes” or “no”. Additionally, volunteers were asked about the most preferred group (consumer preference) and liking/rejection of whole-grain bakery products.

2.10. Processing of Data and Statistical Analysis

Each group (Bread-0, Bread-5, Bread-10, and Bread-15) was baked in triplicate, and each parameter was determined in triplicate, for a total of n = 9 in each group (except for loaf volume and mass, which were determined once). All data are expressed as mean ± standard deviation (SD). All statistical analyses were performed using SPSS software for Microsoft (IBM SPSS Statistics 20), and the significant level was established at α = 0.05. Analysis of variance (ANOVA) was conducted to test differences between treatments, and Tukey´s post hoc multiple comparisons were performed to determine where significant differences existed among the groups. Data from the sensory evaluation were analyzed using the Kruskal–Wallis test, and frequency analysis was performed using the chi-squared test. The Spearman correlation coefficient (ρ) was estimated to determine the linear association between some test variables with an interval scale, while Pearson correlation coefficient was used for variables with an ordinal scale. The results were interpreted according to the degree of association as very high (ρ = 0.9–1), high (ρ = 0.7–0.9), moderate (ρ = 0.5–0.7), or low (ρ = 0.2–0.5), and statistical significance was set at p < 0.05.

3. Results and Discussion

The results for each determination and the corresponding discussion are presented in the following sections.

3.1. Effect of FFP on Bread Physical Properties and Color Attributes

The effects of different FFP substitution levels on the physical properties and color attributes of bread were analyzed ( Figure 1 ). Loaf peak height was negatively affected by FFP and significantly decreased at replacement levels greater than or equal to 10% ( p < 0.001). A similar effect has been previously described for composite flours [ 10 ] or distillers’ dried grains with solubles (DDGS) [ 54 ], and it was associated with decreased swelling index and dilution of gluten protein, respectively.

Effects of different replacement levels (%) of flour by FFP on the ( A ) peak height, ( B ) length, and ( C ) width of bread loaves. Different letters indicate significant differences ( p < 0.05 and p < 0.001). FFP: Fermented food product.

With respect to loaf width, only Bread-5 differed from the control (Bread-0) ( p < 0.05). Loaf length was unaffected by FFP, and no significant differences were observed between the groups.

Color indices were studied to determine the effect of FFP on the physical characteristics of the experimental pieces of bread and revealed that the treatments significantly influenced the crust and crumb color ( Table 2 ). In the crust, the three color attributes decreased as the FFP level increased in the bread formula, indicating a darker (lower L *), less red (lower a *), and yellow (lower b *) appearance with FFP inclusion (all p < 0.001). Compared with Bread-0, L *, a *, and b * decreased 25.1, 24.1, and 35.8% in Bread-15, respectively. We also found a darker (lower L *), and yellow (lower b *) appearance in the bread crumb, with decreases of 30.9 and 2.8%, respectively, in Bread-15, compared with Bread-0 ( p < 0.001 in both). The a * parameter was profoundly altered by FPP and markedly increased with all the substitution levels ( p < 0.001), especially in Bread-10, more than 3.5 times greater than Bread-0.

Effects of different FFP substitution levels on crust and crumb color parameters.

Means ± SD values in the same column followed by different letters are significantly different ( p < 0.001). FFP: Fermented food product.

In agreement with the data presented above ( Figure 1 and Table 2 ), Figure 2 (upper panel) shows that physical appearance differed significantly between the groups. The incorporation of FFP notably influenced bread loaves´ shape, height, and color. The natural color of FFP, which is darker than that of wheat flour, as well as the Maillard browning originating from the reaction between reducing sugars and amino acids during FFP sterilization [ 31 , 39 , 55 ] (See Supplementary Figure S1 ), could explain the darker color found in Bread-5, Bread-10, and especially Bread-15, compared with Bread-0 (control). As the quantity of FFP in the bread formula increased, the loaves became denser and more compact, and these differences were visible in both cell structure and texture ( Figure 2 , lower panel).

Photographs of bread loaves ( upper ) and central transversal slices ( lower ) of the final bread products baked with different replacement levels of FFP. ( A ): Bread-0, ( B ): Bread-5; ( C ): Bread-10, and ( D ): Bread-15. FFP: Fermented food product.

3.2. Effect of FFP on Bread Quality Attributes

The FFP is a source of fermentable residues and postbiotic compounds, such as microbial compounds and metabolites [ 56 , 57 ], factors that can impact the structure of the dough and the bread and change the texture, physicochemical characteristics, and staling properties of bread products [ 28 , 58 , 59 ]. Considering this, the incorporation of FFP in bread formulation may alter bread rheology and quality attributes. As shown in Figure 3 A, the mean mass of all the bread loaves was comparable ( p > 0.05). This was expected because the bread formula was fixed to 1.5 lb loaves. However, in terms of volume, we found differences between groups with FFP levels equal to or greater than 10%, with 18 and 32% decreases in Bread-10 and Bread-15, respectively ( p < 0.001) ( Figure 3 B).

Impact of different replacement levels (%) of flour by FFP on bread ( A ) mass, ( B ) volume, and ( C ) specific volume. Different letters indicate significant differences ( p < 0.001). FFP: Fermented food product.

SV is commonly used to define bread size [ 10 ] and quality [ 60 ], and is determined by gas retention capability, which is itself influenced by factors such as ingredients, particle size, and processing process [ 58 , 61 ]. As in the case of volume, SV decreased with greater amounts of FFP volume ( p < 0.001) ( Figure 3 C), and such effect can also be perceived in the pictures in Figure 2 . This result is in good agreement with the observations of Clark et al. [ 61 ] and Li et al. [ 38 ], who observed a negative correlation between the incorporation level of fiber-rich alternative ingredients and loaf volume, and attributed such effect to a lower swelling index due to changes in gas retention capacity. An exception was a study by See et al. that investigated the incorporation of pumpkin flour for bread formulation and found that the lowest fortification level (5%) provoked the greatest SV compared to the control formulation (0%) and other treatments (10 and 15%) [ 25 ].

The amount and type of protein in bread blench, which depends on the ingredients, also impact starch gelatinization and reduce swelling power [ 10 , 60 , 62 ]. This is in line with our results from protein content (See Section 3.3 ), which negatively correlated to SV (ρ = −0.84; p < 0.001).

Bread volume has been related to bread shelf life [ 59 ] and chewiness [ 38 ]. In response, strategies such as changing the presentation of the ingredients, for example, whole seeds vs. flour [ 29 ], or raw vs. popped grains [ 24 ], could be considered to minimize the impact of the treatments on bread volume.

On another note, previous studies investigating the compactness of bread products have reported that it can impact starch digestion and the glycemic response, insinuating that more compacted bread with reduced volume could led to a reduced glycemic impact [ 18 ]. FFP is also a good source of fiber, which also affects the glycemic response [ 63 ].

Considering the above, there is a potential for Bread-10 and Bread-15 to have a gentler glycemic response than control bread. Nevertheless, appropriate experiments are required to confirm this hypothesis.

3.3. Effect of FFP Replacement Level on Bread Protein Content

Regardless of its vegetal origin, FFP has a relatively high contribution of protein (44.5%). As expected, we found that the protein content in Bread-0 (9.4%) increased with the incorporation of FFP, reaching 10.4, 11.3 and 12.1% of protein content in Bread-5, Bread-10, and Bread-15, respectively. This indicated that even the lowest FFP supplementation significantly increased the protein content of the bread loaves ( p < 0.001) ( Figure 4 ). A similar trend was previously reported for Barbari bread supplemented with different levels of DDGS [ 34 ].

Impact of different replacement levels (%) of flour by FFP on bread protein content (%). Different letters indicate significant differences ( p < 0.001). FFP: Fermented food product.

The protein content of the final product is influenced by the nutritional characteristics of the flour and other ingredients used during the bread-baking process. While the use of FFP (in this study), DDGS [ 34 ], and legumes (soy, fava beans) would have a positive effect on protein content, the incorporation of other foods less rich in protein, such as pumpkin flour [ 25 ] or some cereals [ 26 ], did not raise protein content in the final bread loaves. Nevertheless, other authors have pointed out that additional factors during baking, such as temperature, fermentation characteristics, or Maillard reactions, could also have a symbolic effect on bread protein content [ 37 ], and should therefore be considered.

According to the nutrition claims by the European Commission in Regulation (EC) No. 1924/2006, only food with at least 12 and 20% of the energy value by protein can be considered a “source of protein” and “high in protein”, respectively [ 64 ]. Considering that, only Bread-15 can be considered a source of protein. Previous authors, however, agreed that only bread types presenting 15–20% protein could be considered protein-rich [ 21 ]. The significant increase in protein content with FFP had little or meaningless effect on the nutritional characteristics of the final bread. Notwithstanding, considering the importance of the protein fraction on the glycemic food load and subsequent glycemic control [ 65 ], any strategy to increase the protein content in a food product is valuable and should not be despised. This is particularly important when the products contain white flour, which is a risk factor for type 2 diabetes and other noncommunicable diseases [ 66 ]. Indeed, incorporating 10 and 15% FFP led to a protein content similar to that observed in bread products elaborated with whole-grain flour [ 40 ].

3.4. Image Analysis of Experimental Breads

Image analysis data showed that FFP significantly affected the cellular structure of bread. A proportional decrease in slice brightness was found with increasing levels of FFP ( Figure 5 A; p < 0.001), which is in good agreement with data from color parameters, where FFP dramatically darkened crumb and crust color ( Table 2 and Figure 2 ). The Spearman correlation coefficient confirmed a positive and significant high linear correlation between slide brightness and L* value (ρ = 0.99; p < 0.001).

Effect of different replacement levels of FFP (0, 5, 10 and 15%) on ( A – F ) crumb properties determined by image analysis. High-resolution images of the bread slices with 0 ( G ), 5 ( H ), 10 ( I ) and 15 ( J ) % of FFP captured using the C-Cell software version 2.0. Different letters indicate significant differences ( p < 0.01 and p < 0.001). FFP: Fermented food product.

Both the cell area and the number of cells were only affected by FFP levels equal to or greater than 10% (Bread-10 and Bread-15), with a negative and positive effect, respectively ( Figure 5 B,C; p < 0.001). Indeed, we identified a very high negative correlation between the two parameters (ρ = 0.98; p < 0.001). In previous studies, however, incorporating novel ingredients, such as orange pomace [ 67 ] and distillers’ dried grains (DDG) [ 38 ] in bread and Chinese steamed bread, respectively, had the opposite effect and decreased the number of cells.

Cell volume increased in Bread-5 and Bread-10, while Bread-15 remained similar to the control (Bread-0) ( Figure 5 D; p < 0.001). The incorporation of FFP had a different effect on cell diameter and wall thickness, which were significantly reduced at replacement levels of 15% ( Figure 5 E,F; p < 0.01). No significant linear correlation was identified between cell volume and cell diameter (ρ = 0.40; p > 0.05) and wall thickness (ρ = 0.22; p > 0.05). A previous study investigated the impact of different replacement levels of breadfruit flour on bread quality parameters [ 61 ], and the authors also found a negative impact of this ingredient on slide area, cell diameter, and wall thickness. Contrary to our results, these treatments positively affected the cell volume.

Lastly, in disagreement with data from SV ( Figure 3 C), the main length and slice area were unaffected by any treatment ( Supplementary Figure S2 ). Similarly, we did not find a linear correlation between SV and main length (ρ = 0.004; p > 0.05). As shown in the digital photographs, there were also physical differences in the appearance of the bread slices within the groups. In addition to presenting a more irregular shape, bread with FFP ( Figure 5 H–J) had a more porous surface than the controls ( Figure 5 G). A similar effect has been described for bread enriched with DGG [ 34 ]. In addition, previous studies have shown a negative association between SV and bread loave density [ 68 ]. FFP is fermented and contains viable microorganisms (bacteria and yeasts) [ 52 ], and even though it was autoclaved, it is possible that some microorganisms survived the inactivation or formed spores. This could contribute to the formation of carbon dioxide during bread preparation, thus leading to a greater cell volume in the experimental bread loaves with FFP. On top of that, metabolites and products generated by microorganisms during baking can also significantly impact the physicochemical characteristics of bread and the rate of staling, potentially influencing consumer acceptance [ 59 ].

3.5. Consumer Sensory Study of Experimental Breads

Table 3 presents the mean data from the consumer panel evaluations and Figure 6 helps visualize the sensory study results. Overall, we found significant differences between the groups for all the sensory characteristics tested (all p < 0.001). The control (Bread-0) was liked significantly more in all the sensory parameters than the other experimental bread groups (Bread-5, Bread-10, and Bread-15), and obtained the best scores in general appearance and overall liking. Besides that, most scores were equal to or greater than 5 in all the parameters and groups, except for the general appearance in Bread-15 (4.1, “Just a little bad”). We can conclude that adding FFP to bread affected consumer evaluation, but consumers still accepted all products. When the mean scores were calculated for all parameters, the best punctuation (7.8, “Very good”) was registered for both crumb and crust color in Bread-0.

Effect of different replacement level with FFP (Bread-0, Bread-5, Bread-10, and Bread-15) on sensory attributes of bread loaves as evaluated using a 9-item hedonic scale. FFP: fermented food product.

Means of sensory attributes of bread fortified with different levels of FFP.

Means ± SD. Values in the same column followed by different letters are significantly different ( p < 0.001). Scores on the 9-point hedonic scale were 1 = terrible; 2 = very bad; 3 = bad; 4 = just a little bad; 5 = maybe good, maybe bad; 6 = just a little good; 7 = good; 8 = very good; 9 = great. FFP: fermented food product.

In Figure 6 , we can see that the hedonic ratings of Bread-0 and Bread-5 were closer in most parameters, though they significantly differed. Notably, the impact of FFP on odor was adversely affected even at the lowest replacement level (5%) ( p < 0.001). Despite this, all groups received punctuation closer to or greater than 5, which indicated that most panelists had a neutral or positive perception. This effect is likely due to the soya present in the FFP. Soya and other ingredients, such as chickpea [ 69 ] or sorghum [ 70 ], can significantly impact food taste, and in some cases, cause a bitter or beany taste that may compromise consumer acceptability mentioned [ 42 , 71 ]. Indeed, taste and aftertaste perceptions were also significantly affected by FFP incorporation ( p < 0.001 in both). Organoleptic properties, particularly taste and olfaction, strongly influence consumer behavior and food acceptance [ 72 ]. Therefore, additional ingredients could be incorporated during the production process to mask undesired organoleptic attributes in bakery products with FFP to develop more appealing bread products attractive to consumers.

Bread color was significantly affected by FFP incorporation, as shown in Figure 2 , Figure 5 and Figure 6 . Accordingly, the panelists’ perception of crust and crumb color was also affected by FFP ( p < 0.001), even at the lowest incorporation level (Bread-5). We believe this might have biased other organoleptic attributes and had an important effect on the perception of the sensory characteristics. Previous studies have indicated the impact of color on sensory evaluations, particularly in bakery products [ 9 , 43 ]. Darker bread is commonly associated with whole-grain bread varieties [ 40 ], which can influence consumer acceptance. In our study, however, when panelists were asked about their liking of whole-grain products, most (92.5%) confirmed that they like this category of bakery products.

On another note, other authors have concluded that pH, which is influenced by the fermentation process, significantly affects bread crumb color [ 61 ]. Although there was no information on the pH value of the experimental bread loaves in this study, it is plausible to find differences based on the composition of FFP.

The treatment also altered hardness perception, and its hedonic evaluation decreased with increasing FFP content ( p < 0.001). This could be due to the higher percentage of humidity in bread loaves containing FFP. Unfortunately, the bread texture analysis was not performed.

At the end of the sensory test, panelists were asked to rate their group of choice, and most of them indicated Bread-0 (70.7%), clearly surpassing the other groups ( p < 0.001). Curiously, Bread-10 (17.1%) was preferred over Bread-5 (9.8%), and only a small minority chose Bread-15 (2.4%).

Food neophobia can dramatically alter consumers´ acceptance of food. It can be affected by demographic, cultural, and social factors [ 44 , 73 ], as well as by individual factors (genetics, age, gender, and personality) [ 45 , 72 ]. Moreover, it has been described that the level of nutritional knowledge of the potential consumers is of great relevance as well [ 74 ]. Interestingly, clusters of consumers with similar sociodemographic profiles share food preferences. A previous study examined the different factors influencing the decision to purchase functional foods among a Polish population. The authors found important sex and age differences and concluded that women and older men have a greater interest in the product´s health properties and nutritional value than young men [ 75 ]. Similarly, the same authors reported that women and old men prefer cereal-based functional products, while young men are more attracted to meat-based products. According to the authors, a potential market niche for bread supplemented with FFP would be women, old men, and subjects with a university education. Nevertheless, we did not find any linear correlation between the preferred group and age (ρ = 0.17) and sex (ρ = 0.24) (all p > 0.05). Similarly, there was no significant correlation between the preferred sample and nationality (ρ = 0.10; p > 0.05); however, we found that the individuals who preferred Bread-10 and Bread-15 were from countries whose traditional gastronomy is full of flavored and tasty food (Brazil, Cameroon, Iran and Mexico). Therefore, it is likely that bread enriched with FFP would be better accepted in specific countries or cultures.

Health is an important motivation for consumers when choosing a functional food, and so does price [ 74 ]. Previous research indicates that consumers´ willingness to pay is impacted by their trust in functional foods, which in turn is affected by the food matrix [ 76 ]. Another study with Russian and German participants revealed an important cultural effect and country differences regarding consumer acceptance and food neophobia [ 73 ]. Alternatively, findings from one study on a Lithuanian population suggest that motivating factors other than health consciousness, such as social factors like conspicuous consumption, perceived self-control motivation, and susceptibility to descriptive normative influence, can also impact consumers´ preferences for FF [ 77 ]. Therefore, all the above-mentioned factors should be strategically studied and addressed to identify the target population of bakery products enriched with FFP.

3.6. Further Research

There is abundant space for further progress in analyzing the incorporation of FFP in bread products. More experiments should address key aspects such as FFP dosage, safety, and stability of the final product, and alternative delivery systems or additional ingredients should also be considered.

In the formulation, we used extra-virgin olive oil for shortening, which is the main source of dietary fat in the Mediterranean diet and is associated with many health benefits [ 78 ]. Because the shortening effects of oil are determined by the fatty acid composition of the fat source [ 79 ] and its oxidative stability [ 80 ], the use of olive oil may provide anti-retrogradation activity different from that of bread formulated with regular shortening. Therefore, it would be interesting to study the staling properties of bread with different levels of FFP and verify whether it alters bread predisposition for retrogradation and staling. If FFP incorporation would lead to fast staling, incorporation of antistaling agents into the formulation should be considered to preserve freshness.

On another note, FFP is a good source of fiber, and its incorporation as an ingredient can change the carbohydrate digestibility, sugar content, and glycemic index [ 81 , 82 ] in bread. Similarly, FFP is a plant-based product that is highly likely to contain antinutritional factors. Interactions between nutrients [ 83 ] and other aspects of industrial production such as fermentation and drying are known to influence protein digestibility [ 84 ]. These aspects can be further tested and predicted using different indices and scores, such as the predicted glycemic index [ 28 ], the contribution of digestible starch [ 81 ], and in vitro protein digestibility [ 27 ]. This is relevant because protein and carbohydrate digestibility can significantly impact microbiota composition [ 84 , 85 , 86 ], and carbohydrate and fiber assimilation can also significantly affect the glycemic response [ 28 , 63 ].

Regarding the sensory properties of the experimental pieces, they could be fully explored through a sensory analysis by trained panelists with sensory experience in evaluating different types of bakery products, including more texture and sensory scores [ 9 ]. It would help develop the most suitable recipe. Similarly, a texture profile analysis of the crumb using a texture analyzer like previous work [ 61 , 87 ] would help to evaluate the texture properties of the end-products.

Lastly, FFP is expected to provide many bioactive compounds in the baked products; however, the functional effects of bioactive components is definitely determined by their viability, which in turn is influenced by the matrix, food processing, storage, and digestion process [ 30 ]. For this, well-designed and controlled preclinical experiments and clinical trials are mandatory before making any health claim for novel food products with functional components.

4. Conclusions

This study is the first attempt to incorporate FFP into a food matrix to develop enriched food for human consumption. Our study aimed to create a bread product with improved nutritional properties that can generate interest in regular consumers of FF products. We evaluated three different substitution levels (5, 10, and 15%) of wheat flour for FFP and compared them with control (Bread-0). The quantity of FFP significantly affected all the studied parameters, including physical characteristics, color indices, quality attributes, protein content, and bread structure. The results showed that incorporating FFP into bread formulation could be a valuable strategy for increasing bread protein content. However, future studies should analyze the proximate composition of the final bakery products to determine whether there are also relevant differences in dietary fiber and other macronutrients such as fats and carbohydrates. Besides, the microbiological analysis of loaves with FFP could clarify whether spores or partially activated microorganisms interfere with dough fermentation.

In addition, FFP affected the sensory properties of bread, as indicated by panelists. Sensory tests revealed that the consumers perceived significant differences in the palatability and sensory attributes of the groups. Bread with different levels of FFP had worse sensory scores than the control in most of the studied characteristics, although consumers accepted all groups. On top of that, some panelists indicated their liking for some attributes in Bread-10 and Bread-15, suggesting a market niche for this type of product.

In conclusion, FFP could be included as a functional ingredient in bread or related products to decrease the use of wheat flour, thus potentially increasing the nutritional and functional properties of bread products. Whether the doses (5, 10, and 15% FFP) and the format of FFP (milled and autoclaved) used in this study could lead to significant health improvements in consumers remains unexplored, and could be evaluated in future trials.

Acknowledgments

The authors gratefully acknowledge South Dakota State University for using the facilities and equipment, and the Dairy and Food Science Department and South Dakota Agricultural Experiment Station for providing the resources needed. We are grateful to all volunteers who participated in the consumer panel. We thank Elena Albanell and Leyby Guifarro, for their assistance with the sensory test. We also thank Pentabiol S.L for kindly supplying the FFP.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods12152817/s1 , Table S1. Nutritional and physicochemical profile of the FFP and bread flour; Figure S1. Images of (A) unprocessed, (B) milled, (C) milled and autoclaved FFP; Figure S2. Effect of FFP on bread (A) main length and (B) slice area; Figure S3. Questionnaire facilitated to the panelists for the sensory evaluation of the bread groups; Figure S4. Visual 9-point hedonic scale facilitated to panelists to evaluate the sensory parameters of the test samples.

Funding Statement

Miriam Cabello-Olmo was granted by the Industrial Ph.D. program (Navarre Government) [Reference: 001114082016000011] and by the postdoctoral fellowship “Ayudas para la Recualificación del Sistema Universitario Español para 2021–2023, UPNA, Modalidad Margarita Salas”, funded by the European Union—NextGenerationEU.

Author Contributions

Conceptualization, P.G.K., M.R. and M.C.-O.; methodology, M.C.-O. and P.G.K.; formal analysis, M.C.-O. and M.O.; investigation, M.C.-O.; resources, P.G.K., M.R., M.B. and J.V.D.; writing—original draft preparation, M.C.-O.; writing—review and editing, M.C.-O., M.O., M.A., M.B. and M.R.; supervision, P.G.K., M.R. and M.B. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Conflicts of interest.

Author J.V. Diaz was employed by the company Pentabiol S.L.He contributid by providing the product used in the scientific paper, and he reviewed the manuscript and agreed for its publication. Neither J.V. Diaz nor the company Pentabiol SL have been involed in the study desing, analysis, discussion nor conclusions. Therefore J.V. Diaz’s participation did not affect the authenticity or objectivity of the experimental results in this research paper. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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