1. Introduction
Lately, there has been an increasing interest surrounding celiac disease (CD) due to a rise in diagnoses. Recent estimates indicate about 1–2% prevalence of CD worldwide [
1]. Currently, there is no treatment available for the disease, other than following a lifelong strictly gluten-free diet [
2]. With the advancement in scientific knowledge and processing technology, there has been tremendous growth in the number of gluten-free (GF) products and especially bakery products available to consumers in specialty stores and supermarkets [
2]. Nonetheless, the concerns about the nutritional profile of the GF products persists due to a range of deficiencies, including fiber, protein, calcium, folate, iron and vitamins B12 and D [
3,
4,
5]. Another important nutritional issue of GF foods is related to the relatively high glycemic index (GI), which is mainly affected by available carbohydrates but also depends on multiple ingredients, including fibers and proteins and fat [
6].
Nevertheless, the development of GF products is still a technological challenge, the use of naturally GF ancient and minor cereal (e.g., teff, emmer, spelt) might be a valuable option due to their high nutritional value and functional properties. Indeed, these have been shown to be well suited to making highly nutritious, modern and innovative baked goods meeting functional and sensory standards in terms of nutritional added value, palatability, convenience (extended shelf life) and easy handling during processing.
Among all, teff (
Eragrostis tef Zucc.) is gaining popularity around the world mostly due to its attractive nutritional properties [
7]. Teff is a GF grain and has great potential to be formulated into a range of food/beverage products to aid people with celiac disease [
7]. Due to the very small grain size, teff milling necessarily provides a whole grain flour. Therefore, the flour is rich in fiber due to the incorporate of the bran components. It is also a source of bioactive compounds such as polyphenols [
8]. As a result of the unique chemical composition and the whole grain form, a range of health benefits have been associated with teff that is, in vitro anti-oxidative activities and prevention of incidence of anemia and diabetes [
7,
9]. Despite the high nutritional value, the high content of dietary fiber, the presence of the anti-nutritional factors (e.g., phytic acid) and the absence of gluten might represent a limit to produce bakery products with acceptable technological, nutritional and sensory profiles [
10].
The sourdough technology has largely been proposed as tool to overcome such drawbacks related to the use of wheat-alternatives flours in bakery production thanks to the direct and indirect activity of the autochthonous microbiota composed of lactic acid bacteria (LAB) and yeast [
11].
The present study aimed at optimizing a biotechnological protocol to produce a gluten-free muffin with high nutritional value and optimal technological and sensory properties. Thus, teff type-I sourdough has been produced and propagated till the biochemical stability was achieved. Biochemical and microbiological properties of sourdough were investigated. The sourdough was used to fortify teff muffins. The biochemical, nutritional, structural and sensory characteristics of the fortified muffins were evaluated and compared to those of a muffin produced without sourdough.
4. Discussion
Since the only treatment for CD is a lifelong GF diet [
40], the production of GF foods has increased significantly in the last years and nowadays the GF market can boast a wide selection of products, such as bread, pasta, cookies and cakes. Nevertheless, defects in the final food product, due to the elimination of gluten, are still pressuring researchers and industries to produce food that could meet consumers’ demands in terms of sensory and nutritional quality, as well as sustainable costs. Nutritional deficiencies (e.g., dietary fiber, proteins and minerals) and excesses (e.g., fat and high GI) are the main concerns regarding the GF products. Indeed, enhancing the nutritional quality of GF products is an unquestionable and concomitant task along with the improvement of both their technological and sensory properties [
41].
The use of GF ingredients, rich in nutritional components, flanked by the optimal technological process might represent a valuable option to produce GF products with optimal features and being accepted by the consumers. Maize and rice are the main cereals used as wheat-alternatives in bakery products, however the research and the industry are moving toward the rediscover of pseudo-, minor- and ancient- (amaranth, quinoa, teff, etc.) cereals due to their nutritional quality and interesting technological properties [
10].
Teff contains many proteins (providing all essential amino acids, including lysine), slowly digestible complex carbohydrates (causing satiety), fibers (improving gut health) and more bioavailable minerals (among which calcium and iron) [
7,
42]. These properties make teff an interesting product for human consumption thus a functional food for the health development and prevention of diseases. Indeed, the interest in teff cultivation is spreading to many western countries of the world [
43]. Nevertheless, due to the poorly appreciated sensory profile, biotechnological approaches, that is, sourdough fermentation, have been proposed to improve the aroma quality of teff baked goods [
44,
45].
In the present study, teff was used to produce type-I sourdough through back-slopping procedure in order to be used to fortify gluten-free teff muffins. According to the microbiological and biochemical characteristics, the type-I sourdough achieved the biochemical stability after 9 refreshments, with ratio between lactic acid bacteria and yeast stabilized at 100:1 in mature sourdough, as previously reported [
46].
L. plantarum dominated since the beginning, however, mature sourdough was characterized by the concomitant presence of
Li. fermentum. The prevalence of
Li. fermentum and L. plantarum have already been reported as part of the dominant microbiota of teff flour [
47] and sourdough [
46]. Due to the lactic acid bacteria fermentation, increases of lactic and acetic acids concentrations were found, especially during the first three days of propagation. Nevertheless, if the former stabilized from t3, the latter varied through the propagation reaching the highest concentration after t9. The increase of the acetic acid might be ascribed to the appearance (between t9 and t12) of the strictly hetero-fermentative
Li. fermentum. The balance between homo- and hetero-fermentative lactobacilli reflects on the organic acids released and in turn on the development of flavor and the microbial stability of the bread [
48]. The FQ of the mature sourdough was 2.6 within the optimum range (2.0–2.7) as suggested by Hammes and Gänzle [
49].
The mature sourdough was used to fortify GF muffins at three different level of inoculum (M
5%, M
10% and M
15%) and the characteristics were compared to those of a control made without sourdough (M
CT). The biochemical characteristics of the sourdough reflected well on those of the final products, except for pH. The use of a chemical leavening agent to produce muffin might have buffered the acidity although concentrations of the organic acids > 20 mmol/kg were found (
Table 1). The FQ, ranging from 2.3 to 2.9 suggested optimal sensory profile of the sourdough muffins [
49].
Nevertheless, the quantification of the other flavor components as well as the identification of the sensory attributes which better describe the muffins have been performed. Overall, aldehydes and alcohols were found to be more concentrated in fermented samples than M
CT. Several authors already reported that these compounds are generated by lipid oxidation in baked products [
37,
50], further highlighting that lipid oxidation is a process that begins during ingredients mixing and goes on until baking and during the storage [
51]. Nevertheless, since the samples were subjected to the same conditions of kneading and baking, presumably the higher content of aldehydes and alcohols in sourdough containing-muffins could be due to the action of lactic acid bacteria and yeasts that during the fermentation process promote the generation of hexanal, 1-hexanol, hexanoic acid (as confirmed by the highest content in M
10% and M
15%) [
52]. Moreover, compounds having amino acids as precursor (e.g., 2-penthyl- and ehyl-octanoate and carbon sulfide) were more concentrated in sourdough containing-muffins due to the higher concentration of amino acids released during sourdough fermentation and the acidic environment [
50]. Nevertheless, some of these compounds can also be formed during Maillard reaction, enhanced by free amino acids release [
53,
54]. A singular trend was observed for methyl ketones (C5-C9). As already reported in the literature [
55], methyl ketones are formed from incomplete β-oxidation; the steps include β-oxidation of the released FFA to β-ketoacyl-CoA, which are then deacylated into β-ketoacids under the action of the thioesterases and then the keto acids are decarboxylated to methyl ketones. In fact, their formation can be related to β-oxidation of fatty acids carried out by both lactic acid bacteria and yeasts [
55,
56] as well as by seed enzymes [
57]. They reached their maximum levels in M
5% muffins, pointing out the contribute of microbial β-oxidation to the volatile pattern of muffins, while decreasing where higher levels of sourdough were added (
Table 4). This nonlinear trend could be attributed to an easier involvement of their carbonyl functional group in non-enzymatic browning, that is, Maillard reaction, occurring in higher extent in muffins with higher amounts of sourdough, as pointed also by color analysis showing a significant decrease of the luminosity index L* in the crust (
Table S3).
Arendt et al. [
58] indicated that most of the gluten-free bakery products on the market have very poor quality, particularly when compared to traditional wheat flour yeast bread, since they have reduced flavor and a crumbly and dry texture. Sourdough has been shown to improve overall bread quality, enhancing the textural properties and prolonging shelf life [
59]. In this framework, the sensory evaluation and the textural parameters determination represent key points to develop a new gluten-free product. Our results from sensory evaluation highlighted that odor is the main descriptor to discriminate among M
CT and the fortified samples. Indeed, sourdough fermentation has widely been reported as suitable tool to improve the sensorial characteristics of GF baked goods [
60] with lactic acid bacteria generating very specific aroma profiles and odorant compositions [
59].
The highest values of firmness for the control and the samples M
5%, agree with the those obtained from the image analysis, in fact firmness and porosity can be positively correlated, thus revealing information about the structure [
61]. Porosity is caused by the production of the CO
2 by yeast and some heterofermentative lactic acid bacteria and increases with fermentation [
62].
The increase of the phenols extractability thanks to the acidic environment and the microbial enzymatic activity [
63] led to higher radical scavenging activity in muffins containing the type-I sourdough. The effect was in accordance to the level of fortification (
Table 2). Besides the nutritional value of the antioxidant compounds, from a technological point of view, such feature can contribute to the long-term oxidative stability of foods [
64]. As regards the nutritional value of the muffins, high content of protein (>6%) and total dietary fibers (>3%) were found in all samples, thus suggesting the possible labels—“rich in protein” and “source of dietary fibers” [
65].
The presence of high levels of insoluble fiber and high concentrations of antinutritional factors might be responsible for poor protein digestibility and amino acid availability [
66]. Teff is rich in phytic acid, myo-inositol hexakisphosphate, which negatively affects the mineral and protein adsorption at the intestinal level [
67]. This is, also, one of the major concerns in using teff to make staple foods [
68]. As already suggested by the literature [
69], the combined effect of the endogenous phytases activated through the acidification operated by lactic acid bacteria and the microbial activity led to significant decreases (up to 50% lower than control) of the content of the phytic acid in sourdough muffins (
Table 3).
The proteolytic activity of the lactic acid bacteria led to increases of TFAA concentration, with relevant extents in the essential (2-times higher), hydrophobic (4-times) and aromatic (2-times) free amino acids (
Figure 2). Hydrophobic and aromatic amino acids assist in radical scavenging and metal chelating activities. Amino acids with aromatic side groups are assumed to contribute to the strong radical scavenging activities of peptides [
70]. The release of the FAA during sourdough fermentation also contributes to the enhancement of the nutritional value of sourdough and related food products due to their higher absorbance in the intestine [
71,
72]. Indeed, a 35% higher IVPD values were found in sourdough-containing muffins as compared to M
CT.
The high content of fibers as well as the use of sourdough fermentation led to a decrease of the HI of sourdough-containing muffins as compared to the control. The synthesis of organic acids, especially lactic acid has been related to the decrease of the digestibility rate of the starch [
73].
Food quality is a multivariate notion—taste, health and shelf-life need to be improved in parallel. Sourdough-containing muffins showed lower degree of fungal contamination when higher contents of sourdough were used for the fortification. Organic acids (i.e., acetic, phenyllactic) play an important role in terms of rope inhibition and prolonged shelf-life of baked products [
74]. However, the release of antimicrobial compounds during sourdough fermentation (mainly peptides) have also to be considered [
75].