1. Introduction
Barley (
Hordeum vulgare L.) as cereal is more used for production of beer, but in the last few years, there is a growing interest to use it as food—like breakfast cereal or groats. The interest in using hull-less barley is growing because it is easy to use (there is no hull) and has high nutrition value and high content of fibers, β-glucans and arabinoxylans [
1,
2]. Barley is not used in bread baking especially because of the properties of β-glucans, since barley has bad baking performance and sensory quality as bread. However, the research shows that the use of a sourdough system in making bread, especially wholemeal bread, creates a positive effect [
3,
4]. Also, during the fermentation, the fibers become more soluble, and this can have a positive effect on the health of people [
5]. By using wholegrain flour, a more diverse environment is created for the growth of microorganisms, their development and metabolic activity. The available amount of vitamins, minerals, fibers and biologically active components influences the microbiota of the sourdough [
6].
There are descriptions of different types of sourdough fermentation: spontaneous fermentation, fermentation with a starter culture and dried/stabilized after preparation sourdough [
7], together with mixed-type fermentation that has been started with pure culture and then succeeding continuing with backslopping procedure (also called “refreshment”) [
8]. Each of these types is characterized by a different combination of microorganisms.
Sourdough fermentation can start spontaneously not only with starter culture or mother dough. In spontaneous fermentation, the natural microbiota of the flour is used, and it is influenced by the microbiota of the environment and raw materials. The sourdough prepared with the spontaneous method is based on backslopping processes—repeated cyclic backslopping with a new amount of water and flour [
9]. Through the cycles of backslopping, the microorganisms from different genera have adapted depending on available nutrition elements and influencing parameters of the technological process: temperature, time and amount of added water [
10]. The traditional spontaneous sourdough or the type 1 sourdough is used in the artisan bakeries: the time of fermentation is from short to average (6 to 24 h), the temperature is moderate, 20–30 °C, with low DY < 200 (DY = (flour + water) × 100/flour weight) [
8,
11]. The type 1 sourdough is suitable for leavening the dough without additional leavening agents [
7].
There are several protocols to start spontaneous sourdough preparation, the better-known ones are the French system for wheat sourdough, the U.S sourdough from wheat and/or rye flour and the spontaneous rye sourdough preparation, which is traditional for the Northern and Eastern part of Europe [
7]. In Latvia, the traditional rye bread is made from mother’s dough, which is adapted in bakeries [
12]. Spicher and Stephan [
13] describe different 3–4 step (Stufe) systems to start fermentation with different parameters from 68 to 96 h, in all steps together, at 23–35 °C, and 160–400 DY. Kozlinskis [
12] describes the three-phase system of preparing the rye sourdough. A fermentation system with several backslopping steps allows the option of slowly reducing the acidity as the result of activity of the homofermentative lactic acid bacteria in the sourdough and providing a favorable environment for the growth of yeasts and heterofermentative lactic acid bacteria. In this way, the function of type 1 sourdough leavening would be secured. At the end of the last step of fermentation, such sourdough can be seen as a starter culture, which contains diverse microbiota [
7]. But there is still little research about the use of other cereals, especially barley, for preparation of spontaneous sourdough.
To obtain an active and complete type 1 sourdough from wheat or rye, at least three backslopping steps are necessary [
8,
14]. Stable ripe active sourdoughs are characterized by high carbohydrate concentrations, low pH, and higher lactic acid bacteria cell counts >10
8 CFU g
−1 (colony forming units per gram) compared to the yeast cell counts <10
7 CFU g
−1 [
8]. The pH of the well-developed sourdough is in the range of 3.5–4.3 [
7]. Several influencing parameters in this process can be changed in order to achieve the desired result, such as the ratio of flour and water (DY), temperature and time [
8,
15].
The traditional cereals used for spontaneous fermentation such as rye and wheat are mainly described and researched, but barley can be also used as an alternative. The fermentation capacity of sourdough is determined not only by the microbiota of the flour but also by the type of flour that is used (cereal culture, level of milling, activity of enzymes). The β-glucans in the hull-less barley are characterized by the high capacity to absorb water, and this aspect might be considered as an influencing factor in the making of spontaneous sourdough, which helps for successful development of needed microbiota.
In the active sourdoughs of barley, typical diverse cereal microbiota are observed. The barley sourdough with the fermentation parameter characteristics for type 1 sourdough contains
Pediococcus (dominant species),
Lactobacillus curvatus (new name
Latilactobacillus curvatus [
16]),
Leuconostoc mesenteroides [
17],
Lactobacillus plantarum (new name
Lactiplantibacillus plantarum [
16,
18])
, Weisella confusa [
10] bacteria, and
Saccharomyces cerevisiae, Candida humilis, and
Pichia anomala yeast species [
9]. The main role of lactic acid bacteria is acidifying, but yeasts secure leavening by relieving CO
2 [
19]. From this aspect, it is important to develop symbiosis between lactic acid bacteria and yeasts. By changing the influencing parameters, it is possible to determine which microbiota will develop. It is possible to achieve the dominance of heterofermentative or homofermentative microorganisms as well as the development or complete disappearance of yeasts.
There is just a little research about the development of spontaneous microbiota of hull-less barley [
20]. However, it would be useful to research specifically the starting steps of the sourdough of spontaneous fermentation, as well as the optimization of the influencing parameters for the development of the desired ecosystem. A multistep production system of spontaneous sourdough with three backsloppings is used to apply the different optimal conditions for every step, which allows it to be used for specific applications, mainly as a leavening agent for sourdough bread production or as a mother sponge stored till being used for the next bread making stage.
Taking into account that the nature of the fermentation sourdoughs is very diverse and is characterized by simultaneous mutual interference of different factors, the response surface methodology would be the most appropriate method for optimizing the influencing factors [
20,
21].
The goal of the research is to determine the optimal fermentation parameters of the spontaneously fermented active sourdough in a controlled environment in the three phases of fermentation, and to characterize the diversity of microbiota depending on the conditions of fermentation in three stages of the development.
2. Materials and Methods
2.1. Raw Material
Grains of hull-less barley (Hordeum vulgare) cultivar ‘Kornelija’ from the harvest of 2020 grown in Stende (lat. 57.1412° N, long. 22.5367° E) were used in the study. Harvested grains were cleaned in a cleaner (PETKUS Technologie GmbH, Hamburg, Germany) with the rectangular holes sieve (2.2 × 20 mm). The whole grain flour of hull-less barley cultivar ‘Kornelija’ (moisture 12.05 ± 0.42%, protein 17.76 ± 0.26%, starch 57.84 ± 0.46%, soluble dietary fiber 23.3 ± 4.7%, insoluble dietary fiber 3.2 ± 0.8%) were obtained by milling the hull-less barley grains in a Hawos laboratory mill (Hawos Kornmühlen GmbH, Hamburg, Germany). Flour was sieved through AS 200 mesh (Retsch GmbH, Haan, Germany), particle size 160–710 µm, among which 50% were about 450 µm.
2.2. Experimental Design for Sourdough Preparation and Statistical Analysis
Wholemeal hull-less barley flour cultivar “Kornelija” was used. Sourdough was prepared under laboratory conditions (controlled conditions) in the Bread technology laboratory of the Faculty of Food Technology of the Latvia University of Life Sciences and Technologies.
The Box–Behnken design method was used in order to evaluate the simultaneous impact of independent factors (amount of added water, X
1; temperature, X
2; time of fermentation, X
3) to the parameters (responses) (
Table 1), like the pH, the number of lactic acid bacteria and yeasts.
Sourdough was prepared by mixing wholemeal barley flour and tap water. As the base for preparation of spontaneous sourdough, the three-stage strategy with one backslopping procedure for each fermentation step was used. To start the fermentation, 100 g hull-les barley flour and the amount of added water, fermentation time and temperature were taken as indicated in the experimental design (
Table 1). In the 2nd and 3rd backslopping of sourdough preparation, 100 g of previously fermented sourdough was added, mixed with the amount of flour and water as specified in the design of the experiment (
Table 1).
Next, the optimized parameters of the factors of fermentation steps were established in accordance with the defined criteria of each fermentation step.
Equation (1) is a second order polynomial equation:
The minimum and maximum values of the factors influencing the spontaneous fermentation were established beforehand. Three Box–Behnken designs were created—one composite design with 15 runs for each step.
The values of the influencing factors were entered—chosen by Design-Expert version 12 (Statease Inc., Minneapolis, MN, USA). Statistical significance was measured by analysis of variance (ANOVA). To evaluate the adjustment of the models, regression coefficients, the p-values and determination coefficient R2 were considered.
2.3. Determination of Number of Lactic Acid Bacteria (LAB) and Yeasts and Statistical Analysis
Preparation of test samples, an initial suspension and decimal dilutions for microbiological examination were performed according to the ISO 6887-1:1999 [
22].
The enumeration of lactic acid bacteria has been established according to the ISO 15214Standard [
23], using the MRS agar (Biolife) selective medium. The Petri plates were incubated for 72 h at 30 °C in an incubator cabinet.
The yeast numbers have been established according to the ISO 21527-1 Standard [
24], using the malt extract agar (Biolife). The Petri plates were incubated for 72 h at 25 °C.
The horizontal method for the enumeration of microorganisms, Colony-count technique according to EN ISO 4833:2003, was applied. In order to establish the number of the colonies of microorganisms, the automatic counter of colonies aColyte (Topac Inc., Cohasset, MA, USA) with error limit <5% has been used. The total number of microorganisms was showed as colony forming units log CFU g−1.
The data were subjected to one-way analysis of variance (ANOVA). Significant differences (p < 0.05) were determined using Tukey’s Post Hoc test.
2.4. Determination of pH
The pH of sourdough was determined according to the AACC 02-52:1999 method [
25] using a pH meter (Mettler—Toledo GmbH, Giessen, Germany) at 20 ± 1 °C.
2.5. Statistical Analysis
Optimized factors in the three backslopping steps of fermentation and experimentally determined data of pH, LAB and yeasts enumeration were subjected to one-way analysis of variance (ANOVA), and significant differences (p < 0.05) were determined using Tukey’s Post Hoc test.
2.6. Microorganisms Identification
After each step of sourdough fermentation in each step, the samples were lyophilized in freeze-dryer FT33 (Armfield Ltd., Hampshire, UK) in a condenser chamber at 40 °C and 6.4 Pa for 72 h.
For microbial DNA extraction, 5 g of lyophilized sourdough was weighed and put into sterile 50 mL tubes under the laminar flow cabinet, the sterile hypotonic solution was added, and cells were isolated using gradual centrifugation. After the cell collection, gDNA was extracted using a ZR Quick DNA Fungal/Bacterial Miniprep Kit (Zymo Research Corp, Irvine, CA, USA).
The quantity of the DNA was determined by Qubit™ 4 Fluorometer using a dsDNA HS Assay Kit (Thermo Fisher Scientific). All samples were qualified for sequencing (OD 260/280 > 1.8 and OD 260/230 > 1.7) and used for library preparation according to the in-house sequencing protocol [
26] with V4 (F515/R806) primer pair for 16S rRNA sequencing [
27], and ITS 1 (multiplex primers set) [
28] and ITS2 primers for fungi identification [
29] The reaction was carried out with FIREPol
® Master Mix (Solis BioDyne OÜ, Tartu, Estonia) in an Eppendorf Mastercycler (Sigma-Aldrich Chemie GmbH, Germany). For the multiplexing, Nextera XT Index Kit v2 Set A (Illumina Germany, Berlin, Germany) was used. PCR products were purified by NucleoMag
® NGS Clean-up and Size Select Magnetic Beads (MACHEREY-NAGEL, Germany) in accordance with the manufacturer’s manual.
Amplicon sequencing was performed using the iSeq 100 platform (SN FS10000643, Illumina) and iSeq 100 i1 Reagent kit v2 by 2 × 150 bp, dual index setup for 16S rRNA amplicon protocol and single-end approach for the fungal ITS pipeline.
The processing of sequencing reads according to the primer sequences has been performed with in-house scripts. 16S rRNA sequence data were analyzed using the BION-meta program [
30] according to the author’s instructions. First, sequences were cleaned at both ends using a 99.5% minimum quality threshold for at least 18 of 20 bases for the 5’ end and 28 of 3’ end, then joined, followed by removal of shorter read pairs than 150 bp. Then, sequences were cleaned from chimeras and clustered by 95% oligonucleotide similarity (k-mer length of 8 bp, step size 2 bp). Lastly, consensus reads were aligned to the SILVA reference 16S rDNA database (v138) using a word length of 8 and similarity cut-off of 90%. ITS sequence data were analyzed using QIIME2 pipeline. The microbial designation was analyzed at different taxonomic levels down to species if applicable.
Statistical analysis and visualization were done in R version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria).