Amelioration in gliadin antigenicity and maintenance of viscoelastic properties of wheat (Triticum aestivum L.) cultivars with mixed probiotic fermentation

Abstract

Sourdough fermentation of twenty wheat cultivars was carried out using mixed probiotic culture (Lactobacillus acidophilus UNI, Lactobacillus brevis LR/5 and Lactobacillus plantarum ATCC 8014). The gliadin antigenicity was expressed in terms of its content in twenty different wheat cultivars. The gliadin proteins were characterized by SDS-PAGE and structural changes analyzed on FTIR spectrophotometer. Moreover, changes in the viscoelastic character of fermented and non-fermented dough were studied by rheometry. The results showed a remarkable reduction in antigenicity by 60% (average) in all wheat cultivars on sourdough fermentation. This reduction may be due to the synergistic effect of protease secretion by mixed lactobacilli, responsible for gliadin degradation. These changes in gliadins by mixed culture proteolysis were confirmed on SDS-PAGE on observing new gliadin-derived low molecular weight peptides. The results were further validated by FTIR spectroscopy where structural changes of gliadins were analyzed in the fermented dough. The rheological data indicated a higher storage modulus (G′) compared to loss modulus (G″) in both control and fermented flour of all wheat cultivars, however, with a lower efficacy in sourdoughs. The present study thus establishes that mixed culture sourdough fermentation decreases the antigenic potential of gliadins without any change in the rheology and thereby maintaining the baking or viscoelastic properties of the wheat flour.

Introduction

Wheat (Triticum aestivum L.) is a chief cereal grain cultivated and consumed worldwide (IWGSC 2018). It is a staple food in many countries (CGIAR Research Program on Wheat 2017). Wheat consists of four protein fractions viz., albumins, globulins, gliadins and glutenins. Albumin and globulin are water-soluble proteins that have high nutritive value where as gliadin and glutenin together known as (gluten) are water-insoluble protein fractions, known for their viscoelastic and baking properties and hence making them valuable for food industry (Waga 2004; Wang et al. 2015). Although wheat is best known for its high nutritive and viscoelastic properties, nevertheless, if some deleterious element in wheat is implicated as a risk factor for some biological complication, it would have serious consumer health connotation. Consequently, there are many reports that link consumption of this cereal with the implication of various diseases (Elli et al. 2015; Kolasinska et al. 2018). In this regard, consumption of gliadins in wheat has been correlated with the incidence of many disorders like gluten intolerance (autoimmunity), celiac disease (CD), insomnia and others. The most common symptoms in children include atopic dermatitis (AD) and in adults urticaria and wheat dependent exercise-induced anaphylaxis (WDEIA) (Foschia et al. 2016; Burkhardt et al. 2018). The wheat induced biological complication i.e. celiac disease (CD) is a chronic gastrointestinal tract disorder in which small intestinal mucosa is impaired by an autoimmune mechanism (Scherf et al. 2015; Parzanese et al. 2017). In order to overcome these health complications, the best remedy considered is to remove or lessen this immunological risk factor (gluten) from the diet. Nowadays, the more substantial approaches adopted at large scale is the sourdough fermentation of wheat flour by probiotic lactobacillus strains that is more natural and safe way of reducing gluten antigenicity (Rizzello et al. 2007; Diowksz and Leszczynska 2014) keeping dough viscoelastic properties intact. These lactobacilli bacteria are reported to secrete specific proteases that are capable of hydrolyzing immunogenic gluten peptides involved in gluten causing diseases like CD (De Angelis et al. 2010; Di Cagno et al. 2004). Moreover, the mixed probiotic culture adds to the excellence by producing numerous set of proteases; therefore, confer more effectiveness in reducing immunoreactivity of wheat grain proteins or peptides (Di Cagno et al. 2010; Leszczynska et al. 2012; Gerez et al. 2012). In addition to the efficacy of probiotic specific proteases, sourdough fermentation activates endogenous grain proteases including aspartic peptidases and carboxypeptidases that further reduce gluten antigenicity (Ganzle et al. 2008; Scherf et al. 2018). Overall acidification, proteolysis and activation of a number of enzymes as well as the synthesis of microbial metabolites cause several changes during sourdough fermentation, which affect the dough and baked good matrix, and influence the nutritional/functional quality (Elsanhoty et al. 2016; De Angelis et al. 2010; Di Cagno et al. 2010). These studies therefore clearly demonstrate sourdough fermentation as a promising approach to reduce the antigenic potential of gluten with enhanced nutritional and intact viscoelastic properties. Keeping the above facts under consideration, the present study was designed to investigate the changes in gliadin antigenicity and viscoelastic nature of the wheat flour on sourdough fermentation with three probiotic strains (Lactobacillus acidophilus UNI, Lactobacillus brevis LR/5 and Lactobacillus plantarum ATCC 8014).

Materials and methods

Chemicals

Acrylamide (Sigma-Aldrich Inc., USA), Bisacrylamide (Sigma-Aldrich Inc., USA), Molecular Marker (BLUeye prestained protein ladder-Sigma-Aldrich Inc., USA), Sodium dodecyl sulfate (Sisco Research Laboratories (SRL) Pvt. Ltd., Mumbai), Petroleum ether (S. D. Fine Chem. Ltd, Mumbai), Anti-Gliadin (Wheat) antibody produced in rabbit (Sigma-Aldrich Inc., USA), Bovine serum albumin (Genetix Asia Pvt. Ltd., New Delhi) and Lactobacillus MRS Broth (HiMedia Laboratories Pvt. Ltd., Mumbai).

Materials

Wheat cultivars

Twenty leading wheat cultivars WH-1021, PBW-175, DBW-17, HD-2888, HD-2932, HD-2864, GW-322, PBW-596, HD-2781, C-306, RAJ-4083, DBW-14, RAJ-4120, PBW-396, HD-2987, K-0307, HI-1531, RAJ-3765, RAJ-3077 and RSP-561 were procured from Directorate of Wheat Research (DWR), Karnal, Haryana, India. Freshly harvested wheat grain samples were sun-dried and ground to fine powder for further analysis.

Lactobacillus strains

For sourdough formulations, three Lactobacillus strains viz. L. acidophilus UNI, L. brevis LR/5 and L. plantarum ATCC 8014 were procured from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India.

Methods

Proximate analysis of wheat flour

For proximate analysis, different parameters (moisture, fat, ash and protein) of 20 cultivars of wheat flour expressed in terms of percent dry matter (% dm) were determined according to the standard methods of AOAC (1990).

Moisture content

Aluminum moisture dish was dried in an oven at 130 °C followed by cooling in an airtight desiccator and the weight of the dried dish was recorded. Sample (2 g) was taken in this dried dish kept in hot air oven uncovered (with an opening for ventilation) at 130 ± 3 °C for 1 h. After 1 h, the dish was covered with lid and transferred to airtight desiccator. The dish containing the sample was then allowed to cool to room temperature and the weight was recorded. The loss in weight of the sample was calculated as follows:

$$\text{Moisture}\text{content}=\frac{\text{Loss}\text{in}\text{weight}\text{of}\text{the}\text{sample}\left(\text{g}\right)}{\text{weight}\text{of}\text{sample}\left(\text{g}\right)}\times 100$$

Fat content

Ground dried samples (5 g) were taken in pre-weighed thimbles covered with the cotton plug. These thimbles were dried, placed in Soxhlet Extractor (Soxtee™ 2043) and then petroleum ether was percolated (2–3 drops per second) over the samples for 3 h. The petroleum ether was distilled off and the pre-weighed aluminum canisters (containing fat) were kept in the oven at 100 °C for 30 min for the removal of residual solvent. The canisters were then cooled and the weight was recorded as follows:

$$\text{Fat}\text{content}=\frac{\text{Weight}\text{of}\text{fat}\left(\text{g}\right)}{\text{Weight}\text{of}\text{sample}\left(\text{g}\right)}\times 100$$

Ash content

The samples (5 g) were taken in pre-weighed porcelain Ashing Dishes and ignited at 550 ± 10 °C for 6 h in a muffle furnace (NSW-102). The ash was then cooled in airtight desiccators and the weight was recorded at room temperature as follows:

$$\text{Ash}\text{content}=\frac{\text{Weight}\text{of}\text{ash}\left(\text{g}\right)}{\text{Weight}\text{of}\text{sample}\left(\text{g}\right)}\times 100$$

Biochemical analysis

The proteins from control and fermented dough were extracted following the method of Dvoracek and Curn (2003). Briefly, albumins and globulins were extracted from defatted grain flour by dissolving in distilled water and NaCl solution (0.5 N) respectively. The gluten fraction (glutenins and gliadins) were then extracted from the residue in a step-wise manner through homogenization using the sodium-borate buffer (pH 10), ethanol (70%) and β-mercaptoethanol (5 mM) respectively. The protein estimation was done by Lowry et al. (1951).

Sourdough fermentation

The probiotic lactobacilli (L. acidophilus UNI, L. brevis LR/5 and L. plantarum ATCC 8014) were propagated for 24 h at 37 °C in sterile MRS broth. The broth cells of each bacterium were harvested at 25,000×g for 5 min at 4 °C. The bacterial cells were collected in sterile saline (0.85%) and then preserved in glycerol (18%) at − 80 °C for further use. The number of cells was measured by serial dilution and counted (CFU/mL) on the plates after growing at 37 °C for 36 h as, CFU/mL = CFU × dilution factor × 1/aliquot. For sourdough preparation, wheat flour (200 g), tap water (70 mL) and 30 mL of the cellular suspension containing 10⁹ CFU of each lactobacillus bacterial species/mL (final concentration in the dough, 5–10⁷ CFU/g) were used to produce 300 g of dough. Sourdough fermentations were carried out at 37 °C for 24 h with a continuous high-speed mixer (60 g, 5 min) at final pH (4.6–5.6). The dough produced without bacterial inoculum served as control.

Estimation of gliadins by indirect ELISA

The gliadin extracts both from fermented and control dough were diluted (1:100) in phosphate buffer saline (PBS) and then mixed (1:1) with carbonate-bicarbonate buffer (0.1 M, pH 9.6). Briefly, the coating of microtiter plates with samples containing gliadins from control and fermented sourdough was performed overnight at 4 °C. Next day the sample solutions were decanted and blocking was done with BSA (2%) followed by washing with PBS. The primary un-conjugated antibody (100 µL, anti-gliadin produced in rabbit, Sigma, USA) was added and incubated for 2 h at room temperature. Then conjugated secondary antibody (100 µL, anti-rabbit IgG whole molecule peroxidase) was added and the plates were incubated at room temperature for 2 h. Then, the substrate solution (TMB, Sigma) was added and plates were incubated for 30 min. The reaction was stopped by adding 50 µl of stop solution for TMB (obtained from Sigma Aldrich) and the output absorbance was measured at 450 nm on ELISA Reader (Multiscan RC reader, Genetix).

Analysis of gliadins by SDS-PAGE

Extraction

The gliadin protein fractions were isolated from the control and sourdough fermented wheat cultivars and analyzed on SDS-PAGE. Protein fractions were extracted by the method described by Weiss et al. (1993). Briefly, 1 g of control and the sourdough sample was extracted with Tris-HCl buffer (50 mM, pH 8.8) for 1 h at 4 °C with occasional vortexing and then centrifuged at 10,000×g for 20 min. The supernatant containing albumins and globulins was removed and the pellet was taken and then treated with ethanol (75%) for 2 h at 25 °C in an orbital shaker and centrifuged (10,000 rpm). The supernatant containing gliadins were collected and stored at − 80 °C for further analysis. The protein content was estimated by following the method of Lowry et al. (1951).

SDS-PAGE

The banding pattern of gliadin proteins extracted from control and fermented wheat samples were detected by the sodium dodecyl sulfate-polyacyrlamide gel electrophoresis (SDS-PAGE) performed according to Laemmli (1970) on 10% separating and 5% stacking gels in vertical electrophoretic unit. Prior to the electrophoresis, the proteins fractions were diluted in the ratio 1:2 (v/v) with the sample buffer, heated at 90 °C for 5 min and cooled to room temperature. An equal amount of protein (40 μg/well) was loaded in each well. Gels were run at 50 mA for 5 h, fixed and stained with methanol, glacial acetic acid, distilled water in the ratio of 4.5:1:4.5. Destaining was performed with methanol, acetic acid and distilled water in the ratio of 1:0.5:3.5. Molecular weights of the polypeptides from control and sourdough fermented flour were estimated by using known molecular weight marker (BLUeye Prestined protein Ladder, Sigma).

Determination of structural changes

The gliadins isolated from the control and fermented dough were lyophilized, blended separately with potassium bromide (KBr) and pressed into tablets before measurement. The tablets were then placed on the disc of the FTIR instrument. The structural changes in the lyophilized gliadins of control and fermented dough samples were then analyzed by FTIR spectroscopy and the spectra were recorded on an FTIR Spectrophotometer (CARY 630, Agilent Technologies, USA) at room temperature. The spectra were recorded within the range of 400–4000 cm⁻¹ and the spectrum of the each sample was recorded in transmittance mode.

Rheology of the wheat dough

Frequency sweeps were performed on a controlled stress rheometer (Anton Paar MCR 301, Ostifildern, Germany) fitted with a coaxial geometry. All measurements were done at 25 °C, using parallel plate geometry (40 mm diameter and 1 mm gap). The dough sample was placed between the plates and the edges were carefully trimmed with a spatula. Amplitude sweeps were conducted to establish the linear viscoelastic regions of the samples. Frequency sweeps from 1 to 50^1−S angular frequency (ω) were performed with a target strain of 10⁻³ (0.1%) at 30 °C. Finally storage (G′) and loss (G″) modulus values were obtained. All the rheological experiments were performed at least twice and their averages were recorded. The storage modulus (G′) and loss modulus (G″) were calculated using the Origin software (version OriginPro 8.5).

The power law model (Eqs. 1, 2) was used to analyze the flow behavior of control and fermented dough samples

$${\text{G}}^{\prime }={\text{K}}^{\prime }{\left(\mathrm{\omega }\right)}^{\eta \prime }$$ 1

$${\text{G}}^{″}={\text{K}}^{″}{\left(\mathrm{\omega }\right)}^{\eta ″}$$ 2

where K′ and K″ indicate consistency coefficient while η′ and η″ represent flow behavior index.

Statistical analysis

Data were analyzed using the Graph Pad Prism (version 5.01). Experimental results are mean ± SEM (standard error mean). Data were subjected to analysis of variance (ANOVA), and the Tukey test was used to separate the means (p ≤ 0.05) that were considered statistically significant. The Fitting of Power-Law Model was performed in R software version 3.4.1 setting level at 0.05.

Results and discussion

Basic characteristics of wheat flour

The basic parameters for dough formation like moisture (13.0% dm), ash (1.8% dm), fat (1.8% dm) and protein (11.0% dm) of wheat flour required for proper dough formation was found in the normal range as per AOAC (1990) as depicted in Table 1.

Table 1.

Proximate analysis in grains of twenty Indian wheat cultivars

Wheat cultivar	Moisture content (%)	Ash content (%)	Fat content (%)	Total protein (%)
HD-2764	13.0 ± 0.3477d	1.8 ± 0.0235a	1.6 ± 0.357b	12.40 ± 2.772bc
RAJ-3765	15.0 ± 0.4012 a	1.6 ± 0.0209b	1.8 ± 0.402a	11.81 ± 2.640f
GW-322	12.5 ± 0.3343e	1.6 ± 0.0209b	1.4 ± 0.313 c	11.35 ± 2.537 l
RAJ-3077	13.5 ± 0.3611c	1.6 ± 0.0209b	1.8 ± 0.402 a	12.49 ± 2.792 a
HD-2781	12.5 ± 0.3343e	1.8 ± 0.0235a	1.8 ± 0.402a	12.44 ± 2.781b
WH-1021	13.0 ± 0.3477d	1.6 ± 0.0209b	1.8 ± 0.402a	11.61 ± 2.596hi
PBW-175	10.5 ± 0.2808 h	1.8 ± 0.0235a	1.4 ± 0.313c	11.86 ± 2.651ef
RSP-561	10.0 ± 0.2675 i	1.8 ± 0.0235a	1.6 ± 0.357b	11.51 ± 2.573jk
HD-2888	12.5 ± 0.3343e	1.8 ± 0.0235a	1.8 ± 0.402a	11.55 ± 2.582i
K-0307	14.5 ± 0.3878b	1.6 ± 0.0209b	1.8 ± 0.402a	10.76 ± 2.406 q
RAJ-4120	10.5 ± 0.2808 h	1.8 ± 0.0235a	1.8 ± 0.402a	11.65 ± 2.605gh
RAJ-4083	12.5 ± 0.3343e	1.8 ± 0.0235a	1.6 ± 0.357b	11.54 ± 2.580ij
HI-1531	11.0 ± 0.2942 g	1.8 ± 0.0235a	1.6 ± 0.357b	11.67 ± 2.609 g
HD-2932	12.5 ± 0.3343e	1.8 ± 0.0235a	1.8 ± 0.402a	11.90 ± 2.660de
HD-2987	12.5 ± 0.3343e	1.8 ± 0.0235a	1.8 ± 0.402a	11.94 ± 2.669d
DBW-17	10.0 ± 0.2675 i	1.6 ± 0.0209b	1.6 ± 0.357b	11.44 ± 2.558 k
PBW-396	10.0 ± 0.2675 i	1.6 ± 0.0209b	1.6 ± 0.357b	11.17 ± 2.497n
DBW-14	11.0 ± 0.2942 g	1.8 ± 0.0235a	1.6 ± 0.357b	11.29 ± 2.524 lm
PBW-596	12.0 ± 0.3210f	1.6 ± 0.0209b	1.8 ± 0.402a	10.98 ± 2.455op
C-306	12.5 ± 0.3343e	1.8 ± 0.0235a	1.8 ± 0.402a	11.03 ± 2.466o
CD (p ≤ 0.05)	0.10	0.03	0.01	0.15

Bold values denote highest and bold italic values represent lowest values

Means with the same letter are not significantly different

Effect on protein contents

The amount of water-soluble (albumins and globulins) and insoluble proteins (gliadins and glutenins) determined in the present investigation before and after sourdough fermentation with their maximum and minimum levels are depicted in Table 2. It is evident that both types of proteins varied inter-genotypically among the twenty wheat cultivars. Results of the present study indicate a decrease in gliadin content in the sourdough fermented cultivars of wheat flour (0.81–1.26%) compared to control (3.52–3.97%). A similar pattern of decline was observed in glutenin content in fermented wheat flour (2.05–2.60%) in comparison to their respective controls (4.22–4.59%). Moreover, results depicted a decrease in gluten content in the fermented wheat flour (3.21–3.82%) compared to the control (7.80–8.53%) with a percent reduction of 54.8% (RAJ-3765) to 61.8% (HD-2781).Water-soluble protein fraction (albumins and globulins) increased in fermented wheat flour (5.60–7.01%) compared to the respective controls (2.94–4.18%) with percent increase ranging from 66.5% (HD-2764) to 92.5% (DBW-17). After sourdough fermentation, the maximum soluble protein content was recorded in RAJ-3077 (7.01%). Overall these results indicated an increase in soluble protein content and a decrease in water-insoluble protein fraction. RAJ-3077 with maximum soluble protein content (7.01%) can be regarded as the best wheat cultivar. The protein levels in different wheat cultivars differ, however, the results are in agreement with the reported protein levels estimated by Mallick et al. (2013). Wheat is a rich source of water-soluble and insoluble proteins that contribute to nutritional and viscoelastic properties (Branlard et al. 2001). Among soluble proteins, albumins and globulins have got paramount significance because of improved biological value and hence considered nutritionally superior. The gluten fraction, comprising of gliadin and glutenin have enough water and gas retention properties that contribute to viscoelastic properties and therefore significant in food industry (Stehno et al. 2008). The data generated in the present investigation depict an increase in water soluble protein content (albumins and globulins) and a decrease in water-insoluble protein content (glutens). Therefore, it could be inferred that sourdough fermentation enhanced the biological value and hence improved the nutritional quality of wheat flour.

Table 2.

Impact of lactobacilli sourdough fermentation on protein levels and antigenicity in different wheat cultivars

Wheat cultivars	Albumin + globulin (% mg)		Gliadin (% mg)		Glutenin (% mg)		Gluten (% mg)		% antigenicity (OD) of gliadins
	Control sample	Fermented sample	Control sample	Fermented sample	Control sample	Fermented sample	Control sample	Fermented sample	Control	Fermented	Decrease (%)
HD-2764	4.00ab	6.66 de ( ±  66.5)	3.91ab	1.13efg (− 71.1)	4.50bcd	2.29jk (− 49.1)	8.41ab	3.39hi (59.7)	0.198 ± 0.0030ef	0.083 ± 0.0014c	58.0
RAJ-3765	3.85bc	6.66de (+ 72.9)	3.67fghi	1.18cd (− 67.8)	4.29fghi	2.42gh(− 40.3)	7.96lmn	3.60 cd (54.8)	0.185 ± 0.0028ghi	0.067 ± 0.0011 j	63.7
GW-322	2.94 p	5.60efg (+ 90.4)	3.97 a	1.26a (− 72.3)	4.56ab	2.56cd (− 43.9)	8.53 a	3.82a (− 55.2)	0.217 ± 0.0033 a	0.087 ± 0.0014 a	59.9
RAJ-3077	4.18 a	7.01a (+ 67.7)	3.87bc	1.13efg (− 70.8)	4.43cde	2.60a (− 41.3)	8.30cde	3.73ab (55.1)	0.205 ± 0.0031bcd	0.071 ± 0.0012hi	65.3
HD-2781	4.02ab	6.73bc (+ 67.4)	3.88bc	1.16de (− 70.1)	4.53bcd	2.05m(− 54.7)	8.41ab	3.21 lm (61.8)	0.203 ± 0.0031cde	0.074 ± 0.0012gh	63.5
WH-1021	3.19mn	6.06op (+ 90.0)	3.87bc	1.03ghij (− 73.4)	4.44cde	2.58bc (− 41.9)	8.31cd	3.81a (− 54.2)	0.211 ± 0.0032ab	0.075 ± 0.0012fg	64.4
PBW-175	3.61f	6.55ghi (+ 81.4)	3.74defg	1.05gh (− 71.9)	4.51bcd	2.59ab (− 42.6)	8.25efg	3.64bc (− 55.9)	0.183 ± 0.0028ij	0.071 ± 0.0012hi	61.2
RSP-561	3.52fgh	6.21mn (+ 76.4)	3.77cdef	1.03ghij (− 72.7)	4.22 ij	2.33hij (− 44.8)	7.99lm	3.36hij (− 57.9)	0.177 ± 0.0027jkl	0.077 ± 0.0013ef	56.5
HD-2888	3.48hij	6.22lm (+ 78.7)	3.82bcde	1.23abc (− 67.8)	4.23ij	2.28jkl (− 46.1)	8.05ij	3.51ef (− 56.4)	0.188 ± 0.0028gh	0.085 ± 0.0014bc	54.7
K-0307	3.04no	5.62ef (+ 84.8)	3.61hijk	1.20bcd(− 66.8)	4.27ghi	2.18l (− 48.9)	7.88no	3.38hi (− 57.1)	0.203 ± 0.0031cde	0.079 ± 0.0013d	61.0
RAJ-4120	3.83de	6.57gh (+ 71.5)	3.52 kl	0.81l(− 77.0)	4.50bcd	2.40hi (− 46.7)	8.02jk	3.21lm (− 60.0)	0.176 ± 0.0027 jkl	0.072 ± 0.0012h	59.0
RAJ-4083	3.54fg	6.09no (+ 72.0)	3.63ghij	1.04ghi (− 71.3)	4.37defg	2.54ef (− 41.9)	8.00jkl	3.58cde (55.3)	0.208 ± 0.0031bc	0.086 ± 0.0014b	58.6
HI-1531	3.38jk	6.03pq (+ 78.4)	3.71efgh	1.14ef (− 69.2)	4.59 a	2.58bc (− 43.8)	8.30cde	3.72ab (55.2)	0.212 ± 0.0032ab	0.078 ± 0.0013de	63.2
HD-2932	3.52fgh	6.47ij (+ 83.8)	3.84bcd	1.24ab (− 67.7)	4.54bc	2.48fg (− 45.8)	8.38bc	3.72ab (55.6)	0.182 ± 0.0027ij	0.077 ± 0.0013ef	57.7
HD-2987	3.84cd	6.77b (+ 76.3)	3.70efgh	1.21bc (− 67.3)	4.40cdef	2.60a (− 40.9)	8.10h	3.81a (− 53.0)	0.192 ± 0.0029fgh	0.078 ± 0.0013de	59.3
DBW-17	3.49hi	6.72 cd (+ 92.5)	3.77cdef	1.05gh (− 72.1)	4.51bcd	2.29jk (− 49.2)	8.28def	3.34ijk (59.7)	0.179 ± 0.0027jk	0.075 ± 0.0012fg	58.1
PBW-396	3.40jk	6.33jkl (+ 86.2)	3.54jkl	1.02ij (− 71.2)	4.33efgh	2.42gh (− 44.1)	7.87no	3.44gh (56.3)	0.182 ± 0.0027ij	0.070 ± 0.0012ij	61.5
DBW-14	3.39jkl	6.34jk (+ 87.0)	3.63ghij	1.00jk (− 72.5)	4.37defg	2.55de (− 41.6)	8.00jkl	3.55de (55.6)	0.195 ± 0.0029fg	0.083 ± 0.0014c	57.4
PBW-596	3.28m	6.19n (+ 88.7)	3.56ijkl	1.13efg (− 68.3)	4.24hi	2.34ij (− 44.8)	7.80 op	3.47fg (55.5)	0.186 ± 0.0028ghi	0.083 ± 0.0014c	55.3
C-306	3.36lm	6.26l (+ 86.3)	3.60hijk	0.97kl (− 73.1)	4.48cd	2.28jkl (− 49.1)	8.08hi	3.25kl (59.8)	0.181 ± 0.0027ij	0.079 ± 0.0013d	56.3
CD (p ≤ 0.05)	0.22	0.24	0.18	0.19	0.18	0.13	0.19	0.22	0.011	0.004

Values of percent increase (+) and percent decrease (−) presented under sourdough within (); bold values denote highest and bold italic values represent lowest values

Gliadin protein fractions used are pure and antigenic reactivity levels for control and sourdough fermented samples are obtained from ANOVA analysis of three replica of each sample

Means with the same letter are not significantly different

Effect of fermentation on gliadin proteins

Removal of gluten is currently the only treatment for the diagnosed patients with gluten intolerance. According to Sapone et al. (2012), the daily consumption of wheat products with a lower level of gluten may have a delaying effect on the susceptibility to gluten related diseases or even absence of symptoms. In order to investigate the effect of mixed lactobacilli fermentation on gliadin proteins, the changes in gliadin proteins after 24 h sourdough fermentation were characterized by SDS-PAGE using 10% separating gels and 5% stacking gels. The band patterns of fermented gliadin proteins of the flour samples of different wheat cultivars are shown in Fig. 1b. The electrophoretogram revealed that there was a significant hydrolysis of wheat gliadin proteins in all the cultivars, resulting in the virtual disappearance of some protein bands and appearance of some new lower molecular weight protein/peptides. The bands observed for gliadins extracted from control samples had a molecular weight ranging from 15 to 110 kDa (Fig. 1a); however after 24 h of fermentation, bands of lower molecular weight from 5 to 110 kDa appeared in the electrophoretogram. From the results it could be inferred that the gliadin proteins were degraded into small peptides by the synergic effect of the peptidases secreted by three lactobacillus strains during fermentation. However, some high molecular weight bands observed in light intensity in control appeared with strong intensities after fermentation. Lactobacillus strains produce peptidases viz. PepX, PepN, PepO, PepQ, which cleave together and simultaneously the proline rich 33-mer or immunogenic polypeptides (Francavilla et al. 2017) and therefore this degradation of gliadin proteins into small peptides may be attributed to the peptidase activity of these lactobacilli bacteria. The results of the present study are further supported with the findings of Di Cagno et al. (2002), Wang et al. (2014) and Socha et al. (2015) who studied that the wheat albumins, globulins and gliadins (but not glutenins) were hydrolyzed to about 50% on fermentation with lactic acid bacteria (LAB), therefore, establishing the gluten-degrading potential of LAB. Further, they concluded that the proteolytic digestion during sourdough fermentation is highly complex due to concurrent effects of acidification and enzymatic activities of cereal peptidases, endogenous flour microorganisms resulting in degradation of immunogenic peptides.

Fig. 1.

SDS-PAGE analysis of gliadin protein fractions a before sourdough fermentation and b after sourdough fermentation (M = Molecular marker, Lane 1: HD-2764, 2: RAJ-3765, 3: GW-322, 4: RAJ-3044, 5: HD-2781, 6: WH-1021, 7: PBW-175, 8: PBW 561, 9: HD-2888, 10: K-030, 11: RAJ-4120, 12: RAJ-4083, 13: HI-1531, 14: HD-2932, 15: HD- 2987, 16: DBW: 17, 17: PBW-396, 18: DBW-14, 19: PBW-596, 20: C-306)

Gliadin analysis

Gluten, a heterogeneous complex of proteins (gliadins and glutenins) responsible for giving viscoelasticity to the dough is reported to be involved in the pathogenesis of many disorders and diseases. Di Cagno et al. (2010) studied that the antigenic reactivity of wheat flour was mainly due to gliadin protein fraction of the wheat. So it has become necessary to assess the leading wheat cultivars in context to antigenicity of gluten/gliadin proteins besides baking quality. Also the effect of sourdough fermentation on the antigenicity of the gliadin proteins was evaluated in the present study. For this purpose the gliadin protein fractions were isolated from grain flour samples before and after sourdough fermentation.

Antigenic potential of gliadin proteins of the wheat cultivars was assayed by ELISA and results were recorded in Table 2. A significant variation in the immunoreactive properties of gliadin proteins was observed in absorbance (OD) as estimated by ELISA using Multiscan RC reader at 450 nm. The highest OD values were recorded in cultivar GW-322 (0.217), followed by cultivar HI-1531 (0.212). Values were much lower in DBW-17 (0.177 OD) and RSP-561 (0.179 OD) but the lowest value was found in RAJ-4120 (0.176). According to two different earlier reports, the antigenic values of gliadins were found to vary from 3.3 to 1.1 and 0.216 to 1.31 OD (Waga and Zientarski 2007; Sherif et al. 2006). In comparison to these studies, the cultivars of this experiment carried lower antigenic values ranging from 0.176 to 0.217 OD and hence all the cultivars of this study were considered to be better in general with respect to lower antigenicity and RAJ-4120 was best among all the cultivars and at par with DBW-17 and RSP-561.

A considerable and significant difference was observed in the immunoreactive properties of sourdough gliadins as recorded by indirect ELISA. As compared to control, lower OD values with lesser antigenicity were observed in all cultivars with a reduction percentage from 54.7 to 65.3%, with maximum decline (65.3%) in RAJ-3077. Good results were obtained for all the twenty wheat cultivars showing an average decrease of about 60% in immunoreactivity. GW-322 which carried the highest immunoreactivity level in control (0.217) was lowered down to value 0.087 OD and therefore a percent reduction of about 59.9%. Similarly, in HI-1531 the value reduced from (0.212) to (0.078) showing about 63.2% reduction in antigenic property (Table 2). Therefore, 24 h lactobacilli fermentation produced very impressive results by lowering down gliadin antigenicity by 60%. These results are supported by earlier findings of Leszczynska et al. (2012) where the application of single and mixed culture probiotic fermentation reduced gliadin levels marginally and significantly respectively. The gliadin content in the different wheat cultivars and their percent reduction on mixed sourdough fermentation maintained the rate accordingly. RAJ-4120, RAJ-3765 and RAJ-3077 having the lowest gliadin levels in the controls demonstrated the highest reduction percent 59.0, 63.7 and 65 respectively. In this regard, these wheat cultivars are considered of a premium quality in context to health-promoting foods and therefore may have enough potential for consumer health and food industry. Therefore, the patients already predisposed to wheat intolerant complications like celiac disease may wish to either remove gliadin rich wheat products or increase consumption of probiotic fermented wheat products in their diet.

FTIR of gliadin proteins

The infra-red spectrum banding pattern for the proteins is represented with amide I, II and III that are positioned between 1580–1720 cm⁻¹, 1480–1580 cm⁻¹ and 1430–1480 cm⁻¹ respectively. These characteristic banding patterns reveal protein secondary structures that are sensitive to various factors (Kong and Yu 2007). The amide I band is unique in that it is very sensitive to small variations in molecular geometry and hydrogen bonding pattern and therefore very significant in terms of protein secondary structural composition and conformational analysis. The amide I band includes secondary structures like α-helix, β-turns and β-sheets, therefore, changes in these structures can be studied in this banding pattern. The α-helix and random coil peak is centered between 1658 and 1650 cm⁻¹, β-turns and glutamine side chains show peak at 1668 cm⁻¹ and the peaks assigned to inter and intramolecular β-sheets between 1612 and 1633 cm⁻¹ (Wellner et al. 2005). In this perspective, in the current study, the structure assignment of the amide bands of gliadins was taken into consideration due to its unusual amino acid composition (40 ± 45 mol% glutamine, 26 ± 32 mol% proline and 7 ± 9 mol% phenylalanine). Proline and glutamine are expected to be the major contributors to the intensity of the amide bands. The representative infrared (IR) absorption spectrum of the control (A) and fermented (B) gliadin proteins for the twenty wheat cultivars represented by the cultivar DBW-17 is shown in Fig. 2a, b. In the present investigation, changes in characteristic banding pattern for gliadin proteins were observed in terms of variations in intensities signifying certain modifications in the molecular bonding parameters on sourdough fermentation. Nevertheless, no significant changes in absorbance frequency were found in fermented compared to control wheat gliadin proteins (Table 3). Irrespective of the fermentation, the amide I and II bands of both fermented and control gliadin proteins were focused around 1654 and 1541 cm⁻¹ in almost all wheat cultivars studied. The peaks around 1650–1649 cm⁻¹ are allotted to random coil and α-helix structures. The present study revealed a shifting of the peak to lower frequency (~ 1653 cm⁻¹) in the fermented cultivars compared to control (1654 cm⁻¹). It is therefore hypothesized that this shifting as a result of fermentation may be a consequence of change in the H-bonding pattern and in turn alterations in protein secondary structures. The bands near 1450 cm⁻¹ to 1410 cm⁻¹ suggested the existence of sulfone. The bands centered at 1410, 1406, 1320, 1027 cm⁻¹ and those at 1150–1100 cm⁻¹ indicated the stretching vibration of C–O bonds of the gliadin protein. Whereas the bands at 1240 cm⁻¹ and 1075–1030 cm⁻¹ suggested C–N stretching of the polypeptide backbone of gliadin proteins. Although the positions of the bands for the protein in the control and fermented wheat cultivars is almost the same, however, banding pattern of the protein in the fermented wheat cultivars depicted a decrease both in the wavenumber and absorbance intensities. Therefore, the results of the current study establish that there is an overall change in the secondary structure of the gliadin proteins. These findings were further validated with the SDS-PAGE analysis of gliadin proteins where low molecular weight peptides were observed on sourdough fermentation. Our results are in agreement with the findings of Hongchao et al. (2018) who observed a decrease in the content of random coil and an increase in the β-turn secondary structures of the gliadin proteins on fermentation. Similarly, Wang et al. (2016) observed remarkably diverse spectra assigned to amide I band analyzed for fermented glutenin macropolymer (GMP). The sourdough fermentation was carried out with L. plantarum M616 (SL) and L. plantarum M616 and yeast (SLY). The fermentation was studied in respect to control dough (CK), sourdough fermented with yeast (SY) and dough with acids (SA). The results revealed that there is a loss in α-helix structure in SL, SLY, and SA on fermentation. Further, results demonstrated a decrease in β-turns and an increase in random coil in SL sourdough. Therefore, these findings established that sourdough fermentation increases GMP flexibility, possibly through its proteolytic degradation by secreted bacterial and cereal enzymes. In conclusion, the results of the present study in agreement with previous studies on fermentation establish that production of proteolytic enzymes by three lactobacilli may lead to degradation of gliadins into low molecular peptides resulting in changes in the secondary structure of gliadin proteins.

Fig. 2.

FTIR spectrum of a control and b fermented gliadin protein in variety DBW-17

Table 3.

Common band positions of control and fermented gliadin proteins as observed in FTIR diagrams of twenty wheat cultivars

Common band positions (cm−1)
Control	Fermented	Assignment
1654	1653	N–H; RNH2; amide I
1634	1628	N–H; RNH2
1541	1541	N–H; RCONH; amide II
1448	1438	S=O
1410	1406	C–O stretch, R–CO
1319	1318	C–O stretch, R–COOH
1243	1241	C–N stretch, R2NH
1273	1272	2NH
1383	1381	CH3
1325	1319	C–O stretch, R–COOH
1101	1103	C–O stretch
1073	1035	C–N, primary amine
1044	1044	C–O stretch

Rheological measurements

The amount of gluten present in the wheat flour is determinant of its baking property (Branlard et al. 2001). Gluten proteins have high water and gas retention properties and therefore possess high viscoelasticity in the dough and hence confer excellent baking properties. The rheological data as a determinant of baking or viscoelastic property of the twenty wheat flour samples (control and fermented) is shown Fig. 3a–j. Results indicated a higher magnitude of storage modulus (G′) magnitude compared to loss modulus (G″) in all the twenty wheat cultivars studied. This indicates a higher elastic compared to viscous behavior (G′ ≫ G″) which is a prerequisite for baking quality of a flour sample. A predominant elastic behavior (G′ > G″) was observed for all the dough samples at a temperature of 30 °C throughout the frequency range studied. Both G′ and G″ showed a slight dependency on frequency and increased with increase in angular frequency. A significant difference was observed between fermented and control dough. In case of sourdough samples, storage modulus was greater than the loss modulus in all the wheat cultivars even after 24-h fermentation which indicated that the elastic behavior of sourdough samples was still maintained but the magnitude was slightly decreased as compared to control. Comparing the storage (G′) and loss modulus (G″) of the control and sourdough fermented samples, all the fermented dough samples showed lower G′ and G″ which would be due to the hydrolysis of the gluten proteins (mainly gliadins) that were responsible for maintaining the viscoelastic nature of the dough (Fig. 3). The flow behavior of twenty wheat dough samples was analyzed by fitting the experimental data with power-law model and consistency coefficient and flow behavior index was determined. The magnitudes of consistency coefficients (K′ and K″), flow behavior index (η′ and η″) and the coefficient of regression (R²) are presented in Table 4. The rheological data obtained showed that all the twenty wheat cultivars displayed a solid-like behavior because the magnitudes of consistency coefficient k′ (17,790–9096) were much greater than those of k″ (11,083–5086), like in cultivar HD-2764. Similar results for k′ and k″ were found in all the other twenty wheat cultivars. The values of flow behavior index (η′ and η″) of the fermented dough were higher compared to control, indicating that after fermentation the flow behavior had increased due to the hydrolysis of the gluten proteins. Since the values of flow behavior index (η′ and η″) (Table 4) were less than unity, it indicated Non-Newtonian behavior of all the twenty wheat cultivars. Similar results were reported by Mohammed et al. (2011) and Koussa-Koffi et al. (2015) who studied the dynamic rheological properties of chickpea and wheat flour dough and observed that storage modulus was greater than the loss modulus in all wheat dough samples, indicating viscoelastic and solid-like behavior of all twenty samples.

Fig. 3.

Mechanical spectra showing frequency dependence of storage modulus (G′) and loss modulus (G″) (a–j) of wheat dough samples. C—Control doughs and F—Fermented doughs

Table 4.

Values of power law model for storage and loss modulus of control and fermented wheat dough samples as a function of angular frequency

Wheat cultivars	Storage modulus (G′)						Loss modulus (G″)
	η′		K′		R2		η″		K″		R2
	C	F	C	F	C	F	C	F	C	F	C	F
HD-2764	0.275	0.287	17,790.78	9096.470	0.99	0.98	0.181	0.239	11,083.93	5086.940	0.98	0.96
RAJ-3765	0.317	0.559	6838.553	1623.309	0.98	0.99	0.229	0.531	4443.567	1060.691	0.97	0.99
GW-322	0.335	0.343	13,952.29	6331.690	0.98	0.93	0.238	0.227	9250.051	4857.189	0.95	0.83
RAJ-3077	0.305	0.324	19,014.22	11,044.29	0.95	0.99	0.232	0.270	11,692.68	6739.904	0.83	0.98
HD-2781	0.332	0.486	10,856.69	2601.725	0.97	0.99	0.245	0.423	7502.329	1879.200	0.95	0.99
WH-1021	0.421	0.427	3964.065	2970.401	0.99	0.99	0.342	0.363	2815.987	2210.719	0.97	0.99
PBW-175	0.257	0.345	15,321.76	5675.300	0.98	0.99	0.187	0.303	8965.643	3569.056	0.96	0.98
RSP-561	0.262	0.272	5867.194	5627.081	0.98	0.98	0.209	0.222	3216.416	3141.005	0.97	0.96
HD-2888	0.306	0.334	5098.898	11,031.18	0.99	0.98	0.255	0.274	3131.990	7557.038	0.98	0.99
K-0307	0.297	0.309	5264.737	9837.585	0.98	0.99	0.227	0.249	3418.137	6214.501	0.98	0.98
RAJ-4120	0.297	0.306	5098.898	3239.172	0.98	0.98	0.255	0.255	3131.990	1974.001	0.97	0.98
RAJ-4083	0.301	0.303	7825.764	3474.998	0.99	0.99	0.263	0.262	4734.926	2084.332	0.97	0.98
HI-1531	0.321	0.307	14,505.62	6333.376	0.97	0.99	0.218	0.284	10,390.26	3800.350	0.93	0.97
DBW-17	0.306	0.297	5098.898	3239.172	0.99	0.99	0.255	0.255	3131.990	1974.001	0.97	0.98
PBW-396	0.303	0.338	10,359.25	2268.297	0.97	0.98	0.224	0.234	6975.682	2913.920	0.97	0.98
DBW-14	0.394	0.486	3855.443	6094.589	0.99	0.99	0.335	0.405	2454.716	4128.062	0.98	0.99
PBW-596	0.326	0.496	17,612.21	3006.416	0.99	0.99	0.251	0.429	11,501.34	2166.986	0.96	0.98
C-306	0.337	0.497	14,543.91	4270.106	0.98	0.99	0.241	0.428	9599.008	2871.916	0.98	0.99

G′ is storage modulus, G″ loss modulus, η′ and η″ flow behaviour index, K′ and K″ consistency coefficient, C and F represent control and fermented wheat dough samples respectively

Conclusion

The results of the present investigation establish that sourdough fermentation of wheat samples with combination of three probiotic lactobacilli showed a significant reduction in antigenicity in all the cultivars with little effect on rheology, therefore, maintaining the baking or viscoelastic properties of the wheat samples. Further, the decrease in antigenicity was proportional to gliadin levels in flour and percent reduction in antigenicity due to fermentation also maintained the rate accordingly. Moreover, wheat cultivars like RAJ-4120, RAJ-3765 and RAJ-3077 were found to have the lowest gliadin levels in the controls and the highest reduction percent in antigenicity 59.0, 63.7 and 65.0 respectively. These cultivars may thus be regarded best with respect to public health and food industry. So, individuals predisposed to the gluten intolerance need to consume gluten free foods or increase the consumption of probiotic fermented wheat products in their diet.