Eighteen (three samples of each day and Bakhar) samples were collected from different villages of West Midnapore and Bankura districts of West Bengal, India. For PCR denaturing gradient gel electrophoresis (PCR-DGGE) analysis, one batch samples was collected randomly and then kept in sterilized container and transported into the laboratory in an ice box and stored in the laboratory at −20°C for further analysis.

Haria was initially diluted with sterile distilled water (1:10, w/v), then centrifuged at 1,000 rpm for 10 min to remove the solid particles, and the supernatant was taken. The supernatant was again centrifuged at 5,000 rpm, and the pellet was washed with 2 ml of buffer (pH 8.0) [100 mM Tris–HCl, 100 mM sodium ethylenediaminetetraacetic acid (EDTA), 100 mM sodium phosphate, 1.5 M NaCl]. The pellet was then suspended in 1,000 μl of buffer (pH 8.0) (100 mM Tris–HCl, 100 mM sodium EDTA, 100 mM sodium phosphate, 1.5 M NaCl). Thereafter, 50 μl proteinase K (10 mg/mL, Sigma) and 25 μl lysozyme (40 mg/ml, Sigma), 1 ml of 1% cetyl trimethylammonium bromide (CTAB) was added and incubated at 37°C for 30 min with inversion of content at 10-min interval. Then, 50 μl of sodium dodecyl sulfate (SDS) (10%) was added, and the mixture was incubated at 65°C for 1 h with gentle shaking in every 20-min interval following centrifugation at 8,000 rpm. The supernatant was collected, and equal volumes of phenol, chloroform, and isoamyl alcohol (25:24:1, v/v) were added. The aqueous phase was recovered after centrifugation at 10,000 rpm for 10 min, and DNA was precipitated with 0.6 volume of 2-isopropanol at 4°C for minimum of 30 min. It was centrifuged again at 10,000 rpm for 5 min, and the pellet was washed with 70% ice-cold ethanol and dissolved in 200 μl of TE buffer. The RNA was digested by adding 2 μl of RNase solution [10 mg of RNase (Sigma) dissolved in 1 ml of Milli-Q water] followed by a 30-min incubation at 37°C. The presence of DNA was verified on a 1% agarose gel.

Extracted DNA was used as a template for PCR amplification. A ~340 bp of the 16S ribosomal RNA (rRNA) gene was amplified using LAB-specific bacterial primers Lact-F (5′-AGCAGTAGGGAATCTTCCA-3′) and Lact-R (5′-ATTYCACCGCTACACATG-3′) (Ritchie et al., 2010). GC clamp was anchored in the 5′ end of the reverse sequence. The PCR reaction mixture of 25 μl volume was prepared by using 2 × PCR master mix for the required number of reactions. In brief, each reaction mixture contained 12.5 μl of PCR master mix, 1 μl of each primer (20 pmol), 1 μl of DNA template (50 ng/μl), and 9.5 μl of sterile Milli-Q water. Samples were amplified in a thermocycler using the following protocol: initial denaturation at 95°C for 5 min; 20 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min with an increase in temperature of 0.5°C in each cycle, extension at 72°C for 1 min; 10 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 72°C for 1 min; and final extension at 72°C for 7 min.

DGGE was performed with a DCode electrophoresis system (Bio-Rad) with gel dimension of 16 cm ×16 cm ×1 mm. PCR products were loaded onto a 38–53% gradient of urea and formamide in a polyacrylamide gel (8%) and electrophoresed at a constant temperature of 60°C and a constant voltage of 60 V for 16 h. Gels were stained with ethidium bromide (0.5 mg/L) in TAE buffer for 20 min, destained in sterile deionized water for 10 min, and viewed by UV transillumination.

The visualized DGGE bands were excised from the original gel and incubated in 100 μl sterile distilled water at 4°C overnight. A 1-μl aliquot of elution was subjected to the PCR amplification using corresponding primers, without GC clamp. PCR products were excised from 1% agarose gel and purified with a HiPurA Quick Gel Purification Kit (HiMedia, India) and then subjected to sequencing. Forward and reverse DNA sequencing reaction of PCR amplicons were carried out with respective primers using BDT v3.1 cycle sequencing kit on ABI 3730xl Genetic Analyzer.

The tests were performed by a texture analyzer (TA-XT Plus, UK). A back extrusion test can provide the means to contain a volume of semisolid material to enable its testing. The vessel containing the sample was centrally located beneath a disk plunger, which then moved down into the sample and extrudes it up and around the edge of the disk. The diameter of the disk was 35 mm, and 75% of the sample holder was filled with test sample. When a 5-g surface triggered was attained (i.e., the point at which the disks lower surface was in full contact with the product), the disk proceeds to penetrate to a depth of 25 mm. At this point, the probe was allowed to return to its original position. The force area of the curve up to this point was taken as a measurement of consistency, and force at this point was the indication of firmness. The maximum negative force was recorded to indicate the cohesiveness.

Solid free liquid was extracted by centrifugation of fermented materials at 5,000 rpm for 5 min. The apparent viscosity was measured on a Bohlin CVO rheometer (Malvern Instrument, Malvern, UK) with a cone and plate geometry (CP 2°/20 mm diameter) maintaining a gap of 70 μm. Viscosity measurements were carried out on beverages previously adapted at 25°C for 1 h.

Water/salt-soluble extract from haria was prepared following the method of Ghosh et al. (2015a). Briefly, 10 g of haria was diluted with 30 ml of 50 mM Tris–HCl (pH 8.8), kept at 4°C for 1 h, vortexing at 15-min intervals, and centrifuged at 20,000 × g for 20 min. The supernatant, containing the water/salt-soluble fraction, was filtered through a Millex-HA 0.22-mm pore size filter (Millipore Co., Bedford, MA) and used for analyses. The extracts were analyzed by high-performance liquid chromatography (HPLC) using an Agilent HPLC system (Agilent Technology, 1200 infinity series). Organic acids were determined by using Zorbax SB C18 column, and elution was carried out at 60°C, with a flowrate of 0.6 ml/min, using 10 mM H₂SO₄ as mobile phase (Coda et al., 2011).

Hydrosoluble vitamins were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) as mentioned by Ghosh et al. (2015b).

Approximately, 20 mg of oven-dried samples (added 100 μg heptadecanoic acid as internal standard) was suspended in 1 ml of 5% methanolic solution of potassium hydroxide and saponified at 70°C for 1 h. Then, the pH of the mixture was adjusted to 2 with HCl. A mixture of 1 ml water and 1 ml chloroform was added, and the samples were shaken vigorously. The chloroform phase was collected after the centrifugation (4,000 rpm, 4°C, 15 min), and the extraction was repeated again from the upper phase with 1 ml chloroform. The collected chloroform phases were pooled and evaporated under stream of nitrogen. One milliliter of 14% methanolic solution of boron-trifluoride was added to the saponified samples and incubated at 70°C for 1.5 h. The fatty acid methyl-esters (FAMEs) were partitioned with hexane, which was evaporated to dryness under nitrogen. Finally, the derivatized samples were reconstituted in 100 μl hexane before chromatographic analysis.

The extracted samples were analyzed by gas chromatography (Agilent Technology, 6890N GC), equipped with a flame ionization detector (FID). A capillary column, HP-Innowax (60 m × 0.25 mm × 0.5 μm) was used. The temperature of the injector and detector was both set to 250°C. The oven temperature was held at 50°C, for 2 min, then programmed to rise from 50 to 200°C, at 20°C/min, and from 200 to 240°C at 3°C/min, and finally held at 50 min at 240°C. Nitrogen was used as carrier gas.

The ripened fermented material (fourth day) was extracted with dichloromethane and then analyzed by following the method of Ghosh et al. (2015a) in a gas chromatography–mass spectrometry (GC–MS) using a PerkinElmer Clarus 600C mass spectrometer equipped with a split/splitless injector and a flame ionization detector (FID). The compounds were identified by Perkin-Elmer inbuilt NIST mass spectral library.

Water/salt-soluble extract from haria was prepared following the method of Ghosh et al. (2015a). Briefly, 10 g of haria was diluted with 30 ml of 50 mM Tris–HCl (pH 8.8), kept at 4°C for 1 h, vortexing at 15-min intervals, and centrifuged at 20,000 × g for 20 min. The supernatant, containing the water/salt-soluble fraction, was filtered through a Millex-HA 0.22-mm pore size filter (Millipore Co., Bedford, MA) and used for analyses. The extracts were analyzed by high-performance liquid chromatography (HPLC) using an Agilent HPLC system (Agilent Technology, 1200 infinity series). Organic acids were determined by using Zorbax SB C18 column, and elution was carried out at 60°C, with a flowrate of 0.6 ml/min, using 10 mM H₂SO₄ as mobile phase (Ghosh et al., 2015b).

Phytase activity in the ferment was measured according to the method of Shimizu (1992) with slight modification. The reaction mixture, containing 150 μl of extract and 600 μl of substrate (3 mM Na-phytate in 0.2 M Na-acetate, pH 4.0), was incubated at 45°C. The reaction was stopped by adding 750 μl of 5% trichloroacetic acid. The released inorganic phosphate was measured spectrophotometrically at 700 nm by adding 750 μl of freshly prepared color reagent. The coloring reagent composed of four volumes of 1.5% (w/v) ammonium molybdate in 5.5% (v/v) sulfuric acid solution and one volume of 2.7% (w/v) ferrous sulfate solution. One unit (U) of phytase activity was defined as the amount of enzyme required to liberate 1 nmol of phosphate per minute under the assay condition.

The contents of free minerals including heavy metals in fermented and unfermented rice were determined by using an atomic absorption spectrophotometer (AAS) [Shimadzu Analytical (India) Pvt. Ltd] following the method described by Ghosh et al. (2015b). Briefly, rice products (5 g) were dissolved in 25 ml of deionized water and homogenized. Then, it was centrifuged at 12,000 rpm for 10 min, and the supernatant was used for analysis.

All the laboratory experiments were carried out for five times, and the values were represented as the mean ± standard deviation (SD). Data were statistically analyzed using one-way ANOVA and the Duncan's multiple range test in Sigma Plot 11.0 (USA) to determine the significant relationship between the means.

PCR-DGGE fingerprinting of LAB in haria is shown in Figure 1. Using the NCBI homology search, the sequenced DGGE bands were analyzed to identify the corresponding LAB with higher than 97% nucleotide sequence identities (Table 1). Four different genus were observed in haria: Lactobacillus sp. (bands a, c, h), Lactiplantibacillus sp. (band b), Lysinibacillus sp. (bands d–f), and the uncultured Bacillus sp. (g, i). The starter tablet, Bakhar, contained six types of bacteria (a–f). The “g” and “i” bands reflected the uncultured Bacillus sp., and the “a” and “c” were Lactobacillus brevis. The “d,” “e,” and “f” bands were Lysinibacillus fusiformis, Lysinibacillus sp., and uncultured Lysinibacillus sp., respectively. It is intriguing to note that the Lysinibacillus sp. was not survived in rice during fermentation as evidenced by the disappearance of “e” band after the first day. On the other hand, Lactiplantibacillus argentoratensis, uncultured Lactobacillus sp., and uncultured Bacillus sp. were gradually mounted during the course of fermentation as evidenced by the dominancy of “b,” “g,” and “i” (Figure 1).

Textural properties of the residual rice granules before and after fermentation were measured by a texture analyzer (TA-XT Plus, UK). From the force vs. time graph, firmness, consistency, and cohesiveness were calculated. Firmness, consistency, and cohesiveness were measured from maximum positive force, mean positive area, and maximum negative force of the curve, respectively. The firmness of haria was gradually decreased from 570.1 g force (first day) to 147.9 g force (fifth day) (Figure 2). Consistency and cohesiveness were also gradually decreased during fermentation, and their value was 272.745 and 84.5 g force, respectively, at the end of the fermentation.

Viscosity of the fermented soup after separation of the rice granule was measured on a Bohlin CVO rheometer. From the graph, it was observed that viscosity of the fermented liquid was gradually increased up to third day (0.0347 Pa s) and then decreased (0.0294 Pa s at fifth day) (Figure 2). The decrease in the apparent viscosity of the haria with the increase in shear rate at 25°C temperatures evaluated that the fluid has shear thinning characteristics. Hence, this is non-Newtonian shear thinning fluid.

The organic acids content of the fermented material was quantified by HPLC, and it was observed that the concentration of lactic acid (17.63 mg/g) and acetic acid (0.18 mg/g) was accumulated in highest level on the fifth day of fermentation (Table 2).

Haria contains different types of hydrosoluble vitamins. Folic acid, thiamine, pyridoxine, and ascorbic acid contents were relatively higher in the ripened material than the unfermented one. Folic acid content was highest on the second day (268.33 μg/100 g) and then declined. Besides, thiamine (3.41 mg/100 g), pyridoxine (481.62 μg/100 g), and ascorbic acid (538.24 μg/100 g) content gradually increased during the course of fermentation (Table 2). On the other hand, riboflavin content gradually decreased during fermentation.

Dietary fatty acids play a significant role in the daily caloric intake of the human population in India. Health professionals worldwide recommend a reduction in the overall consumption of trans-fatty acids (TAs) and cholesterol while emphasizing the need to increase intake of n-3 polyunsaturated fats (Daley et al., 2010). The fatty acids profile of haria has been tabulated in Table 3. It was found that palmitic acid (C16:0) was the most abundant saturated fatty acid present in haria. The amount was gradually increased up to the third day (18.84 μg/ mg) and then declined. Oleic acid (C18:1) content reached the highest level of 14.11 μg/mg on the second day. Haria also contained two essential fatty acids: linolenic acid (13.51 μg/mg) and linoleic acid (0.39 μg/mg).

Phytase activity in haria reached the highest level at fourth day (18.93 U/g) and then declined (Figure 3). Simultaneously, it was observed that the quantities of Ca⁺⁺ (0.77 ppm on second day), Fe⁺⁺ (0.43 ppm on fifth day), Mg⁺⁺ (5.42 ppm on third day), Mn⁺⁺ (0.81 ppm on first day), and Na⁺ (0.99 ppm on third day) were profoundly higher than the unfermented rice (Table 3). On the other hand, Cu⁺⁺ and Cr⁺⁺⁺ contents gradually declined during the fermentation, and their quantities were 0.21 and 0.04 ppm, respectively, in the consumable product (Table 3). Pb⁺⁺, Co⁺⁺, and Ni⁺⁺ were not detected in the mature product.

The type and occurrence of volatile compounds were analyzed in GC-MS, which was equipped with Perkin-Elmer inbuilt NIST mass spectral library. Eleven types of volatile compounds were detected in haria by GC-MS (Table 4). These are dichloroacetic acid heptadecyl ester; bicyclo (3.3.1) non-2-ene; ethanone,1-(2,4,6-trihydrophenyl); chloroacetic acid, dodecyl ester; 2h-indene3,3a,4,5,6,7-hexahydro; mucoinisitol, hexaacetate; chloroacetic acid, tetradecyl ester; 1-acetyl-3-(6-methyl-3-pyridyl) pyrazoline; epi-inositol, hexaacetate; N-(3-methyl-4-propionylphenyl) acetamide; and 1h-indene,2,3,4,5,6,7-hexahydro.