Dilute acid hydrolysis of spoiled wheat grains: Analysis of chemical, rheological and spectral characteristics

Bioresource Technology 283 (2019) 53–58

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Dilute acid hydrolysis of spoiled wheat grains: Analysis of chemical, rheological and spectral characteristics

T

⁎

Ranjna Sirohi , Jai Prakash Pandey Department of Postharvest Process and Food Engineering, College of Technology, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand 263 145, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Spoiled grains Acid hydrolysis Rheology Reducing sugars Infrared spectroscopy

In this work, hydrolysis of spoiled wheat grains using dilute acid (5, 10%; 1 N HCl) was investigated and the effect of hydrolysis conditions on reducing sugars, soluble proteins, rheology and infrared spectra of the hydrolysates was determined. Hydrolysis with 10% acid concentration released more quantities of reducing sugar (16.47 mg/mL) at shorter hydrolysis times whereas 5% acid concentration produced higher protein content (28.74 mg/mL) for similar durations. Flow characteristics demonstrated an increased apparent viscosity of the hydrolysates retrieved after 4.5 h of hydrolysis possibly due to breakdown of hemicelluloses and lignin into sugars. Infrared spectroscopy showed release of carbonates after 1.5 h and 5.5 h of hydrolysis perhaps due to oxidation of lignin or a reaction between acid and sugars. The study highlights that acid hydrolysis would be a rapid and cost effective approach to produce fermentable hydrolysates for bio-processing industry applications while generating an avenue for waste grain utilization.

1. Introduction

spoiled food grains as substrates for bioprocesses to produce important industrial products could be a wide and novel area of research. Since agro-residues are recalcitrant, a pre-treatment step is required to break the hemicellulosic fractions into fermentable sugars such as xylose, arabinose, mannose and glucose (Chandel et al., 2012). The effect of different physical, chemical and biological pre-treatment techniques for recovery of sugar and protein fractions from agro-residue has been presented in previous studies. The efficiency of any pre-treatment strategy for hydrolysis lies in its simplicity, economic feasibility, ease of scale-up, cost-effectiveness and its ability to interact with the fraction of interest (polysaccharides and amino acids) in the target substrate. In this regard, dilute acid and alkali hydrolysis, and enzymatic hydrolysis are reported to be the most effective methods for pre-treatment (Ravindran et al., 2018), of which, acid hydrolysis is cost-effective, easy to scale-up to an industrial level and is generally utilized for cereal grains. In acid hydrolysis of cereals, mineral acids such as HCl or H2SO4 are used at lower concentrations to break down the glycosidic linkages in the amorphous starch granules at temperatures lower than the gelatinization temperature of starch, to produce a lower molecular weight chain (Mehboob et al., 2015). Amorphous regions of starch are more susceptible to acid hydrolysis due to loosely packed starch chains as compared to crystalline regions (Wang and Copeland, 2015). Acid hydrolysis would therefore, be a suitable technique for hydrolysis, especially for cereal grains.

India is the second largest producer of wheat and paddy with an annual food grain production of 271.98 million tons (Agricultural Statistics at a Glance, 2017). Of the total food grain produced, about 23–24 million tons of grains are often damaged and/or spoiled (Gangwar et al., 2014) rendering it unsuitable for human consumption. The grains that spoil, though unsuitable, are rich in carbohydrates and proteins and have potential viability for use as feedstock for animals and production of industrially important bio-products. The carbohydrate fraction present in these grains can be depolymerised into simple sugars that can act as a carbon source for microbial bio-catalysis while the protein fraction can be utilized as a nitrogen rich substrate for various bioprocesses. Previous literatures report the production of bioethanol from damaged sorghum, rice (Schaffert, 1995, Suresh et al., 1999a, House et al., 2000), wheat and raw starch (Suresh et al., 1999b), low-grade wheat flour (Neves et al., 2006) and distiller’s grains (Kim et al., 2008). Studies also show that the structural modification in the cereal starch granules enhances its functionality thereby opening up promising applications for the development of bio-degradable nanocomposites, nanocrystals and bio-hydrogen (Wang and Copeland, 2015). Such applications provide an avenue for the use of various spoiled grains while addressing the problem of managing and disposing food grain waste in an environmentally sustainable way. The use of ⁎

Corresponding author. E-mail address: [email protected] (R. Sirohi).

https://doi.org/10.1016/j.biortech.2019.03.068 Received 14 January 2019; Received in revised form 11 March 2019; Accepted 13 March 2019 Available online 14 March 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.

Bioresource Technology 283 (2019) 53–58

R. Sirohi and J.P. Pandey

50 mL beakers were placed in an incubator (Brunswick, Germany) and taken out every 30 min for the first 90 min and at 1 h intervals thereafter. Control samples were also placed under the exact same conditions with Milli-Q water as the hydrolysing medium. Hydrolyzed samples retrieved at regular intervals were neutralized with 1 N NaOH followed by centrifugation at 3000g at 25 °C for 10 min. The supernatant (test sample) thus obtained, was used for analysis.

Various studies for hydrolysis of agro-wastes such as sugarcane baggase, wheat and rice straw and brewers’s spent grain among others, were found, however, the authors did not come across any study for the dilute acid hydrolysis of spoiled wheat grains. Further, limited literatures were available for the characterization of the hydrolysate itself, which is essential for the efficiency of the bioprocesses to follow, like fermentation. Utilization of spoiled grains also serves the purpose of waste management while producing essential by-products, which is a relatively critical area of research and therefore, the major motivation for this work. The aim of this work is to enhance the understanding of the acid hydrolysis of spoiled wheat grain by evaluating the influence of hydrolysis time and acid concentration on the release of reducing sugars and soluble proteins from spoiled wheat grains. The rheological properties and Fourier transform infrared spectroscopy of the hydrolysate were also investigated.

2.5. Analysis of hydrolysate 2.5.1. Estimation of reducing sugars Reducing sugar content was determined by the DNS method (Miller, 1959). Briefly, 3.0 mL of DNS reagent was added to 1.0 mL of test sample. The contents were thoroughly mixed and placed in a water bath at 90 °C for 10 min. The tubes were then cooled immediately (samples were diluted if required). The absorbance was measured using a UV–Vis spectrophotometer (LI- 2904, LASANY, India) at 540 nm. A reference blank was prepared containing 1.0 mL distilled water in place of the sample (hydrolysate). The concentration of reducing sugar in the sample was estimated by computing the absorbance against the standard curve of glucose (0.1–1.0 mg/mL). Standard glucose curve with the equation: A = 0.765 × RS + 0.101; R2 = 0.997, was used for estimation of reducing sugar (RS) in the hydrolyzed samples, where ‘A’ was the measured absorbance and ‘RS’ was the estimated concentration in mg/mL.

2. Materials and methods 2.1. Sample procurement and preparation Spoiled wheat grain samples were procured from the Crop Research Centre of G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand (India). The samples were washed with distilled water and dried in a hot air oven (AIO11E, ALFA Instruments, India) at 60 ± 0.5 °C for 2 h before subjecting it to size reduction. Size reduction was done using a hammer mill (Butex Engineering, India) followed by sieving through 500 μm and 800 μm size mesh to obtain a final grain size in the range of 500–800 μm.

2.5.2. Estimation of soluble protein content Soluble protein was determined using Lowry’s method (Lowry et al., 1951). Standard reagents copper-carbonate-tartarate complex (5.0 mL) and 1:1 diluted Folin-Ciocalteau reagent (0.5 mL) was added to 1.0 mL of test sample. The solution was mixed vigorously and incubated at 30 °C for 30 min in dark for the development of heteropolymolybdenum blue complex from the reduction of phosphomolybdotungstate. Standard curve was prepared using Bovine Serum Albumin (BSA): A = 0.625 × P + 0.175; R2 = 0.999, where A is the absorbance and P is the equivalent protein concentration in mg/mL. Absorbance for the solution was measured at 660 nm. Protein concentrations were estimated from the standard curve of BSA in mg/mL.

2.2. Proximate analysis Samples were analyzed for moisture, protein, fat, crude fibre and ash content using the standard procedure of AOAC (2003). All measurements were carried out in triplicates. Results were reported as mean ± standard deviation. 2.3. Mineral and metal estimation by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) Samples were prepared using wet digestion method (Altundag and Tuzen, 2011) with minor modifications. Briefly, 0.4–0.5 g of sample was taken in a 50 mL beaker. One mL of hydrogen peroxide was added to the sample for oxidation. Sample was digested using 5 mL of conc. nitric acid (69%) with constant heating at 70 °C in a water bath. Digestion was continued for 1.5–2 h till attainment of a pale yellow color. Digested sample was transferred to a 50 mL volumetric flask and the volume was adjusted to 50 mL using Milli-Q water. Metal composition in spoiled wheat grains was determined using a Perkin Elmer ICP-OES (model-Optima 7000 DV). A multi-element standard (Sigma-Aldrich, MO, USA) was used for analysis of heavy metals and minerals. The equipment was calibrated using different concentrations of the standard (5, 10, 25, 50, 100 ppb) in 5% HNO3. Reference blank was taken as diluted HNO3 (5%). Analysis was carried out in triplicates. Results were expressed in mg/kg of spoiled wheat sample (wet weight).

2.5.3. Flow characteristics Rheological study of the hydrolysates was performed using an Anton Paar Rheometer (MCR 52). Flow characteristics were determined on a concentric cylinder geometry. Shear rate was varied from 10-1 s−1 to 500 s−1. Variable shear stress measurement durations were taken with initial measurements every 15 s to final every 2 s with a total of 25 measuring points. All measurements were carried out at 25 ± 1 °C. The data for shear stress (τ ) and shear rate (γ ) were fit to the Ostwald-de Waale equation [Eq. (1)] for determination of flow behaviour index (n) and consistency index (k) (Capitani et al., 2015).

τ = k. γ n

(1)

2.5.4. Fourier transform Infrared (FT-IR) spectroscopy Infrared spectrums for the samples were obtained using a Bruker ECO-ATR FTIR with a ZnSe crystal. The transmittance was measured over a wavenumber (ʋ) range of 4000 cm−1–600 cm−1 and analyzed in OPUS computer software.

2.4. Acid hydrolysis Hydrolysis was carried out with dilute HCl (1 N) concentrations of 5% and 10% (v/v). Previous literature suggested that hydrolysis by HCl yielded higher fermentable sugars in wheat related substrates (Higgins and Ho, 1982). Moreover, the use of HCl results in complete hydrolysis of carbohydrates of plant origin to simple reducing sugars (Arapoglou et al., 2010) and was therefore, chosen as the hydrolyzing medium for this work. Duration of hydrolysis was 6.5 h. 50 mL of dilute acid solution was added to 5.0 g of sample (acid to sample ratio of 10) and incubated at 60.0 ± 0.1 °C at 120 rpm. Multiple identical samples in

3. Results and discussion 3.1. Proximate and metal composition Proximate analysis for the spoiled wheat grain was determined on wet weight basis as: 7.18 ± 0.04% protein, 0.83 ± 0.01% fat, 2.53 ± 0.13% crude fibre, 2.5 ± 0.53% ash and 9.98 ± 0.22% moisture. The moisture, fat and protein content of the spoiled grains 54

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broken down to monosaccharides like glucose and fructose after complete hydrolysis. In the presence of an acidic medium, the fermentable sugars thus formed could undergo conversion to 5-hydroxymethylfuraldehyde (HMF) and some acids under moderate reaction conditions and longer hydrolysis time. Soe and Than (2011) found a similar reduction in RS during acid hydrolysis of rice grains. Daorattanachai et al., (2012) reported that acid catalysis could promote formation of HMF by structural changes in β-glucose. Inspite of the possible formation of secondary products, an increase in recovery rate of RS was observed in phase II with increase in hydrolysis time. At the onset of hydrolysis in Phase I, dilute acid concentrations were able to infiltrate the wheat bran layer which exhibited release of monosaccharides such as xylose, arabinose, galactose and glucose. As the hydrolysis progressed to phase II, the hydrolysing medium could further break through the starchy endosperm thereby releasing more sugars. Zhou et al. (2018) reported that the total starch (amylose and amylopectin) in wheat grain increases from the surface layer to the inner layer which would mean higher release of RS with hydrolysis time due to deeper penetration of the medium into the grain. Results revealed that phase II witnessed the highest RS content for the hydrolysates over the entire duration of hydrolysis which confirms our previous hypothesis. Acid hydrolysis with 10% HCl yielded a maximum RS of 16.47 mg/mL after 4.5 h of hydrolysis, whereas a maximum of 15.64 mg/mL was obtained after 5.5 h in the 5% acid hydrolysed sample. Results indicated that hydrolysis at higher acid content reduced the hydrolysis time while providing higher content of reducing sugars. Higher acid concentration could facilitate penetration into the endosperm of the wheat grains thereby initiating a better interaction between the hydrolysing medium and the amylose and amylopectin of the grain. The presence of greater amounts of active H+ ions in higher acid concentration enables better hydrolysis of the grain thereby releasing more quantities of RS (Ji et al., 2015). Release of polysaccharides from the acid hydrolysed samples could increase the RS in the medium thereby enabling detection by DNS. Results of increased RS yield with acid concentration were consistent with the literature findings (Woiciechowski et al., 2002, Amezcua-Allieri et al., 2017). After 6.5 h of hydrolysis, the RS content for both 5% and 10% acid hydrolysed samples reduced and were comparable (p > 0.05). In this case, a lower hydrolysis time of 4.5 h should be preferred with higher acid concentration of 10% for better yield in reducing sugar. RS in control sample varied within 4.02–7.22 mg/mL indicating lower release of sugars from the starch granules of wheat grain possibly due to sluggish seepage through the grain. Release of reducing sugars with acids could be due to auto-hydrolysis at experimental temperature.

1905.50 5.46

2.54

Cu

0.18

Cr

Mn

10.08

Al

0.12

Ni

30.77

0.79

500

Pb

1000

366.30

651.35

1500

1011.50

1335.00

2000

Cd

Concentration (ppm)

2500

Fe

Ca

K

P

Mg

0

Fig. 1. Mineral composition of spoiled wheat grains analysed by ICP-OES. Data on the bars represent the average of three observations expressed in ppm.

were lower whereas the ash and crude fibre were higher than that reported in literatures (Šramková et al., 2009). Due to spoilage of grain, there could be a possibility of lipid oxidation and protein denaturation, resulting in lower quantities of protein and fat. However, fibres and mineral (in the form of ash) are less susceptible to variation in environmental conditions and hence, are likely to be retained. Fig. 1 shows the metal/mineral composition of the grain. Sample exhibited higher concentrations of potassium, phosphorus, iron, magnesium and calcium with slightly elevated levels of lead. Trace amounts of cadmium and chromium, were also detected in the spoiled grain sample along with a few micro-nutrients such as manganese, copper and nickel. Agricultural practices including fertilizer application and irrigation by wastewater are the likely sources for the presence of micro-nutrients and heavy metals in the grain, respectively. 3.2. Reducing sugars (RS) Fig. 2 shows the variation in RS with time and different acid concentrations. Increase in RS was observed in two phases over the duration of hydrolysis. The first phase extended from 0 to 3.5 h while the second phase was observed from 3.5 to 6.5 h. During the first hour of hydrolysis, the rate of recovery of RS was moderate. After 1 h, an increase in the rate of recovery of RS was observed in both 5% and 10% acid treatments. In Phase I, the maximum increase in RS for 10% acid hydrolyzed samples was observed at 1.5 h whereas 5% acid hydrolyzed samples took an additional hour to reach maxima. Reduction in RS was observed after 3 h of hydrolysis at both acid concentrations. The abrupt decrease in RS could be due to the conversion of fermentable sugars obtained from starch decomposition to other products. Wheat grains contain 65–75 g of starch per 100 g of their dry weight (Lu et al. 2014). Since starch hydrolysis is a decomposition process, starch molecules are

Reducing sugar (mg/mL)

20

Phase I

18

3.3. Soluble protein content Protein is the second largest fraction of the wheat grain after starch. Wheat protein (mostly gluten) is distributed in the outer and inner regions of the grain. Zhou et al. (2018) suggested that the protein content of wheat grains increases radially outward and is lower towards the endosperm cavity. Protein content in control increased gradually with hydrolysis time till 5.5 h of hydrolysis followed by a decrease at 6.5 h (p < 0.05). Acid hydrolysed samples also exhibited an increase in protein content with hydrolysis time. Soluble proteins increased over a 6.5 h hydrolysis, probably due to formation of peptides and enhancement in the functionality of the initial proteins present (Cappelletti et al., 2017). In the initial 1.5 h of hydrolysis, the hydrolyzing medium was able to recover the proteins from the outer and peripheral regions of the grain. The recovery was followed by a brief period of decrement in protein content until 2.5 h of hydrolysis. Reduction in protein could be due to partial loss of the amino acid methionine during acid hydrolysis (Hou et al. 2017). The period of protein degradation extended upto 1 h before it started increasing again at 2.5 h (Fig. 3). This suggests the presence of a low protein belt within the outer and inner layer of the grain that was unable to compensate for the lack of protein in the

Phase II

16 14 12 10 8 6 4

Control 5% HCl 10% HCl

2 0

0

1

2

3

4

5

6

7

Hydrolysis time (h) Fig. 2. Variation in reducing sugar with acid hydrolysis of spoiled wheat grains. 55

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A

35 Control 5% HCl 10% HCl

25 20 15 10 5 0

0.35 Control

0.30 Consistentcy index (k)

Protein content (mg/mL)

30

5% HCl 10% HCl

0.25 0.20 0.15 0.10 0.05

0

1

2

3

4

5

6

0.00

7

Hydrolysis time (h)

B

Fig. 3. Variation in protein content with acid hydrolysis of spoiled wheat grains.

0

1

2

1.8

3 4 Hydrolysis time (h)

5

6

7

3 4 Hydrolysis time (h)

5

6

7

Flow behavior index (n)

1.6

hydrolyzing medium, considering uniformity in hydrolysis. During this interval, the rate of degradation of protein was higher than the rate of protein recovery. After 2.5 h, the protein content started increasing for all the treatments indicating the entry of the hydrolyzing medium into the inner regions of the grain. Samples hydrolysed with 5% HCl solution attained a short-lived maxima in protein content (28.74 mg/mL) at 4.5 h that decreased to 16.87 mg/mL at 6.5 h (p < 0.05). However, the 10% acid hydrolysed samples attained maxima in protein value of 27.05 mg/mL at 6.5 h (Fig. 3). The protein released by lower acid concentration at 4.5 h was comparable (p > 0.05) to that released by higher acid concentration at 6.5 h. In contrast to the trend in RS, higher acid concentration took a longer duration of hydrolysis to achieve higher release in protein content. Tosi et al. (2011) reported that the proteins present near the endosperm are abundant in high molecular weight glutenin and γ-gliadins whereas the outer subaluerone layer is rich in low molecular weight glutenin and α and ω-gliadins. The former was reported to be qualitatively superior in terms of functionality than the latter protein fraction, which could enable better detection by Lowry’s protein assay thus resulting in an increase in protein content.

1.4 1.2 1.0 0.8 0.6 0.4

Control 5% HCl 10% HCl

0.2 0.0

0

1

2

Fig. 4. Trend of flow characteristic of the hydrolysates showing changes in A. consistency index (k) and B. flow behaviour index (n) with time of hydrolysis.

observed in the variation of flow behaviour index with hydrolysis time. A change in viscosity was also observed during the rheological analysis. Fig. 5 shows that there was an increase in viscosity of the hydrolysates in the shear rate range of 100–150 s−1. This phenomenon was clearly observed for the entire duration of hydrolysis. Results depict that the hydrolysates had a region of dilatant tendency probably due to rupture of starch granules with increase in shear rate. Further increase in shear rates > 150 s−1 could increase the homogeneity of the samples thereby resulting in a sharp decrease in viscosity. All samples assumed a brief pseudoplastic region within 200 s−1 shear rate beyond which the viscosity attained a relatively constant value. Final viscosity of the hydrolysates varied from 1.7E to 03 Pa.s to 5.5E−03.

3.4. Flow characteristics The consistency index (k) of control and acid treated samples varied insignificantly (p < 0.05) till 3.5 h of hydrolysis (Fig. 4A). After 3.5 h, k began to increase for acid hydrolysed samples until it reached the maximum value at 4.5 h. For the control, k was unaffected with hydrolysis time and showed only a minor increase at 5.5 h. This showed that acid hydrolysis increased the apparent viscosity of the medium. Acid hydrolysis could have influenced the amorphous and crystalline regions of the wheat starch granule leading to increase in water uptake of the starchy material and leaching of polysaccharides after 4.5 h of hydrolysis resulting in thickening of the hydrolysis medium (Wang and Copeland, 2015). Samples that were hydrolysed with 5% HCl showed an increased consistency index of 0.268 Pa.s0.26 at 4.5 h from 1.02E to 04 Pa.s1.46 at 3.5 h. In the 10% HCl hydrolysed samples, k increased for the same duration from 3.8E to 03 Pa.s1.10 to 0.063 Pa.s0.46 (Fig. 4A). Results showed that the increase of consistency index in the 5% acid hydrolysed sample was higher as compared to the 10% acid hydrolysed sample. After 5.5 h of hydrolysis, the flow behaviour index of the 5% acid hydrolysed sample was > 1 which represents progression towards a dilatant/shear-thickening behaviour (Fig. 4B). On the contrary, the flow behaviour index of the 10% acid hydrolysed sample started decreasing after 5.5 h, showing a pseudoplastic nature. This could have resulted in a higher consistency of the hydrolysate obtained from the 5% acid treated sample over others. An alternating pseudoplastic-dilatant fluid behaviour was seen for all hydrolysis conditions as well as control and no clear trend were

3.5. FT-IR spectrum analysis Spectral images of the hydrolysates were obtained after the first 30 min of hydrolysis and every 1 h thereafter in the transmission mode. Subtle changes were observed in the IR spectra of the 5%, 10% HCl treated samples and the control. Characteristic peaks were obtained at 3853 cm−1, 3740 cm−1, 3302–3328 cm−1, 2360 cm−1, −1 −1 2126–2129 cm and 1637 cm . The peaks above 3000 cm−1 corresponded to the presence of H-bonded OH groups indicating water in the samples. A broad peak was observed at 2126 cm−1 which could indicated the presence of a symmetric eC]Ce stretch along with some nitrogenous compound. A narrow peak at 1637 cm−1 showed the presence of carboxylic acid derivatives (mostly amides). The peak observed at 1637 cm−1 could be associated with water adsorption to the hydrophilic component of the hemicellulosic part of wheat granules (Sun et al., 2000, Hasen et al., 2011) during hydrolysis. With increase in hydrolysis time, there were no significant changes in the peaks at 2126 and 1637 cm−1. However, after 1.5 h of hydrolysis, a distinct peak was obtained at 2360 cm−1 which could arise due the formation of 56

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6.E-03

Control (6.5 h)

8.E-03

5% HCl (6.5 h)

Viscosity (Pa.s)

Viscosity (Pa.s)

1.E-02

10% HCl (6.5 h)

6.E-03 4.E-03 2.E-03 0.E+00 100

7.E-03

200 300 400 Shear rate (s-1)

500

10% HCl (5.5 h)

3.E-03 2.E-03 1.E-03

600

0

Control (4.5 h)

6.E-03

5% HCl (4.5 h)

5.E-03

Viscosity (Pa.s)

Viscosity (Pa.s)

5% HCl (5.5 h)

4.E-03

0.E+00 0

10% HCl (4.5 h)

4.E-03 3.E-03 2.E-03 1.E-03 0.E+00 0

100

4.5E-03 4.0E-03 3.5E-03 3.0E-03 2.5E-03 2.0E-03 1.5E-03 1.0E-03 5.0E-04 0.0E+00

200 300 400 Shear rate (s-1)

500

600

Control (1.5 h) 5% HCl (1.5 h) 10% HCl (1.5 h)

0

100

200 300 400 Shear rate (s-1)

500

100

600

200 300 400 Shear rate (s-1)

4.0E-03 3.5E-03 3.0E-03 2.5E-03 2.0E-03 1.5E-03 1.0E-03 5.0E-04 0.0E+00

500

600

Control (2.5 h)

5% HCl (2.5 h) 10% HCl (2.5 h)

0

Viscosity (Pa.s)

Viscosity (Pa.s)

Control (5.5 h)

5.E-03

100

200 300 400 Shear rate (s-1)

4.5E-03 4.0E-03 3.5E-03 3.0E-03 2.5E-03 2.0E-03 1.5E-03 1.0E-03 5.0E-04 0.0E+00

500

600

Control (0.5 h) 5% HCl (0.5 h) 10% HCl (0.5 h)

0

100

200 300 400 Shear rate (s-1)

500

600

Fig. 5. Time dependent variation in viscosity of spoiled wheat grain hydrolysates.

carbonates (possibly CO2) (Elassal et al., 2011) which subsided in the next IR spectrum observed at 2.5 h. The peak emerged again slightly after 3.5 h of hydrolysis and was prominent after 5.5 h of hydrolysis. Oxidation of lignin present in wheat granules could be responsible for the release of CO2 (Brodeur et al., 2011). Since there was a decrease in RS observed after 3.5 h of hydrolysis (Fig. 2), the formation of carbon di-oxide could also result from a reaction between the acidic hydrolyzing medium and released sugars.

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4. Conclusion It is evident from the results that 5% acid concentration was sufficient for hydrolysis, although 10% acid concentration yielded marginally higher sugars. Hydrolysis of spoiled grains for 4.5 h yielded higher reducing sugar and protein when 10% and 5% HCl solutions were used, respectively. Release of reducing sugars and proteins at 4.5 h also increased the consistency index while enhancing the pseudoplastic (n < 1) tendency of the hydrolysates. Results clearly show that acid hydrolysis provides promising alternates for the utilization of spoiled cereal grains. A large scale techno-economic evaluation could provide a better understanding of the economic feasibility for industrial implementation. References A.O.A.C, 2003. Official Methods of Analysis. Association of Official Analytical. Chemists International, Maryland, USA. Agricultural statistics at a glance, 2017. Government of India, New Delhi, India. Altundag, H., Tuzen, M., 2011. Comparison of dry, wet and microwave digestion methods for the multi element determination in some dried fruit samples by ICP-OES. Food

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