In vitro nutrient digestibility and antioxidative properties of flour prepared from sorghum germinated at different conditions

Abstract

Germination can be used as a bio-processing practice to enhance the digestibility of nutrient and improve the bioactive compounds and rheological properties of food grains. In the present study, effect of germination time 12, 24, 36 and 48 h and temperature 25, 30 and 35 °C on carbohydrate profile, enzyme activity, in vitro nutrient digestibility, antioxidant activity, bioactive components and rheological characteristics of sorghum was examined. As time and temperature for germination progressed, it considerably enhance the activity of diastase enzyme and also the sugar content by hydrolysis of starch and further enhance the in vitro digestibility of starch by 10.50–36.25%. Germinated sorghum had high in vitro protein digestibility and it ranges from 57.50 to 77.91% as compared to native sorghum (54.09%). Germination of sorghum for longer time period at elevated conditions appreciably improve the antioxidant activity by 4.24–52.96%, total phenolic content and flavonoid content by 1.60–4.09 mgGAE/g and 60.30–94.03 mgQE/100 g, respectively Similarly reducing power increased from 29.27 to 47.19 µg AAE/g and metal chelating activity enhanced 19.48–52.09% as period for germination goes from 12 to 48 h and temperature from 25 to 35 °C. Increased enzyme activity during germination degrades the starch and thus lowers down the peak and final viscosity of sorghum. Increased enzymatic activity and higher antioxidant activity also lower down the lightness value by 12.48% while a* was increased by 6.78%. Germination of sorghum thus offers a tool to increase the nutrient digestibility and bioactive potential of sorghum without any chemical or genetic engineering.

Introduction

Sorghum (Sorghum bicolor (L.) Moench), the most drought-tolerant coarse cereal is essential food commodity for majority of populations in semi arid regions of Asia and Africa (Mohapatra et al. 2019). Sorghum is the primary source for weaning foods in developed and developing nations due to its better nutritional compositions as they are served as staple food providing energy supply and nutrients in poor people’s diet (Afify et al. 2012b; Singh et al. 2017). Traditionally, sorghum is used for preparation of alcoholic and non-alcholic beverages, porridge, flat bread and weaning food (Claver et al. 2010; Hassani et al. 2013).

In the present scenario, sorghum gained a perceptive attention amongst health foods owing to its pheloics for healthful life among developed countries (Mohapatra et al. 2019). Peoples suffering from gluten intolerance, sorghum can be used to replace the flour containing the gluten, as it is suggested safe for consumption by the people suffering from celiac disease (Phattanakulkaewmorie et al. 2011). Sorghum thus, offers a good quality source for development of gluten free products like breads, cakes and cookies, snacks and pasta (Sharanagat et al. 2019). It also gained popularity as a nutraceutical and functional food, owing to the presence of significant levels of phenolic acids and its derivatives which exhibits health promoting properties due to their antioxidant nature (Mohapatra et al. 2019). Sorghum is an important source of protein and minerals (Correia et al. 2010), functional health promoting components such as vitamin B group, fibres, antioxidant phenolics and cholesterol-lowering waxes (Hassani et al. 2013).

Germination is a natural, biological processing technique in which seed come out of it legacy state which induced the activity of dormant enzymes, thus improve the functionality of cereal grains (Noda et al. 2004; Hefni and Witthöft 2011). Germination has been used as an alternative to mask undesirable tastes and smells, as well as reduce the presence of anti-nutritional compounds (Sangronis and Machado 2007; Hubner and Arendt 2013), increase the micronutrients, enhance the nutritional, chemical and sensorial characteristics of seeds (Hefni and Witthoft 2011; Adedeji et al. 2014). Several researches showed massive transformation in contents of phyto-chemical during germination, which reveals remobilisation, breakdown and deposition of nutrients (Donkor et al. 2012). Various biochemical activities occurs during germination generate various antioxidants and bio-functional compounds due to enzyme action of the cell wall for instance tocols, phenolic acids, Vitamin C and γ-aminobutyric acid (GABA) and results in enhanced antioxidant capacit (Singh and Sharma 2017). During germination various hydrolytic enzymes were activated (Correia et al. 2010), leading to the degradation of complex starch, polysaccharides proteins and fats into simpler forms, thereby increasing nutrient accessibility, and starch and protein digestibility and bio-availability (Afify et al. 2012a; Sharma et al. 2018). The increased enzymatic activities also results in degradation of starch which lower the viscous properties of the flour.

As sorghum gained lot of popularity in the development of gluten free products and for its better use in the development of variety food products with health enhancing components for all the age groups, there is need to increase its antioxidant compounds and nutrient digestibility through natural processing technology. Germination enhances the antioxidant properties of several food grains. Singh and Sharma (2017) reviewed and reported a prominent increase in antioxidant activities phenol and flavanoid compounds and other bioactive components of wheat, brown rice, barley, buckwheat and millets. However, scanty literature is available on changes in bioactive components and nutrient digestibility of sorghum during germination conditions. Hence, the study was carried out to examine the changes and enhancement in the nutrient digestibility, carbohydrate profile, bioactive components and pasting properties of sorghum at various germination time and temperature conditions.

Materials and methods

Germination of sorghum

Sorghum cultivar SL 44 was procured from Department of Plant Breeding and Genetics, PAU Ludhiana, Punjab (India). The seeds were sorted, washed followed by dipping in distilled water at 25 °C for 10 h, care was taken to change the water in every 5 h interval. Soaked grains were placed on tray encrusted and covered with piece of fabric and placed in incubator (Narang Scientific Works, New Delhi). The grains were germinated for 12–48 h at 25, 30 and 35 °C (90–95% RH). After each germination treatment grains were taken out of the incubator and dried in tray drier to a moisture content of 7 ± 2% to restrict germination. Dried germinated and non-germinated grains were then packed in airtight containers placed at cool and dark place (4 °C) for further investigation.

Carbohydrate profile

Starch and sugar content

Starch present in the given sample converted to sugars by acid hydrolysis and sugars thus released was calculated by measuring the volume of non-measured sugar solution necessary to absolutely reduce a known volume of Fehling’s solution to red unsolvable cuprous oxide (AACC 2000). Sugars (total and reducing) present in the samples were measured by amount of ferricyanide reduced by sugar extract under favourable conditions as per standard AACC (2000) method. Briefly, 5.675 g of sample flour was mixed with 95% ethyl alcohol (2 ml) and 50 ml of acetic acid buffer solution. Shake the contents to bring the flour into suspension and add of 12% sodium tungstate solution (2 ml). Filter the contents and use aliquot for subsequent sugars analysis.

Diastase activity

Diastase enzyme activity which represents the combined action of alpha and beta amylases present in the flour was determined under standard reaction conditions. Enzyme extract was extracted with buffer solution (dissolve 3 ml acetic acid and 4.1 g anhydrous sodium acetate in 1 L distilled water) at 40 °C and reacted with alkaline ferricyanide and the amount of reducing sugars thus formed by amylolytic enzyme action was then estimated by standard AACC (2000) method. Results are expressed as mg maltose produced by 10 g flour in 1 h at 30 °C using reference table.

In vitro nutrient digestibility

Starch digestibility

The protocol of Bernfeld (1955) as described and modified by Sharma et al. (2018) was followed to measure the content of in vitro digestibility of starch, by estimating the amount of maltose produced by the action of dinitrosalicylic acid. Briefly 0.1 g of finely ground sorghum was mixed with phosphate buffer (20 ml/pH 6.9) and then 100 mg of powdered α-amylase was added followed by 2 h incubation of mixture at 37 °C and then centrifugation (3000 rpm/15 min). 1 ml of clear aliquot and 2 ml of dinitrisalicylic acid were mixed together bring to boil (5 min) followed by cooling and volume makeup to 50 ml with distilled water. The OD of the mixture was measured at 540 nm and maltose standard curve was prepared to calculate mg of maltose released equivalent to sample OD.

Protein digestibility

The procedure of Akeson and Stachman (1964) as described and modified by Sharma et al. (2018) was used measure in vitro proteins digestibility. 500 mg of sorghum sample was mixed with 50 ml of pepsin sol. (pH 1.9) made in 0.1 N HCL and incubated at 37 °C for 2 h for first, followed by neutralization of the contents with 0.2 N NaOH. Toluene was added to maintain aseptic conditions. The contents were again mixed with pancreatin solution (pH 8) and incubated at 37 °C for 2 h for 2nd stage proteolysis, followed by centrifugation of the contents * 3000 rpm/15 min). Aliquot was discard, sediments were dried Nitrogen present in the sediments was measured using Macrokjedahl method and in vitro proteins digestibility was computed by ratio of the protein in the sediments to the ratio of protein content in sample before digestion.

Antioxidant activity (DPPH free radical scavenging activity)

Procedure of Brand-Williams et al. (1995) with slight modification was followed to measure the antioxidant activity which require the use of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in methanolic solution. 0.1 ml of acidified methanol extract (80%) was mixed with 3.9 ml of DPPH solution (0.2 mM) prepared in pure methanol and the contents were mixed well, kept for 30 min in dark followed by immediately measurement of the absorbance of the contents at 515 nm. The antioxidant activity was calculated as % discoloration:

$$\text{Antioxidant}\text{activity}(\text{AOA})\left(\text{\%}\right)=1-\frac{Absorbanceofsamplet=30}{Absorbanceofcontrolt=0}\times 100$$

Total phenolic contents

Method of Gujral et al. (2012) which involve the use of Folin–Ciocalteu (FC) reagent was followed to measure the total phenolic content (TPC). 1000 mg of sorghum was mixed with acidified methanol solution containing 0.1%HCl for 2 h at 43 ± 2 °C, followed by centrifugation of the contents. Extraction of the contents was repeated and contents were mixed and final volume is made to 50 ml with acidified methanolic solution (80%). From the volume, 0.5 ml of aliquot and 0.5 ml of methanol is mixed with 10-folds freshly dilute FC reagent (5 ml) followed by addition of saturate solution of NaHCO₃ (4 ml) after 4 min. The reaction mixture was shaken well and kept aside for 2 h at 25 °C followed by measurement of the OD of the contents at 765 nm using spectrophotometer. Results were expressed as mg of Gallic acid equivalents (GAE)/g on dry weight basis.

Total flavanoid contents

Procedure as described by Woisky and Salatino (1998) was employed to measure the total flavanoid content in the samples with minor alterations. 1.0 g of sorghum was mixed with acidified methanol solution containing 0.1%HCl for 4 h at 43 ± 2 °C, followed by centrifugation (3000 rpm/15 min) of the contents. Take aliquot (2.0 ml) and mix it with 10% solution of aluminium chloride solution (0.1 ml) followed by 1.0 M/L solution of potassium acetate (0.1 ml) and 2.8 ml of distilled water. Mix the contents properly and place them in dark form for half hour min and take the OD at 415 nm. Results were expressed as mg Quercetin Equivalent (QE)/100 g on dry weight basis.

Metal chelating (Fe⁺²) activity

The procedure as described by Sharma and Gujral (2011) was employed to calculate the Fe⁺² chelating activity. Sorghum sample (500 µg) was mixed with 80% methanol (5 ml) and extracted for 120 min at orbital shaker (Narang Scientific Works, New Delhi), followed by centrifugation of the contents (10,000 rpm/10 min). Take 0.5 ml of aliquot and put of solution of ferrous chloride (0.05 ml) and then add 80% methanol (1.6 ml) and shake well. After 5 min add 0.1 µl of ferrozine solution, shake the contents and allow them to rest for 10 min at room temperature followed by measurement of OD of the reaction mixture at 562 nm. Metal chelating activity was calculated as followed using methanol as blank:

$$\text{Iron}\text{Fe}\text{chelating}\text{activity}(\text{\%})=1-\frac{Absorbanceofsampleat562nm}{Absorbanceofcontrolat562nm}\times 100$$

Reducing power

The procedure as described by Sharma and Gujral (2011) was employed to calculate the reducing power of the sorghum samples. Sorghum sample (500 µg) was mixed with 80% methanol (6.6 ml) and extracted for 240 min at orbital shaker (Narang Scientific Works, New Delhi), followed by centrifugation of the contents (10,000 rpm/10 min). Take 1 ml aliquot, 2.5 ml of 0.2 M pH 6.6 phosphate buffer and 2.5 ml of potassium ferricyanide solution (1%), mix the contents and incubate at 50 °C for 20 min. Add 10% TCA solution (2.5 ml) in the mixture followed by centrifugation of the contents (10,000 rpm/10 min). Take clear aliquot (2.5 ml) and followed by addition of distilled water (2.5 ml) and ferric chloride solution (0.1%) (0.5 ml) and read the OD at 700 nm. Results were expressed as µg ascorbic acid equivalents (AAE)/g on dry weight basis.

Rheological properties

The changes taking place in the rheological properties of sorghum during germination was assessed with the help of RVA (Rapid Visco Analyzer) (Newport Scientific, Warrie Wood, Australis), using standard 13 min profile. 3.5 g of sample corrected to 14% moisture was put in aluminium canister and then add 25 ml distilled water and paddle was placed in the re mixed test was performed. The pasting properties viz. pasting temperature, peak, holding, breakdown, setback and final viscosity was noted.

Color characteristics

L* (lightness; 0-black, 100-white) a* (+ a*redness, − a*greenness) and b* (+ b* yellowness, − b* blueness) values of the sorghum samples was carried out using Hunter lab colorimeter (CR-300 Minolta Camera, Japan). Following equations was used to calculate the hue angle (h*) and chroma (C) values:

$$\text{h*}=\text{arctan}\left(\frac{b\ast }{na\ast }\right)$$

$$C=\sqrt{{\text{a}}^2+{\text{b}}^2}$$

Statistical analysis

The data given in the tables was mean of three different values along with standard deviation. The collected data was subjected to analysis of variance (ANOVA) and Tukey’s posthoc test with SPSS statistical software (version 16.0, SPSS Inc., Chicago, Illinois, USA).

Results and discussion

Carbohydrate profile of sorghum

Total starch

Data in Table 1 shows the effect of germination conditions on the starch content of sorghum. Germination time (12–48 h) and temperature (25–35 °C), had considerable (p < 0.05) impact on the sorghum starch content. Starch the main reservoir of energy for biosynthesis undergoes degradation during germination which leads to the formation of small dextrin and sugars. The degradation rate of starch was observed to increase as germination period progressed from 12 to 48 h and temperature increased commencing 25–35 °C. Native sorghum has 72.27% total starch content which undergoes hydrolysis as roots starts emerging with increase in metabolic activity of enzymes. Starch was extensively (p < 0.05) decreased by 9.17, 14.12 and 20.26%, when sorghum was germinated for 48 h at 25, 30 and 35 °C, respectively, due to the increased activity of amylases enzyme which hydrolyses the starch, to yield 6-carbon sugars for new germinating seedling (Ayernor and Ocloo 2007). The extent of starch hydrolysis also enhanced as period for germination and temperature elevated owing to higher enzymatic activity.

Table 1.

Effect of germination conditions on the carbohydrate profile and diastase enzyme activity of sorghum flour

Treatment	Temperature (°C)	Time (h)	Carbohydrate profile			Diastase activity (mg maltose/10 g)
			Total starch (%)	Total sugars (% glucose)	Reducing sugars (% glucose)
Control	–	–	72.27 ± 0.76a	3.64 ± 0.18k	2.22 ± 0.09j	273.44 ± 19.66j
Soaked	25	10	71.39 ± 0.21ab	4.94 ± 0.18j	2.56 ± 0.00j	341.67 ± 16.21i
Germination conditions	25	12	70.34 ± 0.38Pb	5.12 ± 0.00Sj	3.07 ± 0.03Ri	372.53 ± 0.00Shi
		24	68.48 ± 0.04Qc	6.33 ± 0.18Rhi	3.41 ± 0.10QRhi	567.68 ± 16.38Rg
		36	67.28 ± 0.21Rd	8.76 ± 0.34Qf	3.91 ± 0.07Qg	709.83 ± 13.04Qf
		48	65.64 ± 0.18Se	10.20 ± 0.00Pe	5.20 ± 0.29Pe	962.65 ± 16.25Pe
	30	12	68.87 ± 0.21Pc	5.82 ± 0.05Sij	3.67 ± 0.00Sgh	446.24 ± 0.00Sh
		24	66.90 ± 0.13Qd	7.23 ± 0.19Rgh	4.32 ± 0.00Rf	742.34 ± 29.43Rf
		36	63.63 ± 0.15Rf	11.50 ± 0.09Qd	6.76 ± 0. + 00Qc	1385.84 ± 35.75Qd
		48	62.06 ± 0.05Sg	16.41 ± 0.00Pb	8.27 ± 0.10Pb	1667.65 ± 19.28Pc
	35	12	65.66 ± 0.20Pe	7.40 ± 0.00Sg	3.84 ± 0.00Sg	740.36 ± 16.11Sf
		24	62.82 ± 0.05Qgf	9.12 ± 0.02Rf	5.97 ± 0.10Rd	1677.76 ± 19.40Rc
		36	60.32 ± 0.42Rh	13.36 ± 0.00Qc	8.17 ± 0.00Qb	2300.95 ± 42.18Qb
		48	57.63 ± 0.25Si	17.71 ± 0.67Pa	9.33 ± 0.00Pa	2415.83 ± 0.00 Pa
Mean#
Germination temperature (°C)		25	67.93p	7.60r	3.89r	653.16r
		30	65.36q	10.28q	5.76q	1060.50q
		35	61.61r	11.89p	6.83p	1783.70p
Germination time (h)		12	68.28p	6.11s	3.53s	519.68s
		24	66.06q	7.56r	4.57r	959.92r
		36	63.74r	11.21q	6.28q	1465.50q
		48	61.77s	14.77p	7.60p	1682.00p

Values represent mean ± standard deviation

Values having different superscripts from a, b, c to k differ significantly (p < 0.05) from each other column wise among different treatments

*Values having different superscripts P, Q, R and S show significant (p < 0.05) differences during different germination time at each germination temperature respectively

^#Means (germination temperature and time) having different superscripts from p, q, r and s differ significantly (p < 0.05) separately for mean values of germination temperature and time

Total and reducing sugars

Starches and complex sugars undergo enzymatic hydrolysis to form free sugars. Data in Table 1 presents the changes in the total sugars and reducing sugars of native sorghum and germinated sorghum. Period and temperature for germination had a significant (p < 0.05) positive influence on the total and reducing sugars of sorghum. Native sorghum has 3.64% total and 2.22% reducing sugars, which showed significant (p < 0.05) increase as germination period increase from 12 to 48 h and temperature from 25 to 35 °C. In the course of varying germination conditions total sugars of sorghum increased significantly (p < 0.05) and ranged between 5.12 and 7.71%, while reducing sugars varied between 3.07 and 9.00%. After 12 h of germination the mean total sugars of sorghum was 6.11% which significantly (p < 0.05) increased by 31.80, 61.75 and 111.98% after 24, 36 and 48 h of germination, respectively. Similar trend for mean noteworthy enhancement in total sugars was also observed when temperature for grain germination elevated from 25 to 35 °C. Reducing sugar content of sorghum germinated at different condition ranged from 3.07 to 9.33%. An increased of 64.44 and 186.67% in reducing sugar was assessed after germination period of 24 and 48 h at 25 °C. When temperature for germination was extended to 30 and 35 °C, reducing sugar content further increased significantly. Saman et al. (2008) as well reported enhancement in total and reducing sugar content of germinated cereals with increase in germination time. The increase in the sugar after germination was due to hydrolysis of starch by amylase enzyme along with combined action of invertase which helps out in the complete hydrolysis of starch and complex sugars to simple and reducing sugar, to yield glucose and fructose (Mohan et al. 2010; Moongngarm and Saetung 2010).

Diastase activity

Diastase activity which combined action of alpha and beta amylases was significantly (p < 0.05) effected by conditions for grain germination (Table 1). Raw sorghum exhibit enzyme activity of 273.44 mg maltose/10 g, which was significantly (p < 0.05) increased as the germination period increased from 12 to 48 h and temperature from 25 to 35 °C and it ranged between 372.53 and 2415.83 mg maltose/10. Interactions among variable conditions for grain germination also had significant influence on enhanced enzymatice activity. Ayernor and Ocloo (2007) also state that the diastase activity enhanced significantly with progress in the germination period.

In vitro nutrient digestibility

Starch digestibility

Germination conditions had significant (p < 0.05) influence on in vitro digestibility of starch. Enhancement in period of germination from 12 to 48 h and temperature from 25 to 35 °C significantly (p < 0.05) enhanced the digestibility of starch in sorghum as compared to control (Table 2). In vitro digestibility of starch of native sorghum was 34.76% and it enhanced appreciably by 4.32% after 10 h of soaking. During germination at variable conditions, in vitro digestibility of starch of germinated sorghum ranged from 38.37 to 47.68% and exhibited a significant (p < 0.05) enhancement of 10.50–36.25% as compared to native sorghum. Interaction between conditions for germination also exhibited a significant (p < 0.05) influence on germinated sorghum starch in vitro digestibility. Germination induces metabolic and structural transformation in the starch molecules and in crystalline form and the remaining components after germination exhibited enhanced inclination to enzyme attack (Srichuwong and Jane 2007), which results in enhanced starch digestibility of germinated sorghum in the present study. Similar results for increase in in vitro digestibility of starch was also reported by Chung et al. (2012) and Xu et al. (2012) in rice due to degradation of starch molecules by action of amylase enzymes.

Table 2.

Effect of germination conditions on the in vitro nutrient digestibility of sorghum flour

Treatment	Temperature (°C)	Time (h)	In vitro nutrient digestibility
			Starch digestibility (%)	Protein digestibility (%)
Control	–	–	34.76 ± 0.76j	54.09 ± 0.96h
Soaked	25	10	36.25 ± 0.57i	54.98 ± 0.84h
Germination conditions	25	12	38.37 ± 0.42Rgh	57.50 ± 0.76Rg
		24	39.86 ± 0.23Rg	59.33 ± 0.17Qef
		36	41.14 ± 0.31Qde	60.22 ± 0.91Qef
		48	42.26 ± 0.12Pd	63.03 ± 0.32Pde
	30	12	40.67 ± 0.60Ref	60.27 ± 0.92Sef
		24	42.86 ± 0.67Qd	63.43 ± 1.07Rde
		36	44.07 ± 0.81Pbc	66.47 ± 0.07Qcd
		48	45.08 ± 0.58Pb	69.96 ± 0.18Pc
	35	12	40.82 ± 0.34Ref	67.19 ± 0.23Scd
		24	44.45 ± 0.21QRbc	70.03 ± 0.88Rc
		36	45.22 ± 0.18Qb	73.52 ± 0.44Qb
		48	47.68 ± 0.15Pa	77.91 ± 0.28Pa
Mean#
Germination temperature (°C)		25	40.41r	60.02r
		30	43.17q	65.03q
		35	44.54p	72.16p
Germination time (h)		12	39.95r	61.65s
		24	42.39q	64.26r
		36	43.47pq	66.74q
		48	45.01p	70.30p

Values represent mean ± standard deviation

Values having different superscripts from a, b, c to i differ significantly (p < 0.05) from each other column wise among different treatments

*Values having different superscripts P, Q, R and S show significant (p < 0.05) differences during different germination time at each germination temperature respectively

^#Means (germination temperature and time) having different superscripts from p, q, r and s differ significantly (p < 0.05) separately for mean values of germination temperature and time

Protein digestibility

Data showing the in vitro digestibility of protein values in grain sorghum as influenced by germination conditions was presented in Table 2. Native sorghum shows an in vitro protein digestibility value of 54.09%. Variable germination conditions hold significant (p < 0.05) positive influence on the in vitro digestibility of sorghum protein and it amplified appreciably as the germination duration progressed from 12 to 48 h and temperature increased from 25 to 35 °C (Table 2). Sorghum germinated at 35 °C for 48 h showed maximum in vitro protein digestibility value (77.915%), while lowest (57.50%) was observed after 12 h of germination at 25 °C. This shows that time and temperature have significant influence on the in vitro digestibility and it was also confirmed from statistically significant interaction effect on protein digestibility. Comparable outcomes were also observed by Elkhalil et al. 2001 and Correia et al. 2010, while working with sorghum. They reported that during germination process proteins were denatured by action of inherent protease enzymes, causing structural changes in sorghum storage protein, which expose the sites of denatured protein and make it more susceptible to enzymatic hydrolysis (pepsin attack) thus increasing the in vitro protein digestibility. The enhanced nutrient digestibility of the germinated sorghum flour increased the market application and utilization of such flour in formulations of processed weaning foods.

Antioxidant activity (DPPH free radical scavenging activity)

Germination conditions significantly (p < 0.05) influenced the antioxidant activity of sorghum as per data shown in Table 3. Statistically significant (p < 0.05) difference was detected in antioxidant activity (DPPH scavenging ability) of native, soaked and germinated sorghum samples. Antioxidant activity of sorghum prior to soaking was 61.11% which reduced marginally to 60.22% after 10 h of soaking. Germination of sorghum for 12–24 and 48 h, showed a significant (p < 0.05) ehancement in mean antioxidant activity by 11.78–20.01 and 40.29%, respectively, as compared to native sorghum. Similar effect was observed in case of change in temperature. Mean antioxidant activity of sorghum was observed to be 69.05% when germination was carried at 25 °C and it increased significantly to 76.70 and 83.57% at 30 and 35 °C of germination temperature. This shows that longer is the germination duration and higher the temperature, superior is the antioxidant activity. Gupta et al. (2013) reported that antioxidative enzymes such as superoxide-dismutases, glutathione-S-transferase, peroxidises and catalases having high antioxidant potential were induced to higher levels during germination at high temperature, which results in enhancement in antioxidant activity of sorghum in the present study as their germination temperature increased from 25 to 30 °C and 35 °C. Similar outcome were also conveyed by studies of Cáceres et al. (2014), they reported germination of brown rice at 34 °C exhibited the elevated antioxidant activity in comparison to when germination at 28 °C.

Table 3.

Effect of germination conditions on the antioxidant activity, total phenolic and flavonoid content of sorghum flour

Treatment	Temperature (°C)	Time (h)*	Antioxidant activity (% inhibition of DPPH)	Total phenolic content (mg GAE/g)	Total flavanoid content (mg QE/100 g)
Control	–	–	61.11 ± 1.57i	1.30 ± 0.08jk	57.68 ± 1.46i
Soaked	25	10	60.22 ± 1.10i	1.19 ± 0.04jk	52.68 ± 1.52j
Germination conditions	25	12	63.70 ± 1.45Rgh	1.60 ± 0.14Rij	60.30 ± 1.08Sh
		24	64.61 ± 0.55Rgh	2.24 ± 0.28QRefg	63.85 ± 0.37Rgh
		36	71.11 ± 1.18Qef	2.58 ± 0.14PQdef	67.56 ± 0.92Qfg
		48	76.81 ± 1.37Pcd	3.21 ± 0.28Pbcd	79.22 ± 0.45Pbc
	30	12	68.06 ± 1.96Sfg	1.76 ± 0.11Rgh	63.47 ± 1.47Sgh
		24	74.31 ± 0.98Rde	2.38 ± 0.08QRdef	67.30 ± 1.16Rfg
		36	77.64 ± 0.20Qcd	2.64 ± 0.28Qcde	74.32 ± 0.47Qde
		48	86.81 ± 1.37Pb	3.59 ± 0.21Pb	81.63 ± 2.23Pbc
	35	12	73.19 ± 0.20Sde	1.84 ± 0.28Sgh	69.58 ± 1.79Sef
		24	81.11 ± 1.18Rc	2.24 ± 0.28Refg	77.36 ± 0.69Rcd
		36	86.53 ± 0.98Qb	3.44 ± 0.28Qbc	83.73 ± 1.10Qb
		48	93.47 ± 0.98Pa	4.09 ± 0.21Pa	94.03 ± 0.26Pa
Mean#
Germination temperature (°C)		25	69.05r	2.41r	67.73r
		30	76.70q	2.60q	71.68q
		35	83.57p	2.91p	81.18p
Germination time (h)		12	68.31s	1.73s	64.45s
		24	73.34r	2.29r	69.50r
		36	78.43q	2.87q	75.20q
		48	85.73p	3.63p	84.96p

Values represent mean ± standard deviation

Values having different superscripts from a, b, c to k differ significantly (p < 0.05) from each other column wise among different treatments

*Values having different superscripts P, Q, R and S show significant (p < 0.05) differences during different germination time at each germination temperature respectively

^#Means (germination temperature and time) having different superscripts from p, q, r and s differ significantly (p < 0.05) separately for mean values of germination temperature and time

Total phenolic content

Data for the total phenolic contents (TPC) of raw, soaked and sorghum germinated for different conditions is given in Table 3. Native sorghum has TPC of 1.30 mg/g GAE, which reduced slightly during soaking due to leaching or solubilisation of phenolic compounds in soaking water (Lu et al. 2007). Germination of sorghum afterwards brought a significant (p < 0.05) perceptible enhancement in TPC and this increase was time and temperature dependent as observed form data presented in Table 3. Sorghum germination for 12 had 33.07% high TPC in comparison to control and TPC levels was additionally enhanced by 76.15–179.23% when period of germination amplified to 24 and 48 h respectively. Similarly when temperature was elevated from 25 to 30 and 35 °C, significant (p < 0.05) enhancement in the TPC was also observed. Similar results were also reported by Pal et al. (2016) during germination of brown rice. Cáceres et al. (2014), also confirms that germination temperature also significantly influenced the accumulation of total phenolic contents in germinated grains. They reported that germination at 34 °C display a higher TPC content than those germinated at 28 °C in different brown rice cultivars, as during germination saccharolytic enzymes hydrolyse the cell wall to release the bound phenolic compounds and thus increasing free phenolics forms which results in higher TPC in germinated grains (Sharma et al. 2012).

Total flavanoid content

Flavonoids having health promoting effects was associated with antioxidant properties and have synergistic consequence with other antioxidants, was significantly (p < 0.05) influenced by variable germination conditions (Table 3). The total flavonoid content (TFC) of native sorghum was 57.68 mg QE/100 g and it ranged from 60.30 to 94.03 mg QE/100 g at different germination process conditions. Progress in germination period from 12 to 48 h and temperature from 25 to 35 °C, the mean TFC in sorghum was increased by 11.88–47.30% and 17.42–40.74%, respectively. This reveals that progress in germination period and temperature drastically enhanced the TFC of sorghum. Interaction between conditions for germination also had noteworthy influence on the TPC of sorghum. During germination the flavonoid pathway was synthesised by activation of metabolic phenylpropanoid pathway, during which acetyl coenzyme A esters (CoA) was generated by the intermediates which are further converted to flavonoids. Diverse kinds of enzymes and co-factors are manufactured during germination which also show the way for production of the flavonoids (Singh and Sharma 2017).

Metal chelating activity

Data in Table 4 shows the % metal chelating activity exhibited by native and germinated sorghum samples at different germination conditions. Native and soaked sorghum exhibits metal chelating activity of 14.91 and 15.67%, respectively. The metal chelating activity of sorghum was appreciably (p < 0.05) affected by variation in germination temperature and time and it ranged from 19.48 to 52.09%. 12 h germination sorghum exhibits 24.43% mean metal chelating activity which was 63.83% more in comparison to raw sorghum and it further enhanced by 104.02–175.57% after 24–48 h of germination respectively. The mean metal chelating activity of sorghum germinated at 25 °C was 23.25% which was significantly (p < 0.05) enhanced to 34.67 and 40.60% when temperature for germination was elevated to 30 and 35 °C. This illustrate that elevated germination temperature and prolonged time results in significantly (p < 0.05) higher metal chelating activity of sorghum. Among all the sorghum samples germinated at variable conditions, highest metal chelating activity (52.09%) was observed after 48 h of germination at 35 °C, while lowest (19.48%) after 12 h of germination at 25 °C. Enhancement in period and temperature for germination cause degradation and structure alteration of the phenolic compounds to different products of maillard reaction that may works as antioxidant and exhibited higher metal chelating activity (Sharma et al. 2012).

Table 4.

Effect of germination conditions on the metal chelating activity and reducing power of sorghum flour

Treatment	Temperature (°C)	Time (h)	Metal chelating activity (%)	Reducing power (µg AAE/g)
Control	–	–	14.91 ± 0.31j	25.24 ± 0.53k
Soaked	25	10	15.67 ± 0.77j	27.05 ± 0.37jk
Germination conditions	25	12	19.48 ± 1.23Pi	29.27 ± 0.74Rij
		24	21.54 ± 0.15QRgh	34.17 ± 0.54Qgh
		36	22.96 ± 0.61QRg	38.88 ± 1.03Pe
		48	29.04 ± 1.23Pde	40.24 ± 0.74Pde
	30	12	24.15 ± 1.08Sfg	31.71 ± 0.42Shi
		24	31.87 ± 0.61Rd	35.02 ± 0.00Rf
		36	36.11 ± 0.46Qc	41.66 ± 0.13Qcd
		48	46.54 ± 1.08Pb	44.75 ± 0.19Pb
	35	12	26.65 ± 0.61Sef	36.15 ± 0.05Sf
		24	37.85 ± 1.05Rc	39.59 ± 0.48Rde
		36	45.89 ± 0.46Qb	42.95 ± 1.18Qbc
		48	52.09 ± 0.61Pa	47.19 ± 1.04Pa
Mean#
Germination temperature (°C)		25	23.25r	35.64r
		30	34.67q	38.29q
		35	40.62p	41.47p
Germination time (h)		12	24.43s	23.38s
		24	30.42r	36.28r
		36	34.99q	41.17q
		48	42.56p	44.06p

Values represent mean ± standard deviation

Values having different superscripts from a, b, c to k differ significantly (p < 0.05) from each other column wise among different treatments

*Values having different superscripts P, Q, R and S show significant (p < 0.05) differences during different germination time at each germination temperature respectively

^#Means (germination temperature and time) having different superscripts from p, q, r and s differ significantly (p < 0.05) separately for mean values of germination temperature and time

Reducing power

Germination period and temperature had a significant (p < 0.05) influence on the reducing power of sorghum which is associated reductones and their antioxidant activity is based on the free radical chain reaction cessation by contributing a hydrogen atom and inhibiting peroxide formation (Sharma et al. 2012). Reducing powers of sorghum significantly (p < 0.05) amplified as germination period increase from 12 to 48 h and temperature from 25 to 35 °C (Table 4). Native sorghum shows reducing power of 25.24 µg AAE/g, which after germination for 12–48 h at 25–35 °C ranged from 29.27 to 40.24 µg AAE/g, showing an increase of 15.97–59.42%, as compared to native sorghum. Interaction between period and temperature of germination also exhibited a noteworthy influence on the reducing power of sorghum. Among different germination condition, highest reducing power was reported after germination of 48 h at each germination temperature condition. During germination some compounds of sorghum donates electron to free radicals to help to stabilize radical chain reactions which in reply results in higher reducing power. Sharma and Gujral (2011) reported that reducing power is mainly due to the phenolics and flavonoids as they possess the capacity to serve as reductones by donating electrons. Therefore, the increase in generation of more phenolic and flavonoids compounds can also be the cause for increment in reducing power after germination in comparison to native sorghum in present study.

Rheological properties

Germination conditions at varying period and temperature significantly (p < 0.05) influence the rheological properties of sorghum (Table 5). Pasting temperature (PT), which provide an indication of the gelatinization time of starch during swelling, was significantly (p < 0.05) influenced by variation in temperature and period of germination. Lowest PT was recorded for native followed by soaked sorghum, which increased considerably (p < 0.05) as germination period and temperature progressed and reached to max of 91.7 after 48 h of germination at 35 °C. When period of germination was upsurge from 12 to 48 h and temperature from 25 to 35 °C, paste temperature increased from 81.5 to 91.7 °C due to gelatinization and fragmentation of starch and protein. Rheological properties of native sorghum differ significantly (p < 0.05), in terms of peak viscosity, hold, final and setback viscosity from sorghum germinated at different conditions. Germination, significantly decrease the peak and final viscosity of sorghum. The decrease was higher as period of germination enhanced from 12 to 24 and 48 h, and temperature increased from 25 to 30 and 35 °C. Native sorghum has intact starch granules which were breakdown into small fragments by enzyme resulting in low peak and final viscosities. The substantial decrease in peak viscosity with increasing germination time and temperature may be attributed to degradation of starch and proteins to simpler units, and higher enzymatic activity (Pal et al. 2016). Native and soaked sorghum has highest hold viscosity which was drastically influenced by germination conditions. Elevation in period of germination from 12 to 48 h and temperature from 25 to 35 °C substantially decreased the hold viscosity of germinated sorghum (Table 5). The hold viscosity of sorghum after 48 h of germination at each temperature was dropped off significantly (p < 0.05) to 444.0, 56.5 and 1.0cp, respectively. During germination starch hydrolyzing enzymes are activated which results in starch fragmentation and thus displayed the reduced viscosity (Zeeman et al. 2007). Final viscosity of sorghum germinated over different germination conditions varied significantly (p < 0.05). Native sorghum had final viscosity of 2652.5 cP which was appreciably higher in comparison to germinated samples. Among all the germinated sorghum samples, the 48 h germinated sorghum had lowest; while 12 h germinated sorghum had highest viscosity at each temperature (Table 5). Decrease in breakdown and setback viscosities were also observed among the sorghum germination at varying conditions as compared to control and lower setback values of the flour samples are useful in the preparation of frozen foods.

Table 5.

Effect of germination conditions on the rheological properties of sorghum flour

Treatment	Temperature (°C)	Time (h)*	Pasting Temperature (°C)	Viscosity (cP)
				Peak	Hold	Final	Breakdown	Setback
Control	–	–	81.2 ± 0.6e	816.5 ± 19.4bc	814 ± 18.4bc	2752.5 ± 30.4b	364.5 ± 5.30ab	1838.5 ± 37.1b
Soaked	25	10	81.4 ± 0.6e	1024.0 ± 24.7a	968 ± 21.2a	2889.5 ± 9.2a	390 ± 5.66a	2021.5 ± 44.9a
Germination conditions	25	12	81.4 ± 0.8Re	901.0 ± 2.1Pb	818.5 ± 5.3Pb	2412.5 ± 13.4Pc	350.5 ± 5.30Pbc	1556.5 ± 25.1Pc
		24	81.9 ± 0.3Rde	869.5 ± 16.6PQb	730 ± 0.7Qd	1915.5 ± 7.8Qf	212 ± 6.36Qd	1185.5 ± 4.6Qe
		36	84.4 ± 0.3Qc	823.5 ± 1.8Qbc	514.5 ± 7.4Re	1222 ± 5.7Rg	93.5 ± 1.06Rf	677.5 ± 11.0Rg
		48	89.7 ± 0.4Pb	721.0 ± 3.5Rd	444 ± 14.1Sf	1127 ± 26.9Sh	82.5 ± 3.18Rfg	624.5 ± 42.8Rg
	30	12	81.9 ± 0.6Rde	897.0 ± 5.7Pb	821.5 ± 6.7Pb	2384 ± 0.7Pd	330.5 ± 3.18Pc	1562.5 ± 20.2Pc
		24	83.6 ± 0.1Rcd	756.5 ± 1.8Qcd	744.5 ± 4.6Qcd	1255.5 ± 16.3Qg	200 ± 0.00Qd	711.0 ± 12.7Qf
		36	89.4 ± 0.6Qb	321.5 ± 31.8Rj	69.5 ± 3.2Rh	434.0 ± 2.1Rf	80.5 ± 1.06Rfg	244.0 ± 3.5Ri
		48	90.0 ± 0.2Pab	314.5 ± 14.8Rj	56 ± 1.4Sh	401.5 ± 12.4Rf	75.5 ± 1.06Rfg	250.5 ± 6.7Ri
	35	12	81.5 ± 0.3Re	865.0 ± 2.1Pb	784.5 ± 3.2Pcd	2188.5 ± 0.7Pe	206.5 ± 3.89Pd	1404.0 ± 3.5Pd
		24	89.3 ± 0.3QRb	514.0 ± 8.5Qd	163.5 ± 3.2Qg	603.5 ± 10.6Qi	147 ± 5.66Qe	340.0 ± 2.1Qh
		36	90.2 ± 0.2Qab	200.5 ± 0.4Rg	58 ± 2.3Ri	237 ± 5.7Rk	22.5 ± 1.06Sg	36.5 ± 3.2Rj
		48	91.7 ± 0.1 Pa	147.0 ± 5.7Sh	31 ± 3.1Ri	154.5 ± 0.7Sk	56 ± 3.54Rg	13.0 ± 2.1Sk
Mean#
Germination temperature (°C)		25	83.75s	828.75p	626.75p	1649.20p	184.62p	1011.0p
		30	84.07r	622.25q	422.88q	1059.50q	171.62q	692.00q
		35	86.32p	431.62r	237.50r	685.88r	108.00r	448.87r
Germination time (h)		12	81.61s	887.67p	808.17p	2315.8p	295.83p	1507.7p
		24	82.35r	698.00q	546.00q	1224.8q	186.33q	745.50q
		36	85.06q	472.67r	195.00r	524.50r	71.33r	319.33r
		48	89.85p	451.83s	167.00s	461.00s	65.50r	294.00s

cP centi Poise

Values represent mean ± standard deviation

Values having different superscripts from a, b, c to k differ significantly (p < 0.05) from each other column wise among different treatments

*Values having different superscripts P, Q, R and S show significant (p < 0.05) differences during different germination time at each germination temperature respectively

^#Means (germination temperature and time) having different superscripts from p, q, r and s differ significantly (p < 0.05) separately for mean values of germination temperature and time

Color characteristics

The color characteristics as well as hue angle and chroma values of germinated sorghum flour as influenced by germination period and temperature are presented in Table 6. Germination significantly (p < 0.05) affected the all colour characteristics. Non-germinated sorghum has highest L value, which decreased notably as period of germination progressed from 12 to 48 h and temperature increased from 25 to 35 °C. However, with enhanced germination temperature and period increased the redness (a*) and yellowness (b*) values of the sorghum increased appreciably. Pal et al. (2016) in their study also reported that germination increased the values of redness and yellowness of brown rice flour while it decreased the Lightness values. Hue and chroma values also increased significantly (p < 0.05) as the germination time and temperature increased. The increase may be due to increase in the pigmentation of the flour due to enzymatic activity and decrease in the L* value of flour samples. Each germination condition affects the colour characteristics of sorghum in different way as sorghum samples germinated at 35 °C exhibits less yellowness in comparison to grains germinated at lower temperature. Similar end results were also reported by Chung et al. (2012), which was due to enhanced activity of polyphenol oxidase and peroxidise enzymes, as during germination oxidative products act together with denatured proteins which assist the enzymatic browning reaction and also due to synthesis of GABA (Moongngarm and Saetung2010), which improves the formation of pigments and thus lowered down the lightness values.

Table 6.

Effect of germination conditions on the colour characteristics of sorghum flour

Treatment	Temperature (°C)	Time (h)*	Colour characteristics			Hue angle (°)	Chroma
			L	a*	b*
Control	–	–	57.65 ± 0.35a	19.90 ± 0.00f	4.01 ± 0.07g	10.95 ± 0.59fg	20.27 ± 0.04ef
Soaked	25	10	57.35 ± 0.21a	19.80 ± 0.00f	3.35 ± 0.03h	9.32 ± 0.20gh	20.07 ± 0.01f
Germination conditions	25	12	56.75 ± 0.35Pb	20.15 ± 0.07Ref	4.14 ± 0.04Rfg	11.36 ± 0.15Rdef	20.55 ± 0.08Sde
		24	56.30 ± 0.28Pb	20.35 ± 0.01QRcde	4.02 ± 0.01Sg	11.26 ± 0.08defR	20.75 ± 0.22Rd
		36	54.30 ± 0.14Qc	20.65 ± 0.03PQbc	5.01 ± 0.02Qcd	13.87 ± 0.42Qb	21.27 ± 0.03Qbc
		48	51.35 ± 0.07Rf	20.85 ± 0.07Pb	5.72 ± 0.00Pa	15.29 ± 0.05Pa	21.62 ± 0.07Pab
	30	12	57.60 ± 0.28Pa	20.35 ± 0.05Qcde	3.14 ± 0.03Rh	8.94 ± 0.42Ri	20.60 ± 0.05Rde
		24	56.25 ± 0.21Qb	20.45 ± 0.07Qcd	4.16 ± 0.02Qfg	11.61 ± 0.42Qcdef	20.88 ± 0.04Qd
		36	54.05 ± 0.21Rc	20.85 ± 0.02Pb	5.38 ± 0.01Pbc	14.00 ± 0.41Pab	21.49 ± 0.03PQabc
		48	52.95 ± 0.21Se	20.95 ± 0.07Pab	5.38 ± 0.02Pab	14.33 ± 0.14Pab	21.62 ± 0.09Pab
	35	12	56.50 ± 0.85Pb	20.45 ± 0.05Rcd	3.97 ± 0.04Sh	10.53 ± 0.42Qfg	20.80 ± 0.04Rd
		24	53.90 ± 1.13PQd	20.70 ± 0.14QRb	4.94 ± 0.02Pdef	12.93 ± 0.64PQbc	21.24 ± 0.09Qc
		36	52.50 ± 0.42QRe	21.00 ± 0.14PQab	4.55 ± 0.05Ref	12.10 ± 0.08Pcde	21.48 ± 0.14PQabc
		48	50.45 ± 0.35Rg	21.25 ± 0.07 Pa	4.71 ± 0.07Qe	12.47 ± 0.04Pcd	21.76 ± 0.07Pa
Mean#
Germination temperature (°C)		25	55.21p	20.50r	4.73r	12.94p	21.05q
		30	54.67q	20.65q	4.49q	12.21q	21.14q
		35	53.33r	20.85p	4.43p	12.00q	21.32p
Germination time (h)		12	56.95p	20.31s	3.68s	14.30p	20.65s
		24	55.48q	20.50r	4.33r	13.33q	20.95r
		36	53.62r	20.83q	4.93q	11.92r	21.41q
		48	51.52s	21.02p	5.25p	10.27s	21.67p

Values represent mean ± standard deviation

Values having different superscripts from a, b, c to i differ significantly (p < 0.05) from each other column wise among different treatments

*Values having different superscripts P, Q, R and S show significant (p < 0.05) differences during different germination time at each germination temperature respectively

^#Means (germination temperature and time) having different superscripts from p, q, r and s differ significantly (p < 0.05) separately for mean values of germination temperature and time

Conclusion

The results showed that germination conditions appreciably enhanced the nutrient digestibility, bioactive components and lower down the pasting viscosities of the sorghum. Germination at higher temperature and duration accelerates the hydrolytic enzymes which results in enhanced starch and protein digestibility in comparison to germination for shorter duration at low temperature. Elevated temperature and germination duration results in the generation of bioactive compounds which results in enhancement in the antioxidant activity, phenolic content and reducing power. Both germination time and temperature are important factor affecting the nutrient digestibility, antioxidantive properties, bioactive components and pasting characteristics of sorghum. Results from the current study will help in delivering the collective information for the selection of optimum time and temperature of germination for the desired quality end product at both commercial and home scale level.