Total Phenolic Content and Antioxidant Activity of Extruded Brown Rice

Abstract

Three paddy cultivars varying in l/b ratio (2.67–4.59) were dehusked to obtain brown rice. The brown rice was germinated for 24 and 48 h, dried, and milled into grit. The grit from controlled (un-germinated) and germinated brown rice was extruded at 100 and 120°C and the total phenolic content and antioxidant activity of the extrudates was determined. The total phenolic content of the control and germinated brown rice varied from 0.803–0.992 mg/g ferulic acid equivalent and germination increased total phenolic content by 8.8–12.0%. The antioxidant activity varied from 6.96–15.86% (decrease in absorbance of 2,2-diphenyl-1-picrylhydrazyl) and germination increased the antioxidant activity by 18.2–37.2%. Upon extrusion at 100°C, the total phenolic content decreased by over 50%. A further decrease of 6–15% in total phenolic content was observed when the extrusion temperature was increased from 100 to 120°C. Similar decrease in the antioxidant activity was observed upon extrusion and rise in extrusion temperature. Significant increase in water solubility, water absorption capacity, and percent expansion of extrudates was observed upon extrusion. The extrudates from germinated brown rice were used to make an instant pudding, which upon evaluation scored higher as compared to the pudding from control brown rice.

Keywords:

• Total phenolic content
• Antioxidant activity
• Germinated brown rice
• Extrusion

INTRODUCTION

Rice (Oryza sativa L.), a major cereal crop, is the staple food source for half of the world population. It is harvested as paddy with the hull comprising approximately 20% of the rice kernel, the bran and embryo about 8–12%, and the endosperm about 70–72%. Paddy after removal of husk gives brown rice that is further polished to remove the bran and germ resulting in white rice. Brown rice is not consumed because it requires longer cooking time, has an undesirable color, and a firm texture. However, in recent years the nutritional significance of brown rice has been recognized and its use is being encouraged. Bran layers of the rice kernel are a rich source of B-complex vitamins, tocopherols, and oryzanols. It contains insoluble fiber that adds bulk to the gastrointestinal tract in humans indirectly promoting cardiovascular health. These biofunctional components exist mainly in the germ and bran layers and are lost once the brown rice is milled into white rice.[1] Cereal grains and their morphological parts, such as germs, are good sources of various phytochemicals. The majority of phytochemicals present in cereal grains are phenolic acids, flavones, phytic acid, flavonoids, glutathione, and phytosterols.[2] Polyphenolic compounds are effective in preventing the oxidative stress, thus they have a protective and beneficial role for the human body.[3]

Germination of brown rice leads to the softening of the outer bran layer and improves water absorption, making the rice easier to cook. Cooked sprouted rice has a sweet flavor because the liberated enzymes break down some of the carbohydrate and protein in the grain.[4] Germination of brown rice increases the gamma amino butyric acid (GABA) content, which has been reported to have several physiological functions.[5] Rice bran is a good source of phenolic compounds that have potent antioxidant properties and free radical scavenging capabilities.[6] These compounds are known to exert various physiological effects in humans, such as preventing oxidative damage of lipid,[7] inhibiting platelet aggregation,[8] and reducing the risk of coronary heart disease and cancer.[9 10] In rice, ferulic acid and p-coumaric acid are the major phenolic compounds.[11] Antioxidants are compounds that slow or prevent the oxidation of other molecules, which produce free radicals that start chain reactions and damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by oxidizing themselves. Though germinated brown rice is good for health, it cannot be compared with white polished rice when it comes to sensory acceptability. Consumers are likely to continue to prefer polished white rice to germinated brown rice. One option is to change the form in which germinated brown rice is consumed, for example, it could be extruded and converted into an instant pudding so that the health benefits of germinated brown rice can still be utilized. Extrusion is a thermal cooking process and it may destroy or remove the phenolic compounds and affect the antioxidant activity.[12 13] The objective of the present investigation was to study the effect of germination and extrusion cooking on the total phenolic content (TPC) and antioxidant activity (AOA) of brown rice. The acceptability of the instant pudding prepared from the germinated brown rice was also studied.

MATERIALS AND METHODS

Preparation of Germinated Brown Rice

Three paddy cultivars, namely IR–8, PR–106, and Sharbati varying in length breadth ratio (2.67, 3.52, and 4.59, respectively) were procured from Punjab Agricultural University, Ludhiana, Punjab (India). The dehusking was carried out in a McGill rice sheller (Rapsco, Brookshire, TX, USA) and brown rice was obtained. Preliminary trials were carried out to standardize the germination of the brown rice. The brown rice was steeped for 24 h and care was taken that water was changed at 2 h intervals and then allowed to germinate in a seed germinator (Caltan, NSW–191–192, Delhi) at 25°C and 100% RH for 24 and 48 h.[14] The germinated brown rice was dried to 12% moisture content in a drier at 45°C to stop germination and stored for further analysis.

Total Phenolic Content (TPC)

TPC was determined by the Folin-Ciocalteu specterophotometric method as described by Singleton and Rossi.[15] The ground sample, 200 mg was weighed and taken in centrifugal tubes along with 4 ml of acidified methanol and extracted for 2 h on a shaker followed by centrifugation for 10 min. The supernatant was collected and 1.5 ml of tenfold diluted Folin-Ciocalteu phenol reagent (Sigma, St. Louis, MO, USA) was added followed by the addition of tenfold diluted 6% sodium carbonate solution (Qualikems Ltd., India). The samples were incubated at room temperature for 90 min and absorbance was read at 725 nm. The TPC was calculated using standard plot of ferulic acid and expressed as mg ferulic acid equivalent/g flour. The following equation was used:

where Y is absorbance and X is concentration of ferulic acid.

Antioxidant Activity (AOA)

AOA was measured by the method described by Brand-Williams et al.[16] Ground sample (100 mg) in 1 ml of methanol was extracted for 2 h followed by centrifugation for 10 min. The supernatant was reacted with 3.9 ml of 6 × 10⁻⁵ mol/L 2,2–diphenyl-1-picrylhydrazyl (DPPH) (Sigma, St. Louis, MO, USA) and then incubated at room temperature for 30 min. Methanol was used as blank. Absorbance of control sample at ‘0’ min was taken at 515 nm. Reduction in absorbance of samples was observed after 30 min.

where t ₀ and t ₃₀ are absorbance at zero time and at 30 min.

Extrusion

The germinated brown rice was coarse ground in a grinder, such that 90% of the grit was retained by 44-mesh sieve (355 μm). The grits were conditioned by adding 4.76 g water/100 g rice and kept for 24 h for moisture equilibration. Extrusion of the grit was carried out at a screw speed of 100 rpm and feed rate of 550 g/min at two different temperatures, namely 100 and 120°C in a lab scale high shear single screw cooking extruder (GL Extrusion Systems, New Delhi, India). The screw had a diameter of 40 mm, L/D (Length/Diameter) ratio of 4:1, and the die diameter was 5 mm.

Bulk Density and Expansion of Extrudates

The bulk density expressed as gram per liter (g/l) was evaluated by measuring the weight of known volume of extrudates. Diameter of extrudates was measured by a vernier caliper and average diameter of ten extrudates was reported.

Water Solubility Index and Absorption Capacity

Ground control (unextruded) or extruded (0.5 g) was taken in the centrifuge tube along with 25 ml water and shaken for 30 min and then centrifuged (3000 rpm) for 10 min.[17] Supernatant was collected in a preweighed aluminium dish and dried at 105°C for 24 h and weighed again. Supernatant was collected to determine solubility index and weight of the remaining sediment was noted to determine absorption capacity.

Instant Pudding Mix

The extrudates were coarsely ground so that 90% passed through 20-mesh sieve (841 μm). The ingredients were optimized (data not reported), the extrudates were mixed with skim milk powder and ground sugar in the ratio 1.7:2:1.5, respectively, to form an instant pudding mix. Warm water was added to the pudding mix in the ratio 1:2 and sensory evaluation was carried out at 40°C using a 9-point hedonic scale, with 1 as dislike extremely and 9 as like extremely. The pudding was evaluated for taste, aroma, mouth feel, appearance, and overall acceptability by a semi-trained panel consisting of ten members from the department.

Statistical Analysis

Analytical and physicochemical tests were carried out in triplicate on dry weight basis. Analysis of variance was carried out and Fishers least significant difference test was used to describe means with 95% confidence using SPSS statistical software (SPSS Inc., Chicago, Illinois, USA).

RESULTS AND DISCUSSION

Total Phenolic Content (TPC)

The TPC in the brown rice from the three cultivars ranged from 0.803–0.885 mg ferulic acid equivalent/g flour (Table 1). The TPC significantly ( p < 0.05) increased in all the three cultivars upon germination for 24 h. Further increase in TPC was observed upon germination for 48 h, however in IR-8 the increase was not significant. The TPC increased by 8.8, 12.1, and 9.1%, respectively, in IR-8, PR-106 and Sharbati upon germination for 48 h. Ohtsubo et al.[18] reported that the total dietary fiber, total ferulic acid, and GABA contents of the pre-germinated brown rice were higher than those of ordinary brown rice or polished rice.

Table 1 Effect of germination duration and extrusion temperature on the total phenolic content of germinated brown rice from different cultivars

Cultivar	Germination time (h)	Control (mg/g)	Extruded 100°C (mg/g)	Extruded 120°C (mg/g)
IR-8	0 h	0.803^aq	0.434^ap	0.408^ap
	24 h	0.846^br	0.529^bq	0.465^bp
	48 h	0.874^bq	0.570^cp	0.537^cp
PR-106	0 h	0.885^ar	0.460^aq	0.409^ap
	24 h	0.943^br	0.490^bq	0.461^bp
	48 h	0.992^cr	0.530^cq	0.504^cp
Sharbati	0 h	0.857^ar	0.541^aq	0.466^ap
	24 h	0.908^br	0.581^bq	0.500^bp
	48 h	0.935^cr	0.614^cq	0.538^cp
a, b, and c superscripts are significantly ( p < 0.05) different column wise within a cultivar, and p, q, and r superscripts are significantly ( p < 0.05) different row wise within a cultivar.

Extrusion and increase in extrusion temperature led to a decrease in the TPC in all the cultivars. A decrease varying from 53.3–85.0% in TPC was observed in the control and germinated samples upon extrusion at 100°C. A further decrease of 6.1–13.7% was observed when the extrusion temperature was increased from 100 to 120°C. The increase in phenolic compounds in germinated brown rice could be explained as an increase in the free forms due to the breakdown of the cell wall during germination. Insoluble phenolic compounds have been found to be cell wall components,[19] which are bound to polysaccharides in the cell wall of rice grains. During germination induced saccharolytic enzymes breakdown endosperm which release bound phenolic compounds.[20] Yin and Mei[21] reported that after long-term germination of legumes the total phenolic and flavonoid content increased and contributed to higher antioxidant activity. Sharma and Gujral[22] reported that germination led to increase in TPC in different barley cultivars. Gumul et al.[23] reported that extrusion cooking resulted in a 40% decrease in the TPC in rye grains. Cooking of common beans in an autoclave at a temperature of 121°C and pressure of 103.4 kPa significantly decreased the TPC in common beans.[24] Heat labile phenolic compounds, such as ferulic acid, could be destroyed during extrusion cooking leading to a decrease in TPC.[25]

Antioxidant Activity (AOA)

The AOA of the three cultivars ranged from 6.9–11.1%. Germination for up to 48 h led to 18.2 to 37.9% increase in the AOA in the brown rice. IR-8 and PR-106 showed higher AOA as compared to Sharbati (Table 2). A significant increase in AOA after germination of barley (DPPH radical scavenging activity) has been reported by Sharma and Gujral.[22] Extrusion at a temperature of 100°C significantly lowered the AOA in all the cultivars. Increasing the extrusion temperature from 100 to 120°C further lowered the AOA. The AOA in IR-8 decreased by 44.67, 65.55, and 55.70% when the control, 24 and 48 h germinated samples were extruded at 100°C. Upon increase in the extrusion temperature, the AOA further decreased by 26.23, 29.07, and 41.29% in the control, 24 and 48 h germinated sample of IR-8. Similar decrease in the antioxidant activity of the control and germinated brown rice from PR-106 and Sharbati upon increase in the extrusion temperature was observed (Table 2). Phenolic compounds have been reported to be responsible for the AOA of grains, vegetables, and other botanical materials.[26] Korus et al.[27] reported that the TPC and AOA of beans decreased upon extrusion. Xu and Chang[28] reported that heat treatments, like boiling and steaming, significantly lowered the TPC and AOA of black beans. The decrease in TPC and AOA due to extrusion of the germinated brown rice could be attributed to the high temperature encountered during extrusion cooking process.

Table 2 Effect of germination duration and extrusion temperature on the percentage of antioxidant activity of germinated brown rice from different cultivars

Cultivar	Germination time (h)	Control	Extruded 100°C	Extruded 120°C
IR-8	0 h	11.14^aq	7.70^ap	6.10^ap
	24 h	13.89^bq	8.39^ap	6.50^ap
	48 h	15.29^br	9.82^aq	6.95^ap
PR-106	0 h	13.41^aq	6.32^ap	5.79^ap
	24 h	14.84^br	7.10^aq	6.01^ap
	48 h	15.86^cr	7.70^aq	6.48^ap
Sharbati	0 h	6.96^aq	4.65^ap	4.07^ap
	24 h	8.71^bq	5.53^ap	4.45^ap
	48 h	9.50^br	7.45^bq	5.38^ap
a, b, and c superscripts are significantly ( p < 0.05) different column wise within a cultivar, and p, q, and r superscripts are significantly ( p < 0.05) different row wise within a cultivar.

Expansion and Bulk Density

Germination and extrusion of the brown rice led to an increase in the expansion of the extrudates. Extrusion of the brown rice from IR-8 germinated for 24 and 48 h significantly increased the expansion of extrudates by 13.32 and 11.07%. The expansion of extrudates increased significantly ( p < 0.05) with rise in extrusion temperature for control (6.49%) and 24 h (4.89%) germinated samples, whereas in 48 h germinated sample the increase of 1.63% was insignificant (Fig. 1). In PR-106, expansion increased significantly ( p < 0.05) with increase in germination duration from 24 to 48 h by 5.53 and 5.30%, respectively. Rise in extrusion temperature did not increase the expansion significantly ( p < 0.05)for the control sample, whereas the 24 and 48 h germinated samples showed significant increase of 6.70 and 19.92%, respectively, with an increase in extrusion temperature (Fig. 1). In Sharbati, the expansion increased significantly ( p < 0.05) with an increase in germination duration by 7.29 and 13.0% (Fig. 1). The expansion increased significantly ( p < 0.05) with rise in extrusion temperature for all germinated samples by 7.83, 9.98, and 6.16%. Kim et al.[14] germinated brown rice for 24 and 48 h and upon extrusion at 120°C and reported that the expansion of extrudates increased as germination time increased. They also reported that the expansion of the extrudates increased as the extrusion temperature increased. Ding et al.[29] reported that a rise in extrusion temperature (100 to 140°C) increased the extrudate expansion and water solubility index and reduced the density.

Figure 1 Effect of extrusion cooking and rise in extrusion temperature on the % expansion of germinated brown rice from different cultivars. (a, b, and c superscripts are significantly (p < 0.05) different row wise within a cultivar, and p and q superscripts are significantly (p < 0.05) different column wise within a cultivar.)

The bulk density decreased as the germination duration increased in all the cultivars. As the extrusion temperature increased, no significant reduction in bulk density was observed for 24 and 48 h germinated samples from IR-8 but a significant decrease from 0.0887 to 0.0755 g/ml in the control sample was observed (Fig. 2). Similar reduction in the bulk density was observed in the extrudates from PR-106 and Sharbati as the extrusion temperature increased. Jha and Prasad[30] reported an increase in expansion and decrease in bulk density of extruded products prepared with blends of rice and mung flours upon rise in extrusion temperature.

Figure 2 Effect of germination and extrusion temperature on the bulk density of germinated brown rice from different cultivars. (a, b, and c superscripts are significantly ( p < 0.05) different row wise within a cultivar, and p and q superscripts are significantly ( p < 0.05) different column wise within a cultivar.)

Solubility Index and Water Absorption Capacity

Increase in the duration of germination increased the water solubility index of the control unextruded brown rice; however, the increase was not significant. During the germination process a significant part of some insoluble complex compounds, such as starch and proteins, are converted to simple soluble compounds leading to increase in solubility Singh et al.[31] Solubility increased significantly upon extrusion in all the cultivars at all germination durations (Table 3).

Table 3 Effect of germination duration and extrusion temperature on the solubility index of germinated brown rice from different cultivars

Cultivar	Germination time (h)	Control	Extruded 100°C	Extruded 120°C
IR-8	0 h	0.039^ap	0.234^aq	0.260^aq
	24 h	0.037^ap	0.263^aq	0.284^aq
	48 h	0.044^ap	0.271^aq	0.353^br
PR-106	0 h	0.040^ap	0.381^aq	0.491^ar
	24 h	0.043^ap	0.426^aq	0.517^ar
	48 h	0.044^ap	0.466^bq	0.519^ar
Sharbati	0 h	0.042^ap	0.316^aq	0.476^ar
	24 h	0.043^ap	0.383^aq	0.533^ar
	48 h	0.055^ap	0.465^bq	0.571^ar
a, b, and c superscripts are significantly ( p < 0.05) different column wise within a cultivar, and p, q, and r superscripts are significantly ( p < 0.05) different row wise within a cultivar.

When the ungerminated, 24 and 48 h germinated samples were extruded at 100°C the solubility increased by 500.25, 500.53, and 516.09%, respectively, in IR-8. Similarly the increase in PR-106 was 833.00, 872.6, and 945.06%, respectively, and in Sharbati the increase was by 652.61, 789.32, and 735.00%, respectively. When the extrusion temperature was increased from 100 to 120°C the solubility increased in all the samples (Table 3). Kim et al.[14] reported that the water solubility increased as the germination time increased from 24 to 48 h in brown rice extruded at 100°C.

The water absorption capacity increased in the control unextruded sample with increase in germination duration, however, the increase was not significant (Table 4). Upon extrusion at 100 and 120°C the absorption increased significantly in all the cultivars at all germination durations. Gujral and Singh[32] reported an increase in the water absorption index of extrudates prepared from brown rice grit. In IR-8 the control, 24 and 48 h germinated samples upon extrusion at 100°C exhibited an increase of 266.12, 254.41, and 257.00%, respectively. Similarly, the increase in PR-106 was 293.84, 310.60, and 288.31%, respectively, and in Sharbati the increase was 180.32, 174.62, and 171.83%, respectively. When the extrusion temperature was increased from 100 to 120°C the absorption increased significantly in all the samples. Kim et al.[14] reported an increase in water absorption of extrudates, which were extruded from brown rice germinated for 48 h. An increase in water absorption capacity with increase in extrusion temperature has been reported by Wang et al.[33] for texturized pea protein. Gutkoski and El-Dash[34] reported that the water absorption index of oat extrudates increased as the extrusion temperature was elevated. Guzman et al.[24] reported an increase in water absorption capacity values in extruded products from three Mexican common bean cultivars.

Table 4 Effect of germination duration and extrusion temperature on the water absorption capacity of germinated brown rice from different cultivars

Cultivar	Germination time (h)	Control	Extruded 100°C	Extruded 120°C
IR-8	0 h	1.24^ap	4.54^aq	5.76^ar
	24 h	1.36^ap	4.82^aq	6.36^br
	48 h	1.52^ap	5.44^bq	6.66^br
PR-106	0 h	1.30^ap	5.12^aq	5.38^aq
	24 h	1.32^ap	5.42^aq	6.30^br
	48 h	1.54^ap	5.98^aq	6.70^br
Sharbati	0 h	1.22^ap	3.42^aq	3.84^ar
	24 h	1.34^ap	3.68^bq	4.10^br
	48 h	1.42^ap	3.86^cq	4.32^cr
a, b, and c superscripts are significantly ( p < 0.05) different column wise within a cultivar, and p, q, and r superscripts are significantly ( p < 0.05) different row wise within a cultivar.

Instant Pudding

The cultivar Sharbati germinated for 48 h and extruded at 100°C was selected for the preparation of the instant pudding since it had the highest level of TPC. It was evaluated against the pudding prepared from control brown rice from Sharbati. The scores obtained for taste and aroma were significantly higher for the pudding made from germinated brown rice (data not reported) than that obtained from control rice. The appearance and mouth feel were not significantly affected, but the pudding made from germinated brown rice scored significantly higher for overall acceptability as compared to control brown rice.

CONCLUSION

The study revealed that both the TPC and AOA increased significantly with the increase in germination duration in all the cultivars. Extrusion and rise in extrusion temperature lowered both the TPC and AOA. The results also suggested that the physical characteristics, such as bulk density, decreased with increase in germination duration and rise in extrusion temperature. The extrudate expansion increased with an increase in germination duration and rise in extrusion temperature. The solubility index and water absorption capacity increased with increase in germination duration and rise in extrusion temperature. Brown rice when consumed in the germinated form delivers higher amounts of TPC having higher AOA but upon extrusion both the TPC and AOA is reduced. The instant pudding made from germinated brown rice was more acceptable than instant pudding made from control brown rice.