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General analysis and Antioxidant study of Traditional fermented drink

Handia, its concentrate and volatiles.

Article · January 2012

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Advances in Life Science and its Applications (ALSA) 54
Vol. 1, No. 3, 2012
Copyright © World Science Publisher, United States
[Link]

General analysis and Antioxidant study of Traditional

fermented drink Handia, its concentrate and volatiles

1
Anupam Roy, 2Kalyani Khanra, 3Anjan Mishra and 4Nandan Bhattacharyya
1,2,3&4
Department of Biotechnology, Haldia institute of Technology, Haldia, Dist. Purba Medinipur, Pin -721657, West
Bengal, INDIA.
4
Author for correspondence and reprint requests, Dean, School of Food & Biotechnology, Haldia institute of
Technology, Haldia, Dist. Purba Medinipur, Pin – 721657, West Bengal, INDIA. Phone: +919434453188.

Email: bhattacharyya_nandan@[Link]

Abstract – In this study, the antioxidant activity of Traditional fermented drink Handia, its concentrate and volatiles
were determined in terms of total antioxidant, reducing capacity, free radical scavenging, metal chelating activity. The
total phenolic compounds were also determined. Results indicated that Handia, its concentrate exhibited similar strong
total antioxidant activity. This was also true for reducing capacity and free radical scavenging activity. For all the cases
volatile components from fermented food Handia exhibits much lower antioxidant activity. Results indicated that active
antioxidants were nonvolatile and resided in the concentrate after evaporation. In that particular drink other components
rather than Phenolics compounds may play a very important role in the antioxidant function. However, identification of
specific antioxidant components should be further investigated.

Keywords – Handia, Antioxidant activity; Reducing capacity; Scavenging activity; Chelating activity

1. Introduction This low cost drink is claimed to be an energetic one

which retards tiredness and offers uninterrupted sleep.
Handia, an indigenous alcoholic-fermented beverage is Besides several therapeutic uses like relief from liver
prepared from parboiled rice by the ethnic tribes Santal, cancer, liver disorder and constipation, treating urinary
Sabar, Bhumij, Paroja, Kondh, kolh, Mundari,Juang etc infections (Dhal et al., 2008) and prevention of cancer
spread among eastern region specially in Orissa and West and cardiovascular disease are also assigned with the
Bengal. It is made by mixing traditional fermenting drink consumer, but there is no epidemiological evidence.
culture- Bakhar with boiled rice and allowing them to
ferment in an earthen pot (Handia) for 2-3 days with We hypothesized that Handia contains some antioxidants
mouth slightly open. Bakhar holds the source of several and active biochemical that may partially contribute the
microorganism prepared by mixing different plant claimed health effect.
ingredient with rice powder, water and giving batter to
the shape of small round balls (Dhal et al., 2008), The objective of this study was to evaluate the general
sometime Bakhar powder from previous batches are also composition and antioxidant properties of Handia, its
added in batter (Hutchinson et al,1925). Then balls are concentrate and volatile compounds (VC) with the
wrapped with leaves, allowed to ferment and dry measurements including the reducing capacity, the
continuously in shade for 3 days. scavenging activity on the free radicals, and the chelating
activity on ferrous ions.
After fermentation, the fermented slurry is filtered and
weak cream-colored product is taken as drink. Handia is a 2. Materials and methods
staple beverage consumed by the people of mentioned
ethnic group both men and women of all ages. It is used 2.1. Chemicals
in day to day life both as a food and an intoxicant
(Chowdhury et al., 2006).This practice of processing and All the chemicals were purchased from Sigma. All other
drinking of Handia is the integral part of mentioned chemicals used were of analytical grade.
ethnic tribes. The consumption pattern increases on
special occasions, like new born, weddings, worshiping 2.2. Samples collection of traditionally prepared
god etc. Handia
Roy et al., ALSA, Vol. 1, No. 3, pp. 54-57, 2012 55

The free radical scavenging activity of Handia, its

Traditionally prepared Handia was collected from a local concentrated sample(cs) and volatile components(vc) was
village of the indigenous ethnic group Jhargram in measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH)
Paschim Medinipur, West Bengal in India. Traditionally using the method of Que, et al., 1986 with slight
prepared Handia was centrifuged at 5000xg for 10 min, modifications Roy, et al., 2012. DPPH solution (0.1
supernatant was collected and stored at 4°c for analysis. mmol/l) in ethanol was prepared. 2ml of this solution was
added to equal volume of water solution containing
2.3 Preparation of concentrated Handia and volatile different amount of sample. After 30 min absorbance was
compounds measured at 517 nm. Lower absorbance of the reaction
mixture indicated higher free radical scavenging activity.
The concentrate of Handia was obtained after it was The % inhibition of the antioxidant was measured as
concentrated to one-ninth of the origin volume by a
vacuum evaporator at 45°C to remove the Volatile The % inhibition
compound (vc). The concentrated sample (cs) was thus
diluted 9 times with distilled water to compensate for the
lost volume prior to analysis. The VC was collected after Where Abs (control) is the absorbance of the control
condensation of evaporated volatiles during (blank, without extract) And Abs (sample) is the
concentration. absorbance in the presence of the sample. From inhibition
data, 50% inhibition or reduction amount (the amount of
2.4 Determination of total antioxidant capacity by a particular concentration of antioxidant that reduces the
phosphomolybdenum method 50% absorbance of DPPH solution) is calculated.

The total antioxidant activity of the Handia, its 2.7 Chelating activity of hardia of Fe2+ ions
concentrated sample (cs) and volatile components (vc)
were evaluated by using phosphomolybdate method as The chelating activity of the sample for Fe2+ is measured
described by Prieto et al. 1999. The assay is based on the according to the methods described by Dinis et al. (1994).
reduction of Mo (VI)–Mo (V) by the test sample and 0.5 ml of sample, 1.6 ml of de-ionized water and 0.05 ml
subsequent formation of a green phosphate/Mo (V) of FeCl 2 (2 mM) is added, followed by the addition of
complex at acid pH. 0.3 ml of fermented broth was 0.1 ml of ferrozine (5 mM) after 15 min gap. Then it was
combined with 3 ml of reagent solution (0.6 M sulfuric incubated 10 min at room temperature. The absorbance of
acid, 28 mM sodium phosphate and 4 mM ammonium the Fe2+ -ferrozine complexes with red or violet color
molybdate). The tubes were capped and incubated in a was measured at 562 nm. One milliliter of distilled water,
boiling water bath at 95°C for 90 min. After cooling to instead of Handia sample was used as a control. Lower
room temperature, the absorbance of the solution was absorbance of the reaction mixture indicated higher
measured at 695 nm using a spectrophotometer. The chelating activity. The chelating anti-oxidant activity for
antioxidant capacity of each sample was expressed as Fe2+ was calculated according to the following formula:
ascorbic acid equivalent using the following linear
equation established using ascorbic acid as standard: [A = Chelating rate (%) = (Ac – As) / Ac × 100
0.0037C + 0.0343; R² = 0.991] where A is the absorbance
at 695 nm and C the concentration as ascorbic acid Where, Ac is the absorbance of the control reaction and
equivalent (μg/ml). The values are presented as the means As is the absorbance of the sample extract.
of triplicate analysis.
2.8 Determination of total phenolic compounds
2.5 Reducing Power Assay
Total phenolic compound of the samples were estimated
The reducing capacities of Handia, its concentrated by the method Sadashivam and Manickam (2004).
sample (cs) and volatile components (vc) were Different aliquots of the samples were pipette out and the
determined by (Oyaizu et al., 1986). Different volumes volume in each tube was made up to 3.0ml with distilled
(50 μl, 100 μl -200 μl) of substrate were taken in 1 ml water distilled water and 0.5 ml Folin –Ciocalteu reagent
water. Then it was mixed with 2.5 ml of 0.2 M sodium was added. After 3minutes, add 2ml of 20% Na2CO3
phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium solution and place the tubes in boiling water bath for one
ferricyanide (w/v). The mixture was incubated at 50°C min, cooled and the absorbance was measured at 765 nm.
for 20 min. After cooling to room temperature 2.5 ml of Standard graph was prepared by using different
10% trichloroacetic acid (w/v) was mixed and mixture concentration of gallic acid.
was centrifuged at 3000 x g for 10 min. The upper layer
(2.5 ml) was mixed with 2.5 ml deionizer water and 1 ml 3. Results and Discussion
of 0.1% of ferric chloride, and the absorbance was
measured at 700 nm. Higher absorbance indicates higher 3.1 Total antioxidant activity of Handia its
reducing power of the sample. concentrated sample (cs) and volatile components (vc)

2.6 DPPH radical scavenging activity The total antioxidant capacity determined after reduction
of Mo (VI) to Mo (V) in presence of the antioxidant
compounds and measuring the green phosphate/Mo (V)
Roy et al., ALSA, Vol. 1, No. 3, pp. 54-57, 2012 56

complex at acidic pH, at 695 nm shows effective (cs). The 18.73 ml volatile component (vc) of Handia
antioxidant activity. From the analysis, the total offers 50% inhibition or reduction amount.
antioxidant content of the Handia was 520μg/ml, whereas
concentrated compounds (cs) exhibited similar strong
antioxidant activity (493 μg/ml), compared to Handia but
the antioxidant activity of Volatile components was much
weaker (21 μg/ml). For all the three cases a linear
equation using ascorbic acid as standard was taken for
analysis. [A = 0.0037C + 0.0343; R² = 0.991where A is
the absorbance at 695 nm and C the concentration as
ascorbic acid equivalent (μg/ml)]. The values are taken as
the means of triplicate analysis.

3.2 Reducing Power Assay

Figure 1 shows the reducing power of the Handia, its

concentrated sample (cs) and volatile components (vc) Figure 2. Free radical scavenging activity of different amounts
using potassium ferricyanide reduction method. Reducing of Handia, its concentrated sample (cs) and volatile components
power is associated with the antioxidant activity (vc) by DPPH radicals. Gallic acid (), Handia (), its
(Amarowicz et al., 2004). The yellow color of the test concentrated sample() and volatile components()
solution changes to various shades of green and blue,
3.4 Metal chelating activity
depending upon the reducing power of sample. The
presence of ductant (Antioxidants) in the fermented broth
Ferrozine form complexes with Fe 2 +. Presence of
causes the reduction of Fe3+/ Ferric cyanide complex to
chelating agents disrupts the complex formation,
ferrous form. Therefore Fe2+ complex is monitored by
resulting in a decrease in the red color of the complex.
measuring the formation of Perl’s prussian blue at 700nm
Measurement of color reduction therefore allows
(Hancock et al., 2007). The reducing capacity of Handia
estimating the metal chelating activity of the coexisting
and concentrated sample (cs) increased with increasing
chelator (Yamaguchi et al., 2000; Gulc ın et al., 2003).
amount, of the substrate and the reducing capacity of
Handia, its concentrated sample (cs) and volatile
Handia was seen to be slightly higher than that of
components (vc) showed limited chelating activity on
concentrated samples. However, volatile compounds (vc)
Fe2+ ions. The standard EDTA (100 μg/ml ) of 0.5 ml
showed little reducing capacity.
volume shows a chelating rate of 39.119% where as
Handia, its concentrated sample (cs) and volatile
components (vc) of 0.5 ml volume yields only
3.66%.3.25 and 0.12 chelating rate.

3.5 Total phenolic compounds

In this study, 3.92 and 3.21 µg of phenolic compounds

were detected in 1ml of Handia, its concentrated sample
(cs).Total phenolic compound in Volatile components
(vc) of Handia was seen to be 0.4µg, consistent result
with the total antioxidant and reducing capacity.
For all cases total phenolic content was measured as
gallic acid equivalent using the following linear equation
Figure 1. Reducing capacity of different amounts of Handia, its established using gallic as standard: [A = 0.210C + 0.292;
concentrated sample (cs) and volatile components (vc) by R²= 0.997] where A is the absorbance at 765 nm and C
potassium ferricyanide method. Handia(), its concentrated the concentration as gallic acid equivalent (μg/ml).
sample() and volatile components()
4. Conclusions
3.3 DPPH radical scavenging activity
The antioxidant property of Handia, its concentrated
Handia and its concentrated sample (cs) showed similar sample (cs) and volatile components (vc) results a similar
significant effects on the DPPH radical scavenging at all trend as compared to Chinese yellow wine, its
amounts (Fig. 2), but volatile compounds (vc) of Handia concentrate and volatiles as reported by Que., et al.
had limited DPPH radical scavenging activity. These Results showed a similar result that Handia and the
results were consistent with total antioxidant activity concentrate owned effective antioxidant activity, but the
(Fig. 1). 50% inhibition or reduction amount (the amount VC had little antioxidant activity for all indicative cases
of a particular concentration of antioxidant that reduces of total antioxidant activity, reducing capacity and free
the 50% absorbance of DPPH solution) of Gallic acid is radical scavenging activity. This might indicate that the
0.3827 ml (concentration 100μg/ml) where as 0.7781 ml antioxidant components resided in the nonvolatile parts
is for Handia and 0.8297 ml for its concentrated sample of the Handia, its concentrated sample. It can Ethanol is a
 Roy et al., ALSA, Vol. 1, No. 3, pp. 54-57, 2012 57

volatile component, and thus the ethanol in the Handia of alcohol use in the Sundarban Delta, West Bengal,
should be evaporated during concentration. That signifies IndiaSocial Science & Medicine ,63 ,722–731.
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Acknowledgements 9. Prieto, P., Pineda , M., and Aguilar, M., 1999.
Spectrophotometric quantitation of antioxidant
The work was funded by Department of Science & capacity through the formation of a
Technology, Government of West Bengal (No. 646/1(8) Phosphomolybdenum Complex: Specific application
(Sanc.)/ST/P/S&T/Misc-14/2009 dated 29-01-2010). to the determination of vitamin E, Anal. Biochem.
269, 337-341.
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