Effect of Traditional Fermentation Time on Total Phenolic Content and In vitro Antioxidant Activity of Kocho: Ethiopian Fermented Food from Enset (Ensete ventricusum welw. Chessman)

Ensete ventricusum, locally known as Enset, is the main crop of a sustainable food source that ensures food security in Central ,South and South West Ethiopia. It has been used as a food and local medicine for thousands of years. Kocho is a product of matured Enset prepared traditionally by fermenting (for few weeks to some months) a mixture of pulverized pseudostem and corm in pit. The objective of this study was to evaluate the effect of traditional fermentation period on the total phenolic content and antioxidant activity of Kocho. The sample was fermented in the pit for 15, 30, 45, 60, 75, 90, 105 and 120 days. Oven dried Kocho powder was extracted with methanol followed by water. Total phenolic content of the extract was determined by the Folinciocalteu method. Antioxidant activities of the extracts were evaluated with 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and total antioxidant activity using phosphomolybdenum assay. The results revealed that Kocho showed greater total phenolic content and antioxidant activities than unfermented product. Methanol extract of Kocho fermented for 45 days showed the highest amount of total phenolic content (9.27 ± 0.44 milligram of gallic acid equivalent per gram of dried extract). In comparison of DPPH scavenging and total antioxidant, methanol extracts were also superior to aqueous extracts. Methanol extract of Kocho fermented for 60 days exhibited the strongest DPPH scavenging activity (IC50 = 0.15 ± 0.02 mg/mL) and total antioxidant activity (0.43 ± 0.04 milligram of ascorbic acid equivalent per gram of dried extract). Thus fermentation offers a tool to further increase the bioactive compounds and antioxidant potential of Kocho.

demonstrated antimicrobial activity against viral, bacteria, fungal and nematodal diseases of humans (Holscher D. and Schneider B. 1998). Some of the Enset varieties are used as medicines by local people for both humans and livestock to cure bone fractures, joint displacement, broken bones, and childbirth problems ie, assisting to discharge the placenta (Balcha, 1990). So far, Biruk et al. (2012) studied Enset starch for industrial uses and found a characteristics of high gelatinization property whereas Gebre- Mariam and Schmidt (1996) obtained its use as a binder and disintegrant for tablets.
Though Enset is widely used as food source and traditional medicine, scarce information is available on the antioxidant activities of food products of this plant (Forsido et al., 2013). To my knowledge no study has been reported on the effect of fermentation period on total phenolic contents and antioxidant potential of Kocho. Therefore, the objective of the present study was to evaluate the effect of traditional fermentation period on total phenolic content and in vitro antioxidant activities of aqueous and methanol extracts of Kocho. In addition, the correlation between total phenolic content and antioxidant activities was also evaluated.

Sample Collection and Preparation
Enset local variety known as ''Adew'' was used for this experiment. The Enset plant was 4 years old grown in 5 Km South of Hawassa town, Sidama zone, South Ethiopia. Woman having traditional experience about the preparation of Kocho was selected to carry out the processing following the traditional farmers' practices. The scraped and pulverized masses ( Figure 1A) were thoroughly mixed with small amount of previously fermented Kocho (as starter of fermentation) and placed in the pit ( Figure 1B), lined with Enset leaves and left for fermentation at ambient temperature. A portion of fermented sample (Kocho) was removed from the pit and the liquid was squeezed out of it, resulting into a moist fibrous Kocho ( Figure 1C). Sample was collected every 15 days for analysis, up to four months from the date of processing until final fermentation stage and the samples were tested for the total phenolic content and antioxidant activities.

Preparation of Kocho Extracts
After oven dried (at 50 o C) sample was ground to fine powder using electric grinder (FM100 model, China). The aqueous and methanol extracts of all were prepared by dissolving 10 g of the samples fine powder separately in 100 mL of each solvent. The contents were kept in orbital shaker for 6 h at room temperature. Thereafter, each extract was filtered using Whatman no.1 filter paper and evaporated to dryness under vacuum at 40 o C by using a rotary evaporator (Buchi, 3000 series, Switzerland). The extraction was done in triplicate for each solvent and 46 the resulting extracts were stored in a sealed plastic container at 4 o C until further investigation.

Total phenolic Contents (TPC)
Total phenolic content was estimated by Folin-Ciocalteu method as described in Shan et al. (2005) with slight modification using gallic acid as standard. To 0.5mL of the extract (5mg/mL), 2 mL Folin-Ciocalteu reagent (diluted ten times) was added and the mixture was left for 5 min and then 1mL (75% w/w) of sodium carbonate was added. The absorbance of the resulting blue color was measured at 765 nm with a UV-visible spectrophotometer (JENWAY, 96500, UK) after incubation for 90 min at room temperature. The total phenolic content was estimated from gallic acid (1-100 µg/mL) calibration curve (y = 0.02x + 0.09, R 2 = 0.99) and results were expressed as milligram gallic acid equivalent/gram of dry extract (mgGAE/g).
2.5. Determination of Antioxidant Activity 2.5.1. DPPH method The capacity of kocho samples to scavenge free radical was measured with the stable radical of DPPH using method as described by Iqbal et al. (2005), , with minor modification. Two mL of freshly prepared DPPH solution (0.06%, w/v) in methanol was mixed with different concentrations ((0.1up to 5 mg/mL) of 1 mL of extract and reference standards (BHT) dissolved in methanol. The mixtures were vortexed and kept in a dark room for 30 min at room temperature. The absorbance of the resulting solution was then taken at 520 nm. Methanol was used as blank. The ability to scavenge the DPPH radical was calculated using the following equation: Where Ac is the absorbance of the control and As is the absorbance in presence of the sample of the extracts. The antioxidant activity of the extract was expressed as IC50. The IC50 value was defined as the concentration of extracts that scavenges the DPPH radical by 50%. The value (in mg/mL) was extrapolated by plotting percentage inhibition of sample against the corresponding sample concentration.

Determination total antioxidant activity by phosphomolybdenum method
The total antioxidant activity of the crude extracts was evaluated by the phosphomolybdenum method (Prieto et al., 1999) with slight modification. The method is based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds or crude extract and subsequent formation of green Mo (V) complexes with a maximal absorption at 695 nm at acidic medium (Mohamed et al., 2011). Plant extract (0.3 mL) was mixed with 3 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The samples were incubated at 95 • C for 90 min, cooled to room temperature and absorbance was measured at 695 nm and methanol (3 mL) was used as blank. The total antioxidant activity was expressed as milligram butylated hydroxyltoluene equivalent/gram of dried extract (mgAAE/g) based on the calibration curve; y = 0.301x + 0.002, R 2 = 0.99.

Statistical Analysis
The data were subjected to analysis of variance (ANOVA) and Duncan's multiple range tests were used for mean separation at p < 0.05. Linear regression analysis was used to calculate IC50 value. Pearson correlations between antioxidant activities, total phenolic contents were considered at p < 0.05.

Total Phenolic Content of Kocho
The total phenolic content of Kocho was expressed as milligram of gallic acid equivalent per gram of dried extract (mgGAE/g) as shown in Figure 1. In all samples methanol extracts had higher total phenolic content than that of aqueous extracts. Phenolic content of methanol Kocho extracts analyzed in the study ranged from 1.32 ± 0.02 mgGAE/100 g for unfermented sample to 9.27 ± 0.9 mgGAE/100 g for Kocho fermented for 45 day. Among the aqueous extracts, Kocho fermented for 60 days had the highest (p < 0.05) phenolic content, while unfermented Kocho sample had the lowest phenolic content. Similarly the total phenolic content of methanol extracts of fermented Kocho increased with increasing the period of fermentation. The total phenolic content of Kocho fermented for 45 days was the highest (p < 0.05). Whereas, the unfermented sample had the lowest total phenolic content (p < 0.5). The result increased up to 45 days. After 60 days of fermentation the total phenolic content decreased gradually but no significance level (p > 0.05) was observed. But the total phenolic content all fermented samples was significantly higher than that of unfermented sample. These results were consistent with findings on fermented Okra seeds (Adetuyi and Ibrahim 2014), Cabbage (Sun et al., 2009), and Jaruk tigarun (Nazarni et al., 2016), traditional fermented food from South Borneo Indonesia, where fermentation caused increase in the total phenolic contents. In natural form, phenolic compounds are combined or bound with sugar 47 which reduces their availability to organism. Many studies indicated that deglycosilation of phenolic compounds could be performed through microbial fermentation due to glycosyl hydrolase family activities (Huynh et al., 2014). These enzymes are well known for their deglycosylation capability by hydrolyzing the different glycosidic bonds existing between sugars and phenolic compounds, causing a significant increase in the amount of different free phenolic compounds (Cho et al., 2009;Lee et al., 2013;Di Gioia et al., 2014 ).

DPPH Free Rradical Scavenging Activity
Scavenging the stable DPPH radical model is a widely used method to evaluate in vitro antioxidant activity. DPPH is a stable free radical with characteristic absorption at 520 nm and antioxidants react with DPPH radical and convert it to diamagnetic 2, 2-diphenyl-1-picrylhydrazine molecule. The degree of discoloration indicates the scavenging potential of the antioxidant extract, which is due to the hydrogen donating ability (Von Gadow et al., 1997). Figures 2 and 3 show dose dependence curve for DPPH radical scavenging activity of methanol and water extracts of fermented Kocho which quantified by its absorbance reduction at a wavelength of 520 nm. As concentration of sample increased, the percent of DPPH scavenging increased. Generally, in response to fermentation treatment, there was an increase in relative percentage of radical scavenging activity in all fermentation periods, compared to the control. The result obtained was in accordance with previous research done on red cabbages by Hunaefi et al. (2013), who reported an increase in the percentage of radical scavenging activity in fermented cabbage compared with unfermented cabbage as a control. The increase may be due to the increase in acidic value during fermentation that is liberating bound flavonoid components and making it more bio available (Ashish et al., 2014). Fermentation time did significantly increase the DPPH radical-scavenging activities for all extracts, as compared to unfermented pulverized corm. Contrary to the present result, fermented Kocho sample collected from South Western Ethiopia, showed weaker antioxidant activity than the unfermented pulverized corm of Enset (Forsido et al., 2013). Table 2 shows IC50 value, which is the inhibition concentration of sample extract required to decrease initial DPPH activity by 50%. DPPH scavenging activities were also reflected in the IC50 values where the value for methanol extract of Kocho fermented for 60 days (IC50 = 0.15 ± 0.02 mg/mL) was significantly (p < 0.05) lower (stronger antioxidant activity) than that of all fermented Kocho samples. The unfermented fresh sample showed the weakest (IC50 > 5mg/mL) DPPH scavenging activity. Similarly, the water extract of Kocho fermented for 60 days showed the strongest DPPH scavenging (IC50 = 0.81 ± 0.47 mg/mL) activity. Further increasing the fermentation period (above 60 fermentation days) decreased the DPPH scavenging potential of Kocho extract. BHT as a reference showed stronger DPPH scavenging activity (p < 0.05) than that of all fermented Kocho extracts. Different studies reported (Moktan et al., 2008;Ademiluyi and Oboh, 2011) that prolonged fermentation decreased the available phenolic compounds because of the microorganisms grow using these compounds as substrates (Ehsan et al., 2010). Therefore the optimal fermentation period is important to get the maximum amount of phenolic compounds during fermentation of food products.  1.55 ± 0.18 c 0.33 ± 0.04 abc 120 1.03 ± 0.39 c 0.27 ± 0.08 ab IC50 (BHT) = 0.08 ± 0.02 mg/mL Values are expressed as mean ± SD (n = 3) from triplicate experiments.. Values within a column with different letters are significantly different at p < 0.05.
3.3. Total antioxidant activity using phosphomolybdenum assay Similar to DPPH scavenging, the methanol extract showed stronger total antioxidant activity than that of aqueous extracts (Table 3). Among the methanol extracts, Kocho fermented for 60 days showed the strongest total antioxidant activity with the value of 0.43 ± 0.04 mgAAE/g. whereas unfermented sample showed the significantly the weakest (p < 0.05) total antioxidant. No significant difference (p > 0.05) was found between the total antioxidant activities of Kocho fermented 15 up to 30 days and also these values have no significant difference with Kocho fermented 75 up to 90 days. But these values showed significantly (p < 0.05) stronger total antioxidant activity than Kocho fermented between 105 and 120 days. The total antioxidant activities of all fermented Kocho samples were significantly stronger (p < 0.05) than that of unfermented sample. Similarly, the water extract of Kocho fermented for 60 days showed the strongest total antioxidant activity (p < 0.05).

Correlation analysis
It is well known that the antioxidant activity of a plant extracts are largely depending on both the concentration and the nature of phenolic compounds present in the extract (Catherine et al., 1996;Weng and Huang. 2014). In this study, the relationship between total phenolic content and antioxidant activity Kocho fermented for different period of time extracted by methanol and water solvents was evaluated ( Table 4). The results showed good linear correlation in the cases of DPPH scavenging activity (R 2 = 0.53, p > 0.05) and total antioxidant activity (R 2 = 0.52, p > 0.05) with total phenolic content of methanol extracts of Kocho fermented up to 120 days. In case of aqueous extracts the total phenolic was weakly correlated ((R 2 = 0.48, p > 0.05) with total antioxidant activity but moderately correlated (R 2 = 0.63, p > 0.05) with DPPH scavenging activity of all Kocho samples.

Conclusions
This study demonstrated that fermentation caused a marked increase in total phenolic content which then enhanced DPPH radical-scavenging ability and total antioxidant activity of the Kocho extracts. Fermentation time affected the antioxidant activities of Kocho and fermentation for 60 days seemed to be applicable as exemplified by the least IC50 value of DPPH radical-scavenging ability and highest value of total antioxidant power. The methanol extract showed the strongest DPPH scavenging and total antioxidant activity. Whereas, in all extraction periods aqueous extracts exhibited weaker DPPH scavenging and total antioxidant activities.
In conclusion, extending of fermentation time had resulted differences in total phenolic contents and antioxidant activity of Kocho. Therefore, fermentation played a significant role in Kocho preparation by enrichment of the product through development and release of phenolic compounds due to the action of cell wall-degrading enzymes produced through fermentation of bioactive components and enhancing antioxidant capacity. Generally, the selection of appropriate Enset varieties, improved processing methods, and recommended fermentation time are good practice for the processing of tradition food products, such as Kocho an Ethiopian traditional fermented food. This work was carried out with the analysis of crude Kocho extract. However, further studies are needed to evaluate the in vivo experiments of various solvent extracts and individual bioactive compounds in various animal models.