Article International Journal of Food Nutrition and Safety, 2014, 5(1): 16-23 International Journal of Food Nutrition and Safety Journal homepage: www.modernscientificpress.com/journals/ijfns.aspx ISSN: 2165-896X Florida, USA Effect of Fermentation Time on Quality Attributes of Zobo Drink Prepared from Hibiscus Sabdariffa Linn Emmanuel O. Nwafor *, Olubunmi O. Akpomie Department of Microbiology, Delta State University, Abraka, Nigeria * Author to whom correspondence should be addressed; E-Mail: obiomanwafor@yahoo.com; Tel.: +2348035878792. Article history: Received 17 January 2014, Received in revised form 21 February 2014, Accepted 23 February 2014, Published 12 March 2014. Abstract: Investigation on the effect of fermentation time on the quality attributes of Zobo drink was conducted at room temperature by examining the changes in ph, titratable acidity, carbohydrate, protein and vitamin C contents and total soluble solids (TSS). Sensory attributes of aroma, taste, visual appearance (colour) and overall acceptability were also assessed. Saccharomyces cerevisiae and Aspergillus niger were used for controlled fermentation for a period of 48 h while analyses for the various parameters were carried out at 12 h intervals. Results showed various degrees of changes in quality attributes compared with fresh product. Zobo drink produced following fermentation for a period of 12 h did not differ much from the fresh product. Similarly, sensory qualities did not show any appreciable preference as regards overall acceptability within the same period. Fermentation by S. cerevisiae yielded products which scored higher for aroma, visual appearance, taste and overall acceptability than those by A. niger. Acceptability declined from the 24th hour. Fermentation for 12 h with Saccharomyces cerevisiae is recommended when fermentation is to be employed in zobo drink production. Keywords: fermentation; quality; attributes; Zobo; Hibiscus sabdriffa; drink. 1. Introduction Zobo drink is obtained from the calyces (flowers) of Hibiscus sabdariffa. It is a refreshing non-
17 alcoholic beverage that now plays very important role in the dietary pattern of most Nigerians. The calyces of Hibiscus sabdariffa are known to be very rich in vitamins and other antioxidants (Ogiehor and Nwafor, 2004). The method of preparation has been widely described, accepted and adopted (Maggi Family Menu Cook Book, 1986; Nwafor and Ikenebomeh, 2009). This method involves very little or no fermentation process. In the fermentation of foods, a complex mixture of carbohydrates, proteins, fats and other similar components undergo modifications simultaneously under the action of a variety of microorganisms and enzymes present. The nature and extent of these changes depend on the food, types of microorganisms present and environmental conditions affecting their growth and metabolic patterns (Jokotagba and Amoo, 2012). Previous reports have indicated the presence of reasonable quantities of carbohydrates, proteins and fats in Zobo drink (Ogiehor and Nwafor 2002; Uche et al., 2012). It is envisaged that if controlled fermentation is allowed to take place in Zobo drink for a given period of time, it may result in the improvement of its quality attributes. The present study was therefore undertaken to determine the effect of fermentation time on the quality attributes of Zobo drink at ambient temperature. 2. Materials and Methods Dried calyces (petals) of Hibiscus sabdariffa were obtained from Igbudu Market, Warri, Delta State. Zobo drink was prepared following modification of the recipe in Maggi Family Menu Cook Book (1986). Microorganisms used for the fermentation were Saccharomyces cerevisiae and Aspergillus niger. The dried petals were thoroughly rinsed in sterile water and divided into 5 different portions of 200 g each. Each portion was placed in a 2.00L wide mouth flask of boiling water and left for 5 min. The mixtures were then allowed to cool at room temperature. There were two sets of the experiment. Thereafter 1.00 ml of 48h old broth culture of Saccharomyces cerevisiae and Aspergillus niger were separately inoculated into the remaining flasks containing the mixture (A. niger in 4 flasks and S. cerevisiae in other 4). The content of one flask each from the two sets were filtered into another flask and to the filtrates was added a syrup made by boiling 5 g of sugar in 0.50L of water for 10 min. The quality attributes were then assessed for each. These attributes include ph, protein, carbohydrate and vitamin C contents, titratable acidity and sensory quality were also assessed. Filtration, addition of sugar syrup and quality attributes determinations were repeated for the other flasks at 12 h time intervals for 48 h. 2.1. Determination of Quality Attributes of Zobo Drink The ph of Zobo drink was determined using standardized ph meter (Model 291 Mk2 Pye Unicam, UK). Titratable acidity was determined by titrating 0.10 M sodium hydroxide (NaOH) against
18 10 ml of Zobo drink using phenolphthalein as indicator (AOAC, 1990) and expressed as per percentage lactic acid (Speck, 1984). Vitamin C content was estimated by titrating 2, 6-dichlorophenolindophenol against 5 ml of Zobo drink treated with glacial acetic acid (Plummer, 1978). Total carbohydrate content was estimated according to Plummer (1978), earlier described by Lowry et al. (1951). Percentage total soluble solids content was determined using Abbe hand refractometer (Atago Co. Ltd, Japan) at room temperature and calculated using sucrose conversion table corrected to 20 o C (Person, 1976). 2.2. Sensory Evaluation A trained 10 member panel consisting of students and staff evaluated the samples for visual appearance (colour) aroma, taste and overall acceptability at intervals of time (12 h). A 9-point hedonic scale (where 1 = dislike extremely; 2 = dislike very much; 3 = dislike moderately; 4 = dislike slightly; 5 = neither like nor dislike; 6 = like slightly; 7 = like moderately; 8 = like very much and 9 = like extremely) was used to score the various parameters (Larmond, 1982). 2.3. Statistical Analysis Data obtained were subjected to statistical analysis of mean, standard deviation and analysis of variance (ANOVA). The significant value was determined by t-test distribution using appropriate computer software (Ogbeibu, 2005). 3. Results and Discussion Changes in the quality attributes of Zobo drink following fermentation with Saccharomyces cerevisiae or Aspergillus niger for a period of time are presented in Tables 1 & 2. There were changes in the various attributes of ph, titratable acidity, protein, carbohydrate and vitamin C contents and total soluble solids. Fermentation carried out by S. cerevisiae (Table 1) shows that there was gradual decrease in ph from the initial value of 3.60 ±0.10 through 2.60 ± 0.02 after 24 h and 2.00±0.01 at the end of 48 h. Titratable acidity increased from the initial value of 0.085±0.02% to 0.18±0.4% at the end of 48 h. Protein, carbohydrate and vitamin C contents of the Zobo drink showed increase from 0.34±0.01%, 0.76±0.03% and 0.36±0.20 mg/ml respectively to 0.60±0.03, 1.00±0.02 and 0.63±0.02 mg/ml after 48 h respectively. The total soluble solids however showed a downward trend, decreasing from initial value of 10.80±0.05% brix to 9.80±0.02% brix at the end of 24 h and 8.50±0.03% brix after 48 h.
19 Table 1. Changes in quality attributes of Zobo drink with time following fermentation by Saccharomyces cerevisiae for 48 h Parameters Fermentation time (h) (Attributes) 0 12 24 36 48 ph 3.60±0.10 3.04±0.03 2.60±0.02 2.40±0.03 2.00±0.01 Titratable acidity (%) 0.085±0.02 0.09±0.01 0.10±0.03 0.15±0.02 0.18±0.04 Protein content (%) 0.34±0.01 0.38±0.01 0.45±0.04 0.49±0.03 0.60±0.03 Carbohydrate (%) 0.76±0.03 0.80±0.04 0.90±0.02 0.96±0.01 1.00±0.02 Total soluble solids (%) 10.80±0.05 10.60±0.03 9.80±0.02 9.30±0.01 8.50±0.03 Vitamin C (mg/ml) 0.36±0.20 0.38±0.05 0.40±0.02 0.05±0.03 0.63±0.02 Note: Each value is the mean ± SD of 2replicates. Table 2. Changes in quality attributes of Zobo drink with time following fermentation by Aspergillus niger for 48 h Parameters Fermentation time (h) (Attributes) 0 12 24 36 48 ph 3.60±0.10 3.02±0.03 2.30±0.02 2.10±0.04 1.40±0.01 Titratable acidity (%) 0.085±0.02 0.09±0.01 0.10±0.03 0.19±0.02 1.00±0.02 Protein content (%) 0.34±0.01 0.37±0.03 0.43±0.02 0.48±0.04 0.50±0.02 Carbohydrate (%) 0.76±0.03 0.70±0.03 0.78±0.04 0.80±0.01 0.96±0.02 Total soluble solids (%) 10.80±0.05 10.40±0.02 9.60±0.03 9.00±0.02 8.50±0.03 Vitamin C (mg/ml) 0.36±0.20 0.36±0.02 0.39±0.02 0.40±0.04 0.44±0.03 Note: Each value is the mean ± SD of 2 replicates. The results presented in Table 2 shows the changes due to fermentation by A. niger. The trends are the same as with fermentation by S. cerevisiae. However, in all the cases, the changes were more with A. niger considering the values for the various parameters at the beginning and at the end of the fermentation period. For instance, the ph after 24 h and 48 h were 2.30±0.02 and 1.90±0.01, respectively which are obviously lower than 2.60±0.02 and 2.00±0.01 from fermentation by S. cerevisiae over the same period of time. Total soluble solids contents also showed a decrease from the initial values of 10.80±0.05% brix to 8.30±0.01% brix after 24 h. On the other hand, vitamin C content increased from 0.36±0.02 mg/ml to 0.39±0.02 mg/ml after 24 h and 0.44±0.03 mg/ml after 48 h of fermentation. The changes in sensory qualities of Zobo drink due to fermentation by S. cerevisiae or A. niger
20 is shown in Table 3. Sensory assessment did not show any appreciable preference as regards overall acceptability of Zobo drink within the first 12 h of fermentation. However, fermentation by S. cerevisiae was scored higher for aroma, visual appearance and taste than that by A. niger. Acceptability started declining after 24 h fermentation. Table 3. Changes in sensory qualities of Zobo drink with time following fermentation by Saccharomyces cerevisiae or Aspergillus niger Ferm. Time (h) S. Sensory Qualities Aroma Visual appearance Taste OA A. S. A. S. A. S. A. cerevisiae niger cerevisiae niger cerevisiae niger cerevisiae niger 0 8.40±1.00 8.30±1.10 8.60±1.00 8.50±0.50 8.0±1.30 8.10±1.20 8.80±1.00 8.80±1.20 12 8.80±1.20 8.00±1.10 8.40±0.50 7.50±0.8 8.20±1.00 7.30±0.50 8.40±0.80 7.0±0.60 24 7.00±1.00 6.0±0.80 6.50±0.60 5.60±0.50 6.30±1.10 5.0±0.80 6.0±1.10 4.80±0.50 36 6.30±0.60 5.30±0.60 6.00±1.10 4.90±1.20 5.0±1.20 4.40±1.10 4.80±0.90 4.0±1.10 48 5.30±1.00 4.0±0.60 5.0±1.20 3.90±0.50 3.80±0.60 3.0±0.80 3.10±0.80 2.8±0.60 Note: Each value represents mean ± SD of 10 scores from 10 member panel; OA = Overall acceptability. This study has indicated that controlled fermentation for a given period of time using appropriate microorganisms can bring about an improvement in the quality attributes of Zobo drink at ambient temperature. During fermentation, microorganisms carry out metabolic activities which results in the release of metabolites such as organic acids. This may account for the decreasing ph values recorded and the concomitant increase in titratable acidity obtained in the present study. It agrees with earlier reports (Efiuvwerwere and Ezeana, 1996; Oshuntogun and Abaoba, 2004), the increase in the carbohydrate, protein and vitamin C contents of the Zobo drink may be due to fermentative activities of S. cerevisiae or A. niger. Microbial fermentations have been linked to breakdown of various components of their substrate thereby releasing nutrients (Ogiehor and Nwafor, 2004). This supports the findings in this study. Increase in protein content with fermentation may also be due to the activity of fermentative microorganisms to synthesize amino acid as has been earlier reported (Nansen and Field, 1984; Jokotagba and Amoo, 2002). Vitamin C content is known to decrease with storage depending on the type of fruit and storage method. The retention of vitamin C is used to estimate the overall nutrient retention of a food product as it is by far the least stable nutrient and highly sensitive to oxidation and leaching into water soluble media during storage (Davey et al., 2000.). However, in the present study, there was a progressive increase in vitamin C content which may be due to the fact that the product under study is not a fruit but in liquid form. It means that during fermentation in a liquid
21 medium like Zobo drink, the vitamin C content increases as against what obtains in a fruit with the internal matrix. This is in line with the report of Uche et al. (2012). The reduction in total soluble solid (TSS) of Zobo drink with fermentation time may be attributed to its utilization by fermenting microorganisms especially fungi. This agrees with the report of Efiuvwevwere and Eka (1991), also reported by Nwafor and Ikemebomah (2009). The changes observed were more pronounced with samples fermented by S. cerevisiae than those by A. niger. S. cerevisiae is a kind of yeast, and yeasts are known to demonstrate greater fermentative ability in the presence of sucrose (Champagne et al., 1989). It may therefore account for the better performance of S. cerevisiae here in the presence of sugar. The decline in sensory quality of Zobo drink after 12 h fermentation may be attributed to a decline in the fermentative ability of the microorganisms with time. Yeast has been reported to decline in population with increasing acidic condition to which yeasts are exposed (de Oliva and Yokoya, 1994). Exposure of yeasts to increasing acidic conditions results in the release of amino compounds. Although other factors may be involved, this suggests interactive effects of the micro-environment, reduced oxygen content and presence of growth factors may have stimulated the growth of lactic acid bacteria (LAB) which may result in the decline in the acceptability scores after 12 h. This corroborates earlier finding by Daesehel et al. (1988). On the other hand, the improvement in aroma up till the 12 th hour can be attributed to the production of desirable amino volatiles (without putrefaction).conversely, the decline in visual appearance (colour) scores after 12 h can be attributed to proteolysis and browning. This agrees with an earlier report by Diarar (1993). The 12 h fermentation process in the production of Zobo drink should be adopted because of its desirable sensory attributes and commercial potential arising from reduction in production time. 4. Conclusions Variation in fermentation time during the preparation of Zobo drink has effect on the quality attributes using Saccharomyces cerevisiae and Aspergillus niger. Such attributes as ph, titratable acidity, protein, carbohydrate and vitamin C contents showed different values for different periods of fermentation. Sensory evaluation within thin the first 12 h did not show any appreciable preference as regards overall acceptability. It is suggested that 12 h fermentation could be adopted for optimum value of the drink as it will reduce waste due to improper fermentation. Acknowledgements The authors are grateful to their project students who assisted in some aspects of the study and the panel members for their co-operation in assessing the sensory qualities of the Zobo drink.
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