Effect of Starter Cultures on PH, Cyanide and Protein Content at Different Inoculums level and Fermentation time
pH
There was a significance difference (p˂0.05) in pH of fermented cassava-teff flour due to single starter culture (Table 1,2), size of inoculums (Table 3) and fermentation time (Table 4) except between Lactobacillus plantarum and Lactobacillus coryneformis using the same inoculum level and fermentation time. Cassava- teff flour fermented with each of 1.5 ml of Lactobacillus plantarum, Lactobacillus coryneformis and Saccharomyces cerevisiae at 48 h showed pH change from 6.72 ±0.02 to 3.60±0.01, from 6.70± 0.10 to 3.62 ±0.02 and from 6.72 ± 0.02 to 5.12 ±0.10 respectively (Table 3), while using 1 ml inocula of Lactobacillus plantarum, Lactobacillus coryneformis and Saccharomyces cerevisiae pH reduced from 6.66±0.02 to 3.91±0.06, 6.66±0.10 to 3.93±0.05 and from 6.71±0.10 to 5.43±0.11 respectively after 48 h fermentation time. As fermentation time increases, there was also reduction of pH in all starter cultures expect control (non-fermented sample), differing the extent of reduction depending on types of microorganisms, inoculum level and fermentation time. Similarly, Gunawan et al. [14] showed that the optimum pH condition of Lactobacillus plantarum and Saccharomyces cerevisiae was 3.5-4.5 and 3.5-6.0 respectively indicating that cassava fermentation by the action of a single species of micro-organisms can result in a significant reduction in pH. The decreases of pH during the fermentation of cassava-teff flour results from the production of an organic acid by lactic acid bacteria on the carbohydrate content of cassava root [17,27,28,30].
Table4. pH, protein and cyanide at different times and microbes (inoculum 1.5 ml for all).
Contents
|
Treatment with different microbes at different time
|
Control
|
Lactobacillus
plantarum
|
Lactobacillus coryneformis
|
Saccharomyces
cerevisiae
|
24 h
|
48 h
|
24 h
|
48 h
|
24 h
|
48 h
|
24 h
|
48 h
|
pH
|
6.62±0.02a
|
6.45±0.19a
|
5.02±0.02d
|
3.60±0.10e
|
5.01±0.20d
|
3.62±0.02e
|
5.70±0.00b
|
5.12±0.01c
|
Protein (%)
|
4.23±0.01e
|
4.22±0.01e
|
5.96±0.37d
|
6.98±0.01c
|
5.90±0.03d
|
7.02±0.01c
|
7.24±0.02b
|
13.31±0.02a
|
Cyanide(mg/kg)
|
156.46±0.02a
|
151.84±0.16a
|
56.38±0.38c
|
5.54±0.06e
|
55.19±0.46c
|
5.29±0.50e
|
67.57±0.88b
|
9.64±0.03d
|
Value represents the mean ± Standard deviation (n=3).Within the raws, the values are significantly different (P≤0.05).
Cyanide
Addition of single starter culture, inoculum level and time of fermentation exhibited significant (p
< 0.05) differences on cyanide content of fermented cassava-teff flour, but there was no significant difference between Lactobacillus plantarum and Lactobacillus coryneformis(Table 2,3 and 4). The cyanide content of all fermented samples was reduced to lower levels compared to control (non-fermented). However, the extent of reduction varied with the type of microorganisms, size of inoculum and fermentation time. The cyanide content of the cassava-teff flour fermented at 48 h with 1.5 ml inoculums of Lactobacillus corneformis was the lowest (5.29±0.50 mg/kg) and followed by Lactobacillus plantarum (5.54±0.06 mg/kg). The variation in the reduction of cyanide content within given microorganism is attributed to differences in time of fermentation and the size of inoculum used. In cassava-teff flour fermented with 1.5 ml inoculum of Lactobacillus plantarum at the 24 h, cyanide contents reduced from 159.00±1.17 mg/kg to 56.38 ± 0.38 mg/kg and subsequently reduced to 5.54 ± 0.06 mg/kg after 48 h.
This shows that further more fermentation of cassava-teff flour with Lactobacillus plantarum and Lactobacillus coryneformis for 48 h caused significant reduction of cyanide content. The reduced cyanide content of fermented cassava- teff flour by Lactobacillus plantarum and Lactobacillus coryneformis were below the safe level recommended by WHO [32]. These findings are consistent with the results of Kobawila et al. [17], who reported cyanide reduction drastically from 1158 to 339.6 mg/kg after 48 h of fermentation which corresponds to 70.67 % reduction only the difference is initial cyanide content.
Fermentation of cassava flour by selected microorganisms result in microbial growth was shown to be essential for the efficient elimination of cyanogens [20,28]. From our investigation, Lactobacillus plantarum and Lactobacillus coryneformis appear to play an important role in cyanide detoxification, as already reported by Labri et al. [18] and Tefara et al. [28]. This indicates that it is possible to significantly reduce the residual HCN content of cassava through fermentation using Lactobacillus coryneformis and Lactobacillus plantarum. The reduction in cyanide content could be attributed to the ability of the inoculated microorganism (Lactobacillus plantarum and Lactobacillus coryneformis) to produce linamarase which can hydrolyze linamarin and result in degradation of cyanogenic glycosides in to HCN which is subsequently converted in to formamide which is used as both a nitrogen and carbon source [17,23].
According to Table 1, 2, 3 and 4 there was high efficiency of the Saccharomyces cerevisiae, next to Lactobacillus plantarum and Lactobacill coryneformis, in abating the cyanide levels that are seen after 24 and 48 h compared to control which contains high levels of cyanide (151.84±0.16 to 156.46±0.02 mg/kg), whereas, in cassava-teff flour inoculated with 1.5 ml Saccharomyces cerevisiae, cyanide level dropped from 159.00+1.17 mg/kg to 67.57±0.88 mg/kg and 9.64±0.03 mg/kg after 24 and 48 h respectively. This shows that Saccharomyces cerevisiae is capable of utilizing cyanogenic glycosides and the breakdown products, thus explaining why they are natural flora involved in cassava fermentation [21,22]. The degradation might be due to enzymes linamarase, hydroxynitrile lyase and cyanide hydratase that catalyze the sequential degradation of cyanogenic glycosides into HCN which is subsequently converted into formamide which is used as both a nitrogen and carbon source [23].
Table5. Comparison of the effect of the microbial fermentation using 1.5 ml inoculums at 48 h and boiling on cyanide content of cassava–teff flour.
Treatment
|
Cyanide content
|
Final concentration (mgHCN/kg dw)
|
HCN reduction (%)
|
Lactobacillus plantarum
|
5.54±0.06d
|
96.51
|
Lactobacillus coryneformis
|
5.29±0.05d
|
96.670
|
Saccharomyces cerevisiae
|
9.10±0.03b
|
94.40
|
Average
|
6.38±0.01c
|
95.90
|
Boiling
|
96.10±0.70a
|
47.77
|
A value represent means±SD (n=3).Within the column, different letters indicate significantly different values(P≤0.05).
Comparison of the Microbial Fermentation and Boiling on Cyanide Contents
The results indicate that the effect of microbial fermentation on cyanide content was significantly different(p˂0.05) from boiling as boiling accounted for about 47.77% of cyanide reduction whereas, the fermentation by the single starter cultures for 48 h accounted for 94.40 to 96.67% (Table 5). These results are consistent with and Cardosan et al. [7] Nambisan [20] that boiling (cooking) method could only reduce cyanide content 20-50%, suitable for the processing of sweet variety which contains small cyanide. The result was also in agreement with the results of Abebe et al. (2014) who reported that the maximum and minimum percentage reduction of cyanide for fermented cultivars of Gamo and 5553-19, and boiled cultivar of Hayek was observed to be 98
% and 51% respectively. This may be in boiling (cooking) enzymatic breakdown of linamarin is small due to heat denaturation of linamarase [7]. Whereas using microbial fermentation was a very efficient process for elimination of cyanide suggests that this method needs to be used for processing of the variety containing a high amount of cyanide [18,24,27].
Crude Protein
Single starter cultures, inoculums level and time of fermentation had a significant effect (p˂0.05) on the crude protein content of fermented cassava-teff flour. The protein content of fermented cassava-teff flour is significantly higher than that of control (Table 2, 3 and 4). The crude protein content of cassava-teff flour fermented with each of 1.5 ml of Lactobacillus plantarum and Lactobacillus coryneformis increased from 4.23± 0.03 % to 6.98 ± 0.01 and 7.02± 0.01 % respectively after 48 h. The extent of increasing protein content depends on types of microorganism, inoculum level and fermentation time which is consistent with the earlier report by Tefera et al. [28] that fermentation of cassava-based food by Lactobacillus Plantarum could have increased protein content up to 4.31%. Okafor et al. [23] also given his observation that cassava mash fermented by Lactobacillus coryneformis increased lysine content 1.2 to 2.45 % after 48 h of fermentation.
The difference is only initial protein content in cassava root that it would appear that the organisms may definitely play some role in increasing the protein content of fermented cassava teff flour because the protein content in the control (non-fermented) cassava- teff flour was consistently lower than the cassava-teff flour inoculated with Lactobacillus plantarum and Lactobacillus coryneformis. The increase in protein content of fermented cassava-teff flour may be because of some microorganisms which degrade cassava pulp by readily could have secreted some extracellular enzymes (proteins) in the cassava pulp [21]. Growth and proliferation of bacteria during fermentation time in the form of single-cell proteins increases that may be possibly accounted for the apparent increase in protein content [5].
The protein content in cassava-teff flour fermented with 1.5 ml inoculums of Saccharomyces cerevisiae (13.31±0.02 %) was higher than that of Lactobacillus plantarum and Lactobacillus coryneformis (Table 4). In our observation, further fermentation of cassava-teff flour with Saccharomyces cerevisiae for 48 h caused a significant (p ˂ 0.05) increase in the protein content. The crude protein content of fermented cassava-teff flour by Saccharomyces cerevisiae showed in Table 3 and 4 was lower than that reported by Boonnop et al. [5] who demonstrated that fermentation of cassava chips with Saccharomyces cerevisiae could increase crude protein content from 2 % to 32.4 %. The difference could probably attribute to the size of inoculums used and fermentation time. Similarly, the increase protein content also agrees with earlier reports [23] that fermentation of cassava with Saccharomyces cerevisiae would increase protein content, indicating that Saccharomyces cerevisiae had the highest capability to enrich the crude protein content of cassava products.
The increase in protein content in fermented cassava-teff flour could be attributed to the ability of Saccharomyces cerevisiae to secret some extracellular enzymes such as amylases, linamarase, and cellulase into cassava mash during their metabolic activities which could lead to yeast growth [5]. This high protein cassava product could very well serve as a protein source in animal diets provided it is economically viable.
Table6. Sensory acceptability scores of injera at different times and microbes (using 1 ml inoculum for all).
Quality attributes
|
Treatment with different microbes at different time
|
Control
|
Lactobacillus
planetarium
|
Lactobacillus
coryneformis
|
Saccharomyces
cerevisiae
|
24 h
|
48 h
|
24 h
|
48 h
|
24 h
|
48 h
|
24 h
|
48 h
|
Flavor
|
2.33±0.29d
|
2.33±0.29d
|
3.13±0.13c
|
3.80±0.20b
|
3.07±0.12c
|
3.77±0.38b
|
3.90±0.17b
|
4.53±0.06a
|
Color
|
4.17±0.29a
|
4.17±0.29a
|
4.30±0.26a
|
4.43±0.40a
|
4.33±0.06a
|
4.47±0.06a
|
4.50±0.50a
|
4.67±0.29a
|
Taste
|
2.60±0.17d
|
2.60±0.17d
|
3.87±0.12b
|
4.67±0.31a
|
3.83±0.06b
|
4.57±0.40a
|
3.13±0.23c
|
3.83±0.15b
|
Texture
|
2.27±0.23d
|
2.27±0.23d
|
3.07±0.40c
|
3.87±0.12b
|
3.07±0.06c
|
3.83±0.29b
|
3.90±0.17b
|
4.47±0.42a
|
Overall
acceptability
|
2.77±0.25d
|
2.77±0.25d
|
3.56±0.06c
|
4.17±0.29b
|
3.53±0.06c
|
4.17±0.29b
|
3.93±0.12b
|
4.40±0.34a
|
Value represents the mean of three replicates ± Standard deviation (n=3). Within the raws, a different letter indicates significantly different values (P≤0.05).
Table7. Sensory acceptability scores of injera at different times and microbes (using 1.5 ml inoculum for all).
Quality attributes
|
Treatment with different microbes at different time
|
Control
|
Lactobacillus plantarum
|
Lactobacillus
coryneformis
|
Saccharomyces cerevisiae
|
24 h
|
48 h
|
24 h
|
48 h
|
24 h
|
48 h
|
24 h
|
48 h
|
Flavor
|
2.33±0.29d
|
2.33±0.29d
|
3.43±0.21c
|
4.17±0.29b
|
3.4±0.17c
|
4.13±0.23b
|
4.17±0.29b
|
4.87±0.23a
|
Color
|
4.17±0.29a
|
4.17±0.29a
|
4.43±0.21a
|
4.17±0.29a
|
4.40±0.17a
|
4.53±0.06a
|
4.67±0.29a
|
4.83±0.28a
|
Taste
|
2.60±0.17d
|
2.60±0.17d
|
4.27±0.25b
|
4.90±0.17a
|
4.27±0.23b
|
4.87±0.23a
|
3.47±0.57c
|
4.23±0.25b
|
Texture
|
2.27±0.23d
|
2.27±0.23d
|
3.40±0.10c
|
4.17±0.47b
|
3.40±0.20c
|
4.17±0.29b
|
4.13±0.23b
|
4.73±0.11a
|
Overall
acceptability
|
2.77±0.25d
|
2.77±0.25d
|
3.87±0.12c
|
4.43±0.21b
|
3.87±0.23c
|
4.40±0.34b
|
4.13±0.23b
|
4.67±0.12a
|
Value represents the mean of three replicates ± Standard deviation (n=3). Within the raws, a different letter indicates significantly different values (P≤0.05).
Sensory Evaluation of Cassava Based Food (Injera)
Flavor
The sensory acceptability showed significance (p˂0.05) differences between single starter culture and control on the flavor of produced injera. Analysis of variance also had shown significance (p˂0.05) differences among single starter cultures (Table 6) at different fermentation time (Table 7) and inoculums level (Table 8) except between Lactobacillus plantarum and Lactobacillus coryneformis. The means scores of the flavor test of samples are appeared to improve with a longer period of fermentation of cassava-teff flour for each starter culture, the highest values being attained around 48 h of fermentation time. The score given to flavor acceptability were highest for injera produced from 1.5 ml inoculum of Saccharomyces cerevisiae after 48 h fermentation time, which was 4.87±0.23. Regarding the inoculum level, the highest score observed in injera produced from a 1.5 ml inoculum level for all starter cultures.
The flavor acceptability score of the control (none fermented) 2.33 ± 0.29 is lower when compared with injera produced from the fermented cassava-teff flour. The microbial activities which increased as fermentation continued might have accounted for the perceived differences in the flavor of the product fermented for different lengths of fermentation time. The flavor of food depends on the balance of volatile compounds those produced during fermentation. A vast number of volatile compounds may be synthesized and modulated by Saccharomyces cerevisiae during fermentation. In line with this finding, Tefera et al. [28] reported that Saccharomyces cerevisiae was able to produce compounds such as organic acids, alcohols, aldehydes, and carbonyls which have imparted appealing flavor to the fermenting cassava.
A previous study by Hasan et al. [15] on fermented rice also showed an increase in volatile compounds due to fermentation by yeasts and lactic acid bacteria. Similar to the current study it has been reported that Saccharomyces cerevisiae contributes to flavor development while fermenting rice for injera production.
Color and Taste
Regarding Color of cassava-based food (injera) the highest score was 4.83 ± 0.28 and 4.67 ± 0.29 by 1.5 and a 1ml inoculum of Saccharomyces cerevisiae respectively at 48 h fermentation time, while the least score 4.17±0.29 was for control. However, Analysis of variance showed that using a single starter culture (Table 6), time of fermentation (Table 7) and addition of inoculum level (Table 8) had shown no significant difference (P ˃0.05) on the color acceptance of injera. Color acceptability scores of the single starter culture and the control (non- fermented) were all in the range of 4.17±0.29 and 4.83 ± 0.28 indicating that using single starter culture at different inoculums level and fermentation time does not change the color of injera.
The sensory score of taste appears greater than three in all cassava-based food (injera) produced by fermentation except for control (non- fermented). Analysis of variance showed that using single starter culture, addition of inoculums level and fermentation time had shown significant (p˂0.05) difference regarding taste attributes of cassava-based food, but no significant difference (p˃0.05) was detected between Lactobacillus plantarum and Lactobacillus coryneformis on taste attributes of cassava-based food (Table 6,7 and 8). This indicates that all organisms seem to be responsible for the taste of the cassava-based food as suggested by Odibo and Umeh [22] and confirmed by the present study. However, panelists rated the sample fermented with 1.5 ml inoculums of Lactobacillus plantarum and Lactobacillus coryneformis at 48 h as having the best taste with the score of 4.90± 0.17 (98%) and 4.87 ± 0.23 (97%) respectively, but the sample fermented with 1 ml inoculums of Saccharomyces cerevisiae at 24 h fermentation time as having lower taste with the score of 3.13±0.23 (62.6%).
As the fermentation time increased, the scores for taste also increased in each starter culture. This result agrees with earlier reports by Tefera et al. [28] that fermentation of cassava-based food (chike) by Lactobacillus mesenteroides result in 72% score of taste, only a difference is inoculum level used. This might be possibly attributed to the fact that Lactobacillus plantarum and Lactobacillus coryneformis converts the sugars in fermenting substrate (primarily glucose and fructose) to lactic acid, acetic acid, ethanol, CO2 and other flavor compounds [24,25,31].
Texture
As shown in Table 6, 7 and 8, there was a significant difference (p˂0.05) in the texture acceptability scores between control and starter cultures as well as among starter cultures, inoculum levels and fermentation time of cassava-based food. However, the least scores 3.07±0.4 and 3.07±0.06 were for the cassava-based food fermented with 1 ml inoculums level of Lactobacillus plantarum and Lactobacillus coryneformis respectively at 24 h. The highest mean score 4.73±0.11 was observed for the cassava-based food fermented with 1.5 ml Saccharomyces cerevisiae at 48 h fermentation time followed by 1.5 ml Lactobacillus plantarum (4.17±0.47) and Lactobacillus coryneformis (4.17±0.29). This showed that the starter culture, inoculums level and fermentation time
influences the quality of dough thereby that of the texture of the injera. This indicates that Saccharomyces cerevisiae possesses cellular activities, where also found to be contributed to the modification of cassava texture during fermentation [21,29].
Table8. Sensory acceptability scores of injera using different inoculum size and microbes (time 48 h for all).
Quality attributes
|
Treatment with different microbes at different inoculum size
|
Control
|
Lactobacillus
plantarum
|
Lactobacillus
coryneformis
|
Saccharomyces
cerevisiae
|
|
1 ml
|
1.5 ml
|
1 ml
|
1.5 ml
|
1 ml
|
1.5 ml
|
Flavor
|
2.33±0.29e
|
3.80±0.20d
|
4.17±0.29c
|
3.77±0.38d
|
4.13±0.23c
|
4.53±0.06b
|
4.87±0.23a
|
Color
|
4.17±0.29a
|
4.43±0.40a
|
4.63±0.15a
|
4.47±0.06a
|
4.53±0.06a
|
4.67±0.29a
|
4.83±0.28a
|
Taste
|
2.60±0.17e
|
4.67±0.31b
|
4.90±0.17a
|
4.57±0.40b
|
4.87±0.23a
|
3.83±0.15d
|
4.23±0.25c
|
Texture
|
2.27±0.23e
|
3.87±0.12d
|
4.17±0.47c
|
3.83±0.29d
|
4.17±0.29c
|
4.47±0.42b
|
4.73±0.11a
|
Overall acceptability
|
2.77±0.25d
|
4.17±0.29c
|
4.43±0.21b
|
4.17±0.29c
|
4.40±0.34b
|
4.40±0.34b
|
4.67±0.12a
|
Value represents the mean of three replicates ± Standard deviation (n=3). Within the raws, a different letter indicates significantly different values (P≤0.05).
Overall Acceptability
Overall acceptability scores exhibited significance (p˂0.05) differences between starter cultures and control (Table 6). The inoculums level (Table 7) and fermentation time (Table 8) also exhibited significance (p˂0.05) differences. However, there was no significance (p˂0.05) difference between the Lactobacillus plantarum and Lactobacillus coryneformis (Table 7 and 8). The highest overall panelists acceptability 4.67±012 was recorded for the sample fermented with 1.5 ml inoculum of Saccharomyces cerevisiae at 48 h fermentation time, followed by 1.5 ml inoculums of Lactobacillus coryneformis and Lactobacillus plantarum which was 4.40±0.34 and 4.40±0.34 at 48 h fermentation time, while the lowest overall panelist acceptability 3.53±0.06 was recorded by 1 ml inoculums of Lactobacillus coryneformis at 24 h fermentation time.
This indicates that pure cultures of isolates had varying contributions to the overall acceptability of injera. This finding is in agreement with Tefera et al. [28] who explained in his report that overall sensory score 3.48 for the sample fermented with Saccharomyces cerevisiae for 48 h with the addition of 0.75 ml inoculums showed preference by panelists compare to samples fermented with Lactobacillus palantarum and Lactobacillus mesenteroides. The difference is only types of food prepared and amounts of inoculum level used. This might be due to the improvement of the sensory quality of the product by Saccharomyces cerevisiae. A previous study by Hasan et al. [15] on fermented rice flour has shown an increase in
volatile compounds due to fermentation by yeasts and lactic acid bacteria. This volatile compound may significantly impact the overall quality of the product. Chelule et al. [8] also explained in his report that fermentation makes the food palatable by enhancing its flavor which makes fermented food more popular than the unfermented one in terms of consumer acceptance.