Preservation of Soybean Daddawa Condiment with Dichloromethane Extract of Ginger

MATERIALS AND METHODS

Collection of Seeds and Soybean Daddawa Preparation
The soybean samples of variety TGX1440-2E used for this research were obtained from the Institute of Agricultural Research and Training (I.A.R and T), Ibadan. Nigeria. Soybean daddawa was prepared according to the methods described by Obatolu et al. (1998).

Preparation of Ginger Extract
Preparation of ginger extract followed the procedures described by Zia-ur-Rehman et al. (2003).

Application of Ginger Extract (GE) and Ascorbic Acid (AA) to Soybean Daddawa
Soybean daddawa samples (5 g) containing 1600 and 2400 ppm ginger extract were prepared separately in sterile cellophane. Ascorbic acid was tested for comparative purpose in the neighbourhood of the legal limit of synthetic antioxidant in foods as reported by Noor and Augustin (1984). Concentrations for the antioxidants used were in accordance with Zia-ur-Rehman et al. (2003). In preparing 1600 ppm of extract, 800 mg of freshly prepared ginger extract was dissolved in 50 mL sterile distilled water, from which 0.5 mL of the resultant suspension was added to 5 g of soybean daddawa. Fifty milliltre sterile distilled water was added to 1200 mg of ginger extract, 0.5 mL of the suspension was added to 5 g soybean daddawa to obtain 2400 ppm. For ascorbic acid preparation 250 mg of ascorbic acid was dissolved in 50 mL sterile distilled water to obtain a standard solution. Half a milliliter of the standard solution was added to 5 g soybean daddawa to obtain 500 ppm concentration. To obtain 250 ppm, the standard solution was diluted twice before adding 0.5 mL of the resultant solution to 5 g of soybean daddawa. Control samples were prepared by adding 0.5 mL of sterile distilled water. All samples of each treatment were prepared in triplicate and stored at ambient temperature (30±2°C) for two weeks and the various analyses were done at two days interval.

Measurement of Antioxidant Activity
The effectiveness of the ginger extract, compared with ascorbic acid (synthetic) as an antioxidant in stored soybean daddawa was tested by the determination of Free Fatty Acid (FFA) content and peroxide value (POV) of the soybean daddawa samples kept at ambient temperature. The FFA content (expressed as % oleic acid) was estimated using an alkali titration method (AOAC, 1990), while the peroxide value (meq kg^-1) of samples was measured by titration with 0.1M sodium thiosulphate solution using starch as an indicator (AOAC, 1990). All determinations were carried out in triplicates and mean values were calculated. The experiments were repeated three times.

Microbial Counts
The method of Omabuvfe et al. (2000) was used for the enumeration of microbial population in the stored soybean daddawa samples. Counts were noted at zero time and two days interval.

Counted bacteria colonies were expressed as colony forming units per gram (cfu g^-1) of samples. Mean values of triplicates plates were recorded.

Statistical Analysis of Data
The data obtained were subjected to analysis of variance(ANOVA). Data were expressed as mean±standard deviation.

RESULTS AND DISCUSSION

Free Fatty Acid (FFA) content of untreated (control) samples increased progressively with time of storage while a corresponding decrease was recorded for samples treated with GE and AA. The difference observed in FFA values of samples treated with GE and those treated with AA was significantly different (p<0.05) compared to the values obtained for untreated (control) samples.

Even though the effect of treatment with antioxidant was more at higher concentrations (2400 ppm of GE and 500 ppm of AA), there was no significant difference (p>0.05) between the GE treated samples at both concentrations and AA treated samples at both concentrations (Table 1).

Observation in this study is in agreement with those of Halliwell and Gutteridge (1985), on the antioxidant properties of ascorbic acid. It has been reported that ascorbic acid plays its antioxidative role by reacting very rapidly with the reactive oxygen species, producing the semi-dehydroascorbic radical which breaks down rapidly in a very complex way eventually producing oxalic and L-threonic acids.

Lipid oxidation is a major cause of flavour deterioration in some pre-cooked foods (Ahn et al., 2007). The formation of organic acids and free fatty acids which is an initial step in fat deterioration was effectively controlled by the addition of GE and AA to the stored soybean daddawa. Although the ability of GE to reduce the rate of fat degradation was not investigated in this study, the report of Xuesong (1997) that identified various antioxidant compounds, especially gingerol, gingerdiol, shogaol and cumcumin in the non-volatile fraction of ginger may be relevant in this regard.

A drastic and progressive increase in peroxide values (POV) was recorded for untreated control samples with increasing storage time. Significant differences (p<0.05) were observed between the control and GE and AA treated samples the concentration of treatment notwithstanding.

Table 1:	Effect of ginger extract and ascorbic acid on Free Fatty Acids (FFA) content and peroxide values of stored soybean daddawa

Table 1:	Continued
Within a column, values with different superscripts are significantly different (p<0.05), Values are mean of 3 replicates± standard deviation, †Free fatty acid as % oleic acid, ‡Peroxide value in meq kg-1

Although for GE and AA treated samples, gradient in concentration did not significantly (p>0.05) affect POV values, significant differences (p<0.05) were observed in POV values obtained for GE treated samples and AA treated samples. Also, it was observed that, by the 8th day of storage, peroxidation in treated samples began to decline.

The initial increase in peroxide values obtained in the present study compares favourably with the pattern reported for soymilk (Iwe, 1991). That peroxide values increased with storage time is an indication that peroxidation is associated with soybean daddawa spoilage. Since an increase in peroxide value is a good predictor of fat deterioration (Zia-ur-Rehman et al., 2003). It may therefore imply that treatment of stored samples with GE and AA was responsible for the observed reduction in peroxidation of stored samples.

Both ginger extract and ascorbic acid reduced the bacterial load of the stored soybean daddawa samples (Table 2) as there was a significant difference (p<0.05) between the microbial load of untreated samples and those treated with antioxidants. However differences were not observed in the ability of the GE and AA to reduce the number of microorganisms present in the treated samples.

The representative bacteria species isolated from the product include Bacillus subtilis, B. lincheniformis, Staphylococcus sp. and Micrococcus sp. These organisms have been reported as the main organisms involved in the fermentation of soybean seeds to soy-daddawa (Omabuvfe et al., 2000) and have been used as starter cultures for the laboratory production of soy-daddawa (Popoola et al., 2007). Their presence is an indication that even after the normal period of fermentation, these bacterial species are still associated with the product in storage.

Table 2:	Total bacteria count (log10 cfu g-1) in stored soybean daddawa samples treated with different concentrations of ginger extract and ascorbic acid
Along a column, values with different superscripts alphabets are significantly different (p<0.05), Data is the means of three replicate samples, *Natural fermentation (Without antioxidant) ** Figures are Viable Count log10 cfu g-1

Although there are no reported cases of any kind of food poisoning due to the consumption of soybean daddawa, the results of this present study has shown that secondary oxidative compounds may have a role to play in the deterioration of soybean daddawa. The ingestion of oxidative products can be a risk to human health. There is therefore a need to achieve oxidative stability in stored daddawa for maintaining safety and quality of the product. The results of this study have shown dichloromethane extract of ginger can effectively play the role of ascorbic acid as an antioxidant for the purpose of preservation and extending the shelf life of soybean daddawa.

Many natural plant extracts primarily contain phenolic compounds which are potent antioxidants (Wong et al., 1995). Some of these compounds are also known to possess antimicrobial effects against food borne pathogens (Elgayyar et al., 2001). Details of the compounds of the extract of ginger which gives it antioxidant and antimicrobial properties as observed in this study is not known at this stage, but Cuppett (2001), reported that polyphenolic compounds are primarily responsible for antioxidant activities in many natural plant extracts.

Recently, the use of more natural preservatives has become more popular as compared to synthetic antimicrobials and antioxidants (Ahn et al., 2007). The growing interest in use of natural products of plant origin therefore gives GE an advantage over AA in developing a practical approach for the preservation of soybean daddawa. The likely adverse effect of high concentration of preservative on organoleptic properties of the condiment does not seem to pose a major problem with the adoption of GE as a preservative for soybean daddawa as there was no major difference between the antibacterial and antioxidant capabilities of GE and AA at the concentrations examined. However, further studies are needed to determine the effective concentration at which GE would achieve antimicrobial and antioxidant activities without adversely affecting organoleptic and related functional properties of stored soybean daddawa.