Abstract

Lipases are glycerol ester hydrolases which hydrolyze esther linkages of glycerides at water-oil interface. The activities of lipase in fermented oil bean seeds (ugba), fermented castor oil seeds (ogiri) and kunu were determined with respect to the levels of free fatty acids produced from each sample. The samples were prepared by local adoption of processing oil bean and castor seeds. The seeds were cooked (100°C) in cooking pot for 4hr, chopped into pieces, washed with clean tap water and covered in a stainless pot and allowed to ferment at room temperature. The lipid peroxidation and lipase activity were also determined on the fermented products using standard methods. The total free fatty acid determined were 349± 7, 1026± 5 and 94± 5mg/ml for ugba, ogiri and kunu respectively. The level of lipid hydroperoxide concentrations of fermented ugba, ogiri and kunu were (2.5 ±0.3) ×10^-4, (1.57 ±0.05) ×10^-4 and (4.5 ±0.3) × 10^-4 mg/ml malondialdehyde respectively. It was observed that the higher the peroxidation or concentration of malondialdehyde in a given sample, the least the lipase activity as determined by the level of free fatty acids. The findings of this research indicate that fermented ugba, ogiri and kunu could be good sources of lipase for industrial application.

1. Introduction

Food fermentation is important aspect of diet worldwide. Fermentation constituted a substantial percentage of African regimens for decades ^{1, 2}. Some of the advantages of fermentation are the preservation of significant amount of food and biological fortification of food substrates with vital nutrients ².

‘Ugba’ is a prevalent native fermented condiments made from legumes and oil seeds by fermenting melon seeds (Citrullus vulgaris), fluted pumpkin (Telfairia occidentlis) and castor oil seeds (Ricinus communis) ³. Different varieties exist depending on the raw material used ³.

Kunu is a popular nonalcoholic drink made from grains and consumed mainly in the northern part of Nigeria ⁴. The beverage drink is mostly produced from millet, sorghum or maize depending on the local availability ⁵. Although a refreshing drink, the locally processed beverage drink has as a challenge of having short shelf life and thus needs appropriate preservatives to improve its shelf-stability and keeping value ⁴. Spicy ingredients like ginger and saccharifying agents are added depending on the locality and taste ^{6, 7}. To improve the shelf life of kunu and present it in a hygienic state different processing methods and studies have been adopted ^{8, 9, 10}.

The term ‘lipase’ represents the enzyme system that is commonly called ‘lipases’ ¹¹. They are hydrolase enzymes that catalyze the hydrolytic cleavage of ester carboxyl bonds in acylglycerol into their constituent free fatty acids and glycerols; which may be employed as structural material and energy source by the organism ¹¹. Lipases are the most common biocatalysts that have significant applications in stimulating several biochemical methods in the industry ¹². They are effective in catalyzing several processes related to the food, pharmaceutical, leather ¹³, cosmetic, detergent, ¹⁴, medical diagnostics, diary, beverage, fatty acid and paper industries ¹⁵.

Lipid hydroperoxides are non-radical intermediates derived from unsaturated fatty acids, phospholipids, glycolipids, cholesterol esters and cholesterol itself. Their formation occurs in enzymatic or non-enzymatic reactions involving activated chemical species known as reactive oxygen species (ROS) which are responsible for toxic effect in the body via various tissue damages. These reactive oxygen species include among others hydroxyl radicals, lipid oxyl or peroxyl radicals, singlet oxygen and peroxynitrite formed from nitrogen oxide, all free radicals.

Significant contributions have been made in microbiology and biochemistry of fermentation of legumes and oil seeds leading to production of fermented condiments such as ugba from African bean seed, ogiri from castor seed or melon seed ¹⁶. Because continuous intake and production of lipase in our daily life is paramount, this research work is therefore, aimed at examining food samples especially fermented food stuffs like ugba, ogiri and kunu to determine their malondialdehyde concentration of free fatty acid through lipase activities.

2. Materials and Methods

African oil bean seeds (Pentaclethra macrophylla), castor seeds (Ricinus communis) and millet seeds (Eleusine coracana) were purchased from local market in Abia State, Nigeria.

The African oil bean seeds (300g) were boiled in water (100°C) over an open fire for 5hours according to the method described by Odunfa and Oyeyiola ¹⁷. The cotyledons were removed from the seed coats and washed after which it was boiled over a low flame for 12hours. It was allowed to cool, drained several times to remove bitter components in the cotyledons and soaked in water for a period of 6 hours. The cotyledons were then cut into long thin slices which were mixed with salt (4.0g), put in a clean pot, covered and fermented for 5 days at room temperature.

Castor seeds (300g) were boiled for 12 hours and allowed to cool for 10 minutes. The seeds were de-hauled by pressing between palms and then the cotyledons were boiled in water for 1hour and allowed to cool after which they were sliced to desirable sizes. The sliced cotyledons were wrapped in jute bag and allowed to ferment for 48hours ³.

This was done as described by Adeyemi and Umar ⁹. Millet seeds (300g) were soaked in distilled water for three days at room temperature, spread in a flat surface and covered with jute bag for another 3 days to enable the seeds germinate. The germinated seeds were blended with sweet potatoes (Ipomoea batatas) (50g), ginger(Zingiber officinale) (5g) and pepper (Capsicum annuum) (1g) to form a sweet paste. The paste was divided into two, one part was placed in a vessel and had boiling water poured on it and stirred to give a mixture. The other part of the paste was then added to the mixture and stirred very well. The mixture was left for 2 days at room temperature for the grain husk to settle. The husk and other sediments were filtered out of the mixture using silk sieve and the filtered liquid is bottled for consumption as kunu.

The lipid peroxidase activities of fermented ugba, ogiri and kunu were determined by the method of Maduka ¹⁸. Aliquot (1ml) of dissolved samples (in distilled water) each was added to test tube and incubated with 3ml KCI buffer (0.02m) pH 7.4 for 30minutes. After the incubation, 0.12ml of 5NHCI was added and mixed thoroughly, followed by addition of 0.35ml of 2% Na-TBA solution (thiobarbituric acid) together with 0.35ml TCA. The tubes were covered with cotton wool and boiled for 10 minutes, allowed to cool and then the color absorbance read at 532nm using UV-visible spectrophotometer.

Each sample (10g) was dissolved in 50ml of distilled water in 250ml conical flask and 4 drops of phenolphthalein indicator was added as described by Maduka ¹⁸. The mixture was titrated against 0.1M NaOH and was constantly shacked until a pink color which persisted for fifteen seconds was obtained. The absorbance was read at 715nm.

Results obtained were subjected to analysis using SPSS version 20.0. The results were presented as mean ± SD and difference in mean were compared using ANOVA at a probability threshold P=0.05.

3. Results

The results of this study are shown in Table 1. It was observed from the research that kunu has the highest peroxidase activity followed by ugba and finally ogiri (P<0.05). This suggests that fermentation causes some level of deterioration of local stored food preparations. The level of lipid peroxidation was statistically higher.

Following the result of this research, ogiri had the highest lipase activities followed by ugba and finally kunu from the free fatty acid determined (P<0.05) (Table 1). The sample with the highest malondialdehyde concentrations exhibited the least lipase activity suggesting a relationship between malondialdehyde and concentration of fatty acids.

4. Discussion

The fermented foods namely African oil bean seed commonly called ugba, fermented melon commonly called ogiri and fermented local sorghum beverage (kunu) were subjected to biochemical investigation in nutrition. Lipid peroxidation as determined by malondialdehyde was evaluated to access their extent of deterioration and the effect of fermentation as local food preparatory method. The samples contain varied concentration of malondialdehyde, a degradation product of lipid peroxidation pathway. As reviewed by the results kunu contain the highest values. This is fallout of past nutritional evaluation of most of the fermented local samples earlier reported ^{19, 20, 21}. It is also necessary to acknowledge the fact that since malondialdehyde appears to be endogenous DNA adduct in human beings which contribute significantly to cancer linked to lifestyle and dietary factors, it should be at the barest minimum concentration to ensure good health in our society.

The lipase activities as determined by free fatty acid were also investigated in the samples. All the samples contained copious amounts of free fatty acids showing reasonable lipase activities. Lipases catalyze hydrolysis of free fatty acids from triacylglycerides ¹² and can be used in industrial purpose as the source of flavor in production of foods. The catalytic activities are used in the industrial preparation of detergent, pharmaceutical products, agrochemical, cosmetics, bioremediation and biosensoring ¹³. In the research being reported, reasonable lipase activities have been implicated and interplaying in the fermentation processes of the local ugba, ogiri and kunu which are all consumed locally as nutritional supplements and locally nutritional supplements and novel sources of proteins, lipids and vitamin ²².

The samples with the highest MDA concentrations exhibited the least lipase activities suggesting a relationship between MDA and concentrations of fatty acids.

5. Conclusion

The study revealed the fact that fermented food samples can be exploited as source of lipase which can be used for industrial purposes.

Conflict of Interest

The authors have no conflict of interest with regards to this publication.

References