Antimicrobial Activity of Coconut Water, Coconut Oil and Palm Kernel Oil on selected Clinical Enterobacteriaceae Isolates

 Corresponding author: C. Jesumirhewe +2348034648066Email: Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Antimicrobial Activity of Coconut Water, Coconut Oil and Palm Kernel Oil on selected Clinical Enterobacteriaceae Isolates C. Jesumirhewe 1, *, T. O Bamgbose 1 and T. Owolabi 2 1 Department of Pharmaceutical microbiology, Prof Dora Akunyili College of Pharmacy, Igbinedion University Okada, Edo state, Nigeria. 2 Department of Pharmacognosy, Prof Dora Akunyili College of Pharmacy, Igbinedion University Okada, Edo state, Nigeria. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 080-092 Publication history: Received on 25 August 2025; revised on 01 October 2025; accepted on 03 October 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.1.0852 Abstract Antimicrobial resistance and the decreasing efficiency of antimicrobial drugs have resulted in the search for antimicrobial agents as an important strategy for the establishment of alternative therapies in handling difficult infections. This study was carried out to compare and investigate the in vitro antimicrobial activities of coconut water, coconut and palm kernel oil on selected clinical Enterobacteriacae isolates from Edo state, Nigeria. The antimicrobial activities of the samples were screened using agar well diffusion assay methods. The physicochemical properties of coconut and palm kernel oil was determined using previously described methods to characterize the oil samples. Using previously described methods, the proximate analysis and qualitative presence of phytochemical constituents were analyzed in the coconut water sample. The assay of antibacterial activity of the Enterobacteriaceae isolates showed the highest susceptibility to Coconut water. Enterobacter cloacae showed the highest zone of inhibition of 20mm when compared with the positive control. Coconut and palm kernel oil also showed inhibitory effects on the isolates. The phytochemical and proximate tests of the coconut water sample revealed the presence of reducing sugar and terpenoids and also a good proximate composition (ash, moisture, crude lipid, crude fiber and protein). The oil characterization results for coconut and palm kernel oils are acid value (4.348 mgKOH/g, 3.363 mgKOH/g), saponification value (413.036 mgKOH/g, 267.177 mgKOH/g), peroxide value (0.55 mEq/kg, 8.8 mEq/kg) and iodine value (14.657 g/100g, 14.276 g/100g). Our study showed good promising evidence for the antimicrobial effects of the samples and their usefulness in food and manufacturing industries Keywords: Antimicrobial Activity; Coconut; Palm Kernel; Phytochemicals; Proximate Analysis 1. Introduction The emergence of drug-resistant pathogens most especially to the chemically synthesized antibiotics has been of global concerns due to its hindrance to effective and successful treatment of microbial infections and diseases [1]. Antibiotic resistance have impacted negatively economically on the patient by way of prolonged hospital stay as well the search and purchase of more expensive alternatives and the possibility of death after a prolonged treatment. The rising trend of multidrug resistance as a result of the overuse and misuse of antibacterial agents is of significance thus necessitating the need of alternative sources and reduction in the over dependence on conventional antibiotics [2]. Medicinal plants also called medicinal herbs have always been pivotal to the sustenance of traditional medicine and conventional medicine, as these plants contain components that serve as a source for drug lead compounds [3]. The advent of ethno medicine with the frequent use of medicinal plants by 88% member countries of the World Health Organization provides the indices for reference as a readily available alternative with promising outcomes when World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 080-092 81 compared with the conventional antibacterial agents [4]. Medicinal plants can thus be best described as plants that are used to manage or treat specific health conditions [5] as they possess therapeutic natural compounds that exert pharmacological properties on the human and animal body [6]. Natural products offer an untold diversity of chemical structures. These natural compounds often serve as lead molecules whose activities can be enhanced by manipulation through combinations with chemicals and by synthetic chemistry [7]. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids. These metabolites have been found in vitro to have antimicrobial properties. Interest in medicinal plants has increased in recent years. This interest has led to the discovery of new biologically active molecules by the pharmaceutical industry and the adoption of crude extracts of plants for selfmedication by the public [7]. Many plants have been evaluated not only for their inherent antimicrobial activity, but also for their action as a resistance-modifying agent [7]. The enhancement of antibiotic activity or the reversal of antibiotic resistance by natural or synthetic non-conventional antibiotics has led to the classification of these compounds as modifiers of antibiotic activity [7]. Coconut water, coconut oil and palm kernel oil have been found to be pharmacologically useful [8] as well as its use in the treatment of different microorganisms due to their various phytochemical properties [9]. They also show other biological activities such as anti-inflammatory, anti-diabetic, antibacterial, antioxidant, diuretic effects amongst many others thus indicating their pharmacological significance [10, 11]. The prevalence of antibiotic resistance continues to be on the rise thus causing a major setback in the effective control and treatment of bacterial infections and diseases. The discovery of new bacterial organism and the development of mechanism of resistance to the conventional antibiotics have brought about the need for an alternative source of treatment. Ethno medicine and the use of medicinal plants through research done over the years have proven to be a suitable alternative for antibacterial therapy. This study was carried out to compare and investigate the in vitro antimicrobial activities of coconut water, coconut and palm kernel oil on selected clinical Enterobacteriacae isolates from Edo state, Nigeria. 2. Materials and Methods 2.1. Plants collection Coconut fruit, coconut oil and palm kernel oil were purchased at the Okada central market, Ovia north east local government area of Edo state. Nigeria. The purchased items were transported to the pharmaceutical-microbiology laboratory, Igbinedion University, Okada. 2.2. Extraction Process Coconut fruits were first washed with distilled water and surface sterilized using alcohol before opening at one of the eyes on the apical region using a sterile knife. The coconut water was aseptically collected into a sterile glass container 2.3. Micro-organisms used Gram positive and Gram-negative bacteria Bacillus pumilus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Salmonella spp. Klebsiella pneumoniae, Klebsiella variicola, Providencia stuartii, Enterobacter cloacae from different clinical sources (Urine, wound, sputum, catheter tip) were collected from the University of Benin Teaching Hospital, Benin City (UBTH) Edo state and used for the antimicrobial studies. Isolates were sub-cultured on nutrient agar plates to obtain pure colonies. Previously described standard microbiological techniques [12] and Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonik GmbH, Bremen, Germany) analysis was used for species identification. All the isolates were maintained on nutrient agar slants at 4℃. 2.4. Oil Characterization 2.4.1. Acid Value Determination Two grams of the sample was dissolved in 50 cm3 of mixed neutral solvent (25 cm3 diethyl ether with 25 cm3 ethanol carefully neutralized with 0.1M NaOH using 1% phenolphthalein solution). The mixture was titrated with 0.1M NaOH aqueous solution with constant shaking to faint pink colour [13]. Acid value = 𝑉𝑁𝑎𝑂𝐻× 𝑀 × 56.1 𝑊 where VNaOH = Volume of sodium hydroxide titrant used (mL) M = Molarity of NaOH World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 080-092 82 W = Weight of the fatty oil being examined (g) 2.5. Determination of Ester Value The ester value is calculated by subtracting the acid value of oil from the saponification value of the corresponding oils. 2.5.1. Determination of the Saponification Value 0.5 M KOH was prepared in 95 % ethanol, 2g of oil sample was weighed and 25 cm3 of KOH was added, 25 cm3 of the blank solution was also measured into a conical flask. The two samples were then connected to a reflux apparatus and allowed to boil for an hour until the reflux is completed, 1 cm3 of phenolphthalein was added to the mixture and the resulting mixture was titrated while hot against 0.5 M HCL acid solution. The volume of the acid used to attain the end point was recorded, the blank determination was carried out using the same procedure described above until the colour changes from blue to transparent white, then the volume of acid used was noted, the Saponification value was determined using the relationship below [13]. Saponification value = 56.1 ×𝑇 (𝑉0−𝑉1) 𝑀 Where, T= Molarity of the standard KOH solution used (M), Vo = Volume of acid used for the first titration with oil sample (cm3), V1= Volume of acid used for the second titration of the blank solution (cm3), M= Mass of the oil sample used (g). 2.6. Peroxide Value Determination Known weight (2g) of sample was weighed into clean dried boiling tube, 1 gram of potassium iodine (KI) powder was added to the oil and 20 cm3 of the solvent mixture (i.e, glacia acetic acid and chloroform in the ratio 2:1). The boiling tube was placed in boiling water bath so that the liquid mixture boils within 30 seconds and allowed to boil vigorously for not more than 30 seconds, the content after boiling was quickly poured into a flask containing 20 cm3 of 5 % potassium iodine (KI) solution and the tube was washed out twice with 25 cm3 of water. The mixture was titrated with 0.002 M sodium thiosulphate using fresh 1 % starch solution, a blank titration was carried out at the sample time, and the peroxide value was calculated using the relationship below [13]. Peroxide value= 𝑇 × 𝑀 ×1000 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒(𝑔) where T = titre value of Na2S2O3 = Sample titre – Blank titre, M = Molarity of Na2S2O3 2.6.1. Iodine value For Iodine value (Wij’s method) of each sample 2g of oil was dissolved in 15 mL carbon tetrachloride in 250 mL glass stoppered flask. 25 mL of Wij’s solution was added, the flask stoppered and allowed to stand for 2 hours in the dark at 25°C, 20 mL of 10% potassium iodide (kI) solution was added and mixture titrated with 0.2N sodium thiosulphate (Na2S2O3) using starch indicator. A blank determination was carried out and the Iodine value calculated using the formula: Iodine value = 12.69𝑁(𝑉2 −𝑉1) 𝑊 Where N = Normality of thiosulphate V1 = Volume (in mL) of thiosulphate solution 1 used in test. V2 = Volume (in mL) of thiosulphate solution 2 used in blank W = Weight of sample (2g). 2.7. Proximate Analyses of Coconut water 2.7.1. Determination of moisture content The method described by A.O.A.C [14] was adopted, a clean crucible was dried to a constant weight in air oven at 105°C, cooled in a desiccator and weighed (W1). Five grams of sample was accurately weighed into the previously labeled crucible and reweighed (W2). The crucible containing the sample was dried in an oven at 105°C to constant weight (W3). The percentage moisture content was calculated thus: World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 080-092 83 % Moisture content = 𝑊2 − 𝑊3 𝑊2−𝑊1 ×100 2.7.2. Determination of ash content The A.O.A.C [14] method was used. The porcelain crucible was dried in an oven at 105°C for 10 min, cooled in a desiccator and weighed (W1). Five grams of the sample was placed into a previously weighed porcelain crucible and reweighed (W2), it was first ignited and then transferred into a furnace which was set at 550°C. The sample was left in the furnace for eight hours to ensure proper ashing. The crucible containing the ash was then removed; cooled in a desiccator and weighed (W3). The percentage ash content was calculated as follows: % Ash Content = 𝑊3 − 𝑊1 𝑊2 − 𝑊1 ×100 2.7.3. Determination of crude lipid content by soxhlet method A clean, dried 500 cm3 flat bottom flask was weighed (W1) with 300 cm3 petroleum ether poured into the flask filled with soxhlet extraction unit. The extractor thimble with sample weighing five grams was fixed into the Soxhlet unit. The round bottom flask and a condenser were connected to the Soxhlet extractor and cold water circulation was connected. The heating mantle was switched on and the heating rate adjusted until the solvent was refluxing at a steady rate. Extraction was carried out for 4 h. The solvent was recovered and the oil dried in an oven set at 70°C for 1 h. The round bottom flask and oil was then weighed (W2). The lipid content was calculated thus: % Crude Lipid content = 𝑊2−𝑊1 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 ×100 2.7.4. Determination of crude fibre The defatted sample (5 g) was weighed into a round bottom flask, 200 cm3 1.25% sulphuric acid solution was added and the mixture boiled under reflux for 30 min. The hot solution was quickly filtered under suction. The insoluble matter was washed several times with hot water until it was acid free. It was quantitatively transferred into the flask and 200 cm3 of hot 1.25% Sodium hydroxide solution was added, the mixture boiled under reflux for 30 min and filtered under suction. The residue was washed with boiling water until it was base free, dried to constant weight in an oven at 100°C, cooled in a desiccator and weighed (C1). The weighed sample (C1) was then incinerated in a muffle furnace at 550°C for 2 h, cooled in a desiccator and reweighed (C2). The loss in weight on incineration = C1C2 % Crude fibre = 𝐶1−𝐶2 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 ×100 2.7.5. Determination of percentage protein 5 g of the sample were transferred to a Kjeldahl digestion flask and 10 g of anhydrous sodium sulphate, 0.3g of copper sulphate were added. 20 mL of conc. H2SO4 was added in an inclined position and shaken occasionally on a heating mantle for 2 h. The liquid formed was cooled and washed into a 100 mL volumentary flask and made to mark with distilled water. 50 mL of 1% boric acid solution and mixed indicator were added to the receiving flask. The distillation apparatus was collected with the delivery tube deeping below the acid solution. The diluted digest was made alkaline by the addition of 50 mL 45% NaOH solution. About 50 mL of the distillate were collected and back titrated with 0.5 N NaOH. A blank was also titrated under the same condition [14]. % by mass protein = 8.75 × (𝐵 − 𝐴) × 𝑁 𝑀 Where; B = Titre value in blank, A = Titre value in sample, N = Normality of sodium hydroxide (2N), and M = Mass of sample. 2.7.6. Determination of carbohydrate by (difference) The total carbohydrate was determined by difference. The sum of the percentage moisture, ash, crude lipid, crude protein and crude fibre was subtracted from 100. % Total carbohydrate = 100 - (% moisture + % Ash + % fat + % Protein + % Fibre). World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 080-092 84 2.7.7. Qualitative Phytochemical Screening of Coconut water Screening for the presence of secondary metabolites were performed as described by standard methods [15, 16] with slight modifications 2.7.8. Test for Saponin The filtrate (0.5ml) was placed in a test tube and 5mL of distilled water was added and shaken vigorously. Persistent fronting indicates a positive test for saponin. 2.7.9. Test for Flavonoid The filtrate (2ml) was added to 2mL dilute ammonia followed by the addition of 1mL of Conc. H2SO4 acid. A yellow coloration revealed the presence of flavonoid, which disappeared upon standing of the tube. 2.8. Test for Glycosides and Reducing Sugar 2.8.1. Test for Cardiac Glycoside To the filtrate (2ml) was added 2mL of glacial acetic acid, 1mL of 0.1% FeCl3 and 1mL of Conc. H2SO4 acid. A green-blue coloration indicated the presence of cardiac glycoside. 2.8.2. Test for Reducing Sugar Fehling solution A and B (2ml each respectively) was added to 2ml of the sample and heated for 30 minutes. A red coloration indicated the presence of reducing sugar. 2.8.3. Test for Alkaloids A volume of 3mL of 1% HCL was added to 3ml of the sample filtrate and the mixture was steamed for 30 minutes before cooling and centrifuging at 2000-3000rpm for 10 minutes: 2.8.4. Wagner’s Test for Alkaloids One ml (1ml) of wagner’s reagent was added to 1ml of supernatant of sample obtained after centrifugation. A reddishbrown precipitate indicated a positive result for alkaloids. 2.8.5. Dragendorff’s Test for Alkaloids One ml (1ml) of dragendorff’s test was added to 1ml of supernatant of sample obtained after centrifugation. An orange color precipitate indicated a positive result for alkaloids. 2.8.6. Mayer’s Test One ml (1ml) of Mayer’s reagent was added to 1ml of supernatant of sample obtained after centrifugation. A cream color precipitate indicated a positive result for alkaloids. 2.8.7. Test for Phlobotannins Two ml (2ml) of 1% HCL acid added to 2ml of the sample filtrate and heated in a water bath for 30 minutes. A red colored deposit at the base of the test tube indicated the presence of phlobatannins. 2.8.8. Test for Starch/Polysaccharide Iodine solution (6 drops) was added to 2ml of the sample filtrate. A Blue-black coloration indicated the presence of starch. 2.8.9. Test for Steroid To 0.5mL of sample was added 0.5mL of acetic acid anhydride before cooling in ice, thereafter 0.5mL chloroform and 1mL Conc. H2SO4 acid was added carefully using a pipette. A reddish brown ring at the interphase of the two liquid revealed the presence of steroid. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 080-092 85 2.8.10. Test for Tannins Two ml (2mL) of 0.1% FeCl3 solution was added to 2ml of the sample filtrate. A blueblack coloration indicated the presence of hydrolysable tannin and brownishgreen color indicated the presence of condensed tannin. 2.8.11. Test for Terpenoids Six drops (6 drops) of Brady’s reagent was added to 2ml of the sample filtrate. A yellowishorange coloration indicated the presence of terpenoids. 2.9. Preparation of Extracts 2.9.1. For susceptibility testing 5ml of various oil extracts was measured and placed in respective universal bottles. 5ml of dimethylsulphoxide (DMSO) was added to the universal bottles to dissolve the extracts giving a concentration of 100% stock concentration. A twofold serial dilution of the stock concentration was carried out to obtain the following concentrations: 50% and 25%. 2.9.2. Preparation of negative control Negative control was prepared by adding 2ml of dimethylsulphoxide (DMSO) to 2ml of sterile distilled water in a universal bottle for the oil samples while sterile distilled water was used as the negative control of Coconut water sample 2.9.3. Preparation of positive control 1ml of gentamicin (80mg/2ml) was put into 99ml of sterile distilled water to make 400μg/ml concentration. Then 1ml for 400μg/ml was collected and put into 39ml of sterile distilled water to make a final concentration of 10μg/ml. 2.9.4. Antimicrobial Assay. The antimicrobial activities of the oil samples and resulting crude extracts were screened using agar well diffusion assay methods with slight modifications [17]. A volume of 0.1ml of 10-2 dilution of each isolate was used to inoculate the preprepared Muller Hinton Agar plates and surface plated using a sterile swab sticks. The sterile cork borer {8 mm diameter} was used to bore equidistant holes on the plates. Using a Pasteur pipette, 0.1ml of different concentration of the extracts (100%. 50% and 25%) was used for the assay. A volume of 0.1ml of the positive control and negative control were also used. The experiment was carried out in duplicates and examined for zones of inhibition after incubation for 24 hours 3. Results 3.1. Plant Authentication. The fruit was authenticated as the Cocos nucifera linn fruit. This was done at the Dora Akiyuli College of Pharmacy herbarium at the Igbinedion University Okada, Edo state 3.2. Phytochemical screening Table 1.0 summarizes the qualitative phytochemical analyses of Coconut water sample. The result of the screening showed that the extract under investigation contained reducing sugar and terpenoids. Table 1 Phytochemical analysis of Coconut water Phytochemical constituents Coconut water Alkaloids - Phlobatannin - Reducing sugar ++ Cardiac glycoside - Flavonoid - World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 080-092 86 Starch - Tannin - Terpenoids ++ Saponin - Steroid - Keys: ++ = moderately present; - = absent 3.3. Proximate analysis result The following values included in table 2.0 are a summary of the proximate analysis value of the Coconut water. The analysis showed the presence of ash, moisture, crude lipid, crude fiber and protein. Table 2 Proximate analysis of Coconut water Component % composition of the Sample Crude protein 8.4 Ash 5.619 Crude Fiber 0.052 Crude lipids 12 Moisture 29.961 Carbohydrate 43.968 3.4. Oil Characterization result Table 3.0 gives a summary of the oil characterization of palm kernel oil and coconut oil respectively. Table 3 Oil Characterization of Coconut and Palm Kernel oil Test Value for Coconut oil Value for Palm kernel oil Acid value 4.348mg KOH/g 3.363mg KOH/g Ester value 408.688 263.814 Saponification value 413.036mgKOH/g 267.177mgKOH/g Iodine value 14.657gI2/100g 14.276gI2/100g Peroxide value 0.55mEq/kg 8.8mEq/kg 3.5. Antimicrobial Susceptibility Test Results on the antimicrobial activity of Coconut water, coconut oil and palm kernel oil on the clinical isolates are presented in the Figure 1.0 to Figure 3.0. The extracts showed varying activity on the isolates, Gentamicin which was used as the positive control also had activity on the isolates while dimethylsulphoxide (DMSO) had no activity on the isolates. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 080-092 87 Figure 1 Antimicrobial activity of palm kernel oil on isolates Figure 2 Antimicrobial activity of coconut oil on isolates World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 080-092 88 KEY: ZOI-Zone of inhibition Figure 3 Antimicrobial activity of coconut water to isolates 4. Discussion Global drug resistant infections have been on the rise due to antibacterial resistance without effective interventions, which alarmingly have led to pandemic scale like projections [18]. This has led to extensive chemical and biological investigation of ethno medicinal plants with high indices of being curative and clinically potent [19]. This research work was undertaken to carry out an in-depth investigation on the reported usefulness coconut water, coconut oil and palm kernel oil. The phytochemical screening of coconut water showed the presence of glycoside reducing sugars and terpenoids as constituents with potential antimicrobial activity, while the proximate analysis of the coconut water sample indicated a favorable nutritional composition. Sumonsiri [20] reported the chemical composition of coconut water to consist of water (96.11%), sugars (2.7%), proteins (0.25%), lipids (0.51%) and ash (0.43%). In another recent report, coconut water was characterized by a high moisture content of 95.95 ± 0.943%, minimal ash (0.47 ± 0.753%), fiber (0.08 ± 0.292%), and protein (0.51± 0.854%) levels, alongside a moderate carbohydrate percentage of 2.91 ± 0.744%. Additionally, negligible amounts of non-fiber extract (NFE) and crude fat were observed, denoting its low-fat content and richness in essential hydration elements [21]. The slight differences in some of the values obtained in this study could be possibly due to the source of the sample. For example a previous study found the moisture content of 94.96%, carbohydrate at 4.38% and fiber at 0.0355% for street marketed coconut water compared to another that showed moisture content 81.09%, protein 3.96%, fat 12.27% and carbohydrate 5.58% for another sample [22]. The phytochemical constituents of Coconut water, reducing sugar and terpenoids are known to be of great health significance due to their different pharmacological activities. For example terpenoids are known to possess antitumor, anti-malarial, antiviral, anti-oxidative and antibacterial effects [23]. Reducing sugars are known to influence pharmacological activities in relations to other compounds and on its own possess biological activities such as anticancer, anti-inflammatory, antiviral and anti-microbial activities [24]. The oil characterization results for coconut and palm kernel oils are acid value (4.348 mgKOH/g, 3.363 mgKOH/g), saponification value (413.036 mgKOH/g, 267.177 mgKOH/g), peroxide value (0.55 meq/kg, 8.8 meq/kg) and iodine value (14.657 g/100g, 14.276 g/100g). The coconut and palm kernel oil showed slightly higher iodine value compared to previous reports [25, 26]. The iodine value from this study means both the coconut and palm kernel oils has high percentage of unsaturated fatty acid bonds. The determination of the iodine value of oil classifies oil into drying, semi-drying and non-drying oil. The ability of the oil to undergo across link reaction which is oxygen-induced leads to a formation of a solid film known as drying [27]. The higher the iodine value, the more unsaturated fatty acid bonds are present in a fat, hence its high susceptibility to oxidative degradation [28]. Hence, addition of antioxidants may be necessary to prolong the storage ability of the oils. According to Maliki et al. [29], oil with iodine value above 100g/100g is a drying oil and below