Fair value measurement and profitability of listed manufacturing firms in Nigeria

 Corresponding author: Moussa Konate 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. Isolation of acetic acid bacteria with biotechnological uses from wild fruit Diospyros mespiliformis picked in the north Côte d’Ivoire Souleymane Soumahoro 1, Moussa Konate 2, *, Maimouna Liliane Kouame 1, Attawa Campbelle Dongo 1, Abdoulaye Toure 3 and Yadé René Soro 2 1 Laboratory of Biochemistry, Microbiology and Valorization of Agroresources, Agropastoral Management Institute, Peleforo Gon Coulibaly University, Korhogo, Côte d'Ivoire, Korhogo BP 1328. 2 Laboratory of Biotechnology, Agriculture and Valorization of Biological Resources (LBAVBR), Faculty of Biosciences, Felix Houphouët-Boigny University, Abidjan, Côte d'Ivoire, 22 BP 582 Abidjan 22. 3 Laboratory of Biotechnology and Valorization of Agroresources and Natural Substances, Faculty of Biological Sciences, Peleforo Gon Coulibaly University, Korhogo, Côte d'Ivoire, Korhogo BP 1328. World Journal of Advanced Research and Reviews, 2025, 27(01), 2672-2685 Publication history: Received on 22 June 2025; revised on 27 July 2025; accepted on 30 July 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.27.1.2820 Abstract Despite its nutritional value, Diospyros mespiliformis is an underexploited fruit in Ivory Coast. This study was conducted with the aim of enhancing this fruit. Thus, according to the analysis, the precise quantities of fruits were collected in Korhogo (northern Côte d'Ivoire) and the physico-chemical properties determined. Endogenous acetic acid bacteria from the fruit were then isolated on selective medium and identified using phenotypic and biochemical methods. Their acidification capacity and resistance to different stresses were tested using reference methods. The strains with the best technological potential were selected by the ascending classification technique. The results showed that the fruit has an acidic pH (2,85 ± 0,01) with a soluble dry extract of 5°Brix, high water content (74,97 ± 0,86 %) and vitamin C (5,06 ± 2,16 mg/100 g). Out of a set of thirty-two (32) isolates of acetic bacteria, nine (09) or (28.1%) showed a strong ability to produce acetic acid in solid media. However, among these nine (09) isolates, four (04) named BAD11, BAD14, BAD23 and BAD24 presented the best resistance to different stresses. Thus, these four (04) isolates identified as Acetobacter sp. with highest acetic acid productions and best other physiological properties could be used in biotechnological processes, particularly in the vinegar industry. Keywords: Isolation; Influence; Acetic acid; Acetic acid bacteria; Diospyros mespiliformis; Korhogo 1. Introduction Fruits and vegetables are an essential part of a healthy diet. Indeed, a diet rich in fruits and vegetables is likely to reduce the risk of cardiovascular disease and protect against certain types of cancer [10]. Thus, the consumption of 400 to 600 g of fruits and vegetables per day is recommended by the World Health Organization and the Global Research Fund to combat cancer, [1, 27]. Côte d'Ivoire, recognized as one of the leading fruit producers in West Africa, plays a crucial role in food supply and regional economy [11]. The country’s climatic and geographical diversity promotes the cultivation of a variety of tropical fruits, which are not only intended for local consumption but also for export to international markets. Among the most popular fruits, we find mango, pineapple and banana, which contribute significantly to farmers' incomes and the country’s trade balance. World Journal of Advanced Research and Reviews, 2025, 27(01), 2672-2685 2673 Indeed, mango is the third fruit exported by the country behind banana and pineapple [35]. In addition, a large proportion of the fruit grown is still destined for the local market, where it is consumed fresh or processed. This local consumption dynamic is essential to support rural economies and ensure food security [22]. In addition, Côte d'Ivoire is also home to a wealth of easily accessible wild fruits [25] and only for local consumption without resulting gainful activity. Indeed, these wild fruits are consumed directly at the place of picking. Also, these fruit species are traded because of their uses in the agro-food and pharmaceutical industries around the world [17]. Among these fruits, Diospyros mespiliformis, of the family of the Ebenaceae still known as African Ebenier [6]. It is a species of tree that produces fruits whose pulp is very sweet and very appreciated not only by animals that ensure its dispersion in the natural environment, but also by rural populations [3]. Indeed, its edible fruits are used as food supplements during lean periods [7] and are also used to produce juice and alcoholic beverages [9]. Diospyros mespiliformis fruits are rich in sugar. Due to the high carbohydrate content in the pulp, which gives it a sweet taste, they could be an appropriate substrate for research into a fermentative flora of interest for the food industry. Fermentation is the transformation of organic matter by ferments, which leads to changes in the organoleptic properties of a food [5]. Acetic bacteria classified in the Acetobacteria group of the Alpha-Proteobacteria class [14, 23] are microorganisms known for their ability to produce acetic acid, an essential process in vinegar production. Acetic bacteria are distinguished by their ability to oxidize sugars, alcohols into organic acetic acids, ketones and aldehydes through a process known as oxidative fermentation [21]. They are widespread in nature and can be isolated from various sources such as fruits, flowers, sugars and fermented beverages [16]. However, they are considered tedious due to their difficult growth on conventional growing media. Their culture is often weak and highly irregular [33]. However, the isolation of acetic bacteria capable of producing large amounts of acetic acid with better resistance to alcohol has always been of interest. The general objective is to contribute to enhance Diospyros mespiliformis fruits technological value. 2. Material and methods The biological material used in this study consists of Diospyros mespiliformis fruits (Figure 1), still known as African Ebony, harvested at the site of the Peleforo Gon COULIBALY University. Then it is sent to the laboratory of the university for physico-chemical and microbiological analyses. Figure 1 Diospyros mespiliformis fruits 2.1. Determination of physico-chemical parameters of the fruit 2.1.1. Water and dry matter content The AOAC method [2] was used to determine water and dry matter. It consists in dehydrating the samples by drying them in an oven until a constant mass is obtained. 5 grams of pulp is weighed into a glass capsule of known mass (m0). The capsule with the sample (total mass m1) is placed in an oven at 105 °C for 24 hours, then cooled in a desiccator. The moisture content (Mc) expressed as a percentage of wet sample mass is determined by the following relationship: World Journal of Advanced Research and Reviews, 2025, 27(01), 2672-2685 2674 Mc (%) = ((m1-m2) ×100) /(m1-m0) m0: crucible mass with lid (g) m1: mass of the crucible with its lid and containing the sample before drying (g) m2: mass of the crucible with its lid and containing the sample after drying (g). The dry matter content (Dm), expressed as a percentage of the wet sample mass, is calculated as: Dm (%) = 100 - Mc Dm: sample dry matter content (%); Mc: sample moisture or water content (%) 2.1.2. pH The pH was determined by the AOAC [2] method, using a pH meter with an electrode sensitive to hydrogen ions (H+). Ten (10) grams of pulp were homogenized in 100 mL of distilled water, and the mixture was filtered through filter paper (WHATMAN). The pH meter glass electrode (HANNA) was immersed in the filtrate. The pH value was determined on the screen of the pH meter, previously calibrated. 2.1.3. Titratable acidity The titratable acidity was determined by AOAC [2] method that involves measuring the acidity of a product by titrating it with sodium hydroxide solution (NaOH) in the presence of phenolphthalein as a color indicator. Ten (10) grams (me) of the fruit pulp were crushed and homogenized in 100 mL of distilled water. After filtration on Whatman paper, 10 mL (V0) of the filtrate was transferred in an Erlenmeyer to which 3 drops of phenolphthalein were added. A solution of NaOH (0.1N) contained in a burette was then added drop by drop to the mixture until a persistent pink color was obtained. The volume of NaOH (V1) added up to the turning point has been noted on the burette scale. This volume allowed the acid concentration in the initial sample to be calculated, expressed as a percentage of the corresponding acid. The titratable acidity (Tac) was expressed in milliequivalent (meq) per 100 g of fresh matter by the following relation: Tac = (N×V1×104) / (mp×V0) V0: volume (mL) of the test portion; V1: Volume of NaOH added; mp: mass (g) of fresh pulp sample; N= 0.1 (normality of sodium hydroxide solution) 2.1.4. Soluble dry extract The soluble dry extract expressed in degree brix was measured using a digital refractometer as recommended by the device manufacturer. Ten (10) grams of fruit pulp were put into the extractor to obtain the juice. A drop of this juice was placed on the plate of the prism of the refractometer. The value was obtained directly. 2.1.5. Ethanol content The percentage ethanol content was measured with a digital refractometer as recommended by the device manufacturer. Ten (10) grams of fruit pulp were crushed in the extractor. The regrind was used to obtain the pulp juice. A drop of this juice was placed on the plate of the prism of the refractometer. The value is obtained directly. 2.1.6. Vitamine C content The method used to determine vitamin C content was that described by Pelletier [26]. The principle of this method is to stabilize vitamin C by metaphosphoric acid/acetic acid 2, then oxidize with reduced 2,6-dichlorophenol indolphenol. A mass of 10 g of fruit pulp was solubilized in 40 mL of metaphosphoric acid/acetic acid (2%; w/v). The mixture was centrifuged at 3000 tr/min for 20 min. The supernatant is recovered in a 50 mL flask and filled to the gauge with distilled water boiled and cooled off from air. A volume of 10 mL of the contents of the flask was taken and placed into an erlenmeyer (test portion). The test portion is titrated with a 0.5 g/L solution of 2,6-DCPIP (2,6-dichlorophenol indophenol) until it turns persistent pink for 30 s. The 2,6-DCPIP solution is pre-calibrated with a 0.5 g/L vitamin C solution. Either V (mL), the volume of 2.6 DCPIP paid to equivalency. The vitamin C content as a percentage of fresh sample mass is determined by the following relationship: World Journal of Advanced Research and Reviews, 2025, 27(01), 2672-2685 2675 Vitamine C content = [2(VC-VO) / (VE-VO)] *100 VO: volume (mL) of 2,6-DCPIP solution poured into the blank; VE: volume (mL) of solution used for 2.6 DCPIP solution calibration; VC: volume (mL) of 2,6-DCPIP solution used for the test portion. 2.2. Isolation and group distinction test of acetic bacteria 2.2.1. Isolation 25 g of the pulp of Diospyros mespiliformis are taken aseptically into a stomacher bag to which 225 ml of buffered peptonic water are added. The whole was thoroughly mixed by manual stirring giving the stock solution. The purpose of this stirring is to dissolve germs. In test tubes containing 9 mL of tryptone salt (TS), decimal dilutions of the stock solution were performed from 10-1 to 10-5 under aseptic conditions. To do this, 1 mL of the stock solution was taken and placed in a tube containing 9 mL of TS, resulting in 10-1 dilution. Subsequently, 1 mL of this dilution was taken and placed in another tube containing 9 mL of TS, corresponding to 10-2 dilution. The dilutions are thus carried out in the same way, one after another. 100μL of each dilution range were inoculated by spreading on the agar surface. The seeded media were incubated in a 30 °C oven for 72 hours under aerobic conditions. Biochemical tests for the identification of acetic bacteria are Gram stain, catalase test, oxidase test and respiratory type. 2.2.2. Group distinction test for acetic bacteria The group distinction test was performed according to the method described by Soumahoro et al. [32]. The purpose of this test is to subdivide isolates into two groups: those that oxidize the acetic acid into CO2 + H2O and those that are unable to do it. The principle of this group distinction test is based on the ability of strains to deacidify the medium containing acetic acid. Thus, the HS broth was prepared with bromocresol green and placed in a flask. After sterilization at 121°C and cooling to 45°C in a water bath, an amount of acetic acid (1%) as the carbon source was added to the broth. The addition of acetic acid causes the colored pH indicator to turn from green to yellow. The HS broth is distributed in 5 mL hemolysis tubes at a rate of 3 mL per tube. Seeding a colony of the strain to be tested is done in broth with a sterile Pasteur pipette. The seeded tubes are then incubated for 5 days in aerobic conditions at 30 °C. The ability of the strain to use acetic acid results in a deacidification of the broth, causing the medium to go from yellow to green. 2.3. Study of the potential technological properties of acetic bacteria isolates 2.3.1. Analysis of the acidification ability The ability of isolates to acidify the medium was demonstrated using the method proposed by Aydin and Aksoy [4]. The purpose of this method is to evaluate the acidification power of isolated strains. Thus, colonies were inoculated by spot on pre-poured agar in petri dishes. The seeded media were incubated for 5 days at 30 °C in aerobic conditions. During the growth of acetic bacteria, acidification of the medium results in the presence of a clear halo around the colony. Two trials for each isolated strain were carried out on the same container. 2.3.2. Ethanol resistance To evaluate the effect of ethanol resistance, 100 μL of a suspension of isolates (OD600= 0.5, optical density at 600 nm) previously prepared in Tryptone Salt (TS) were inoculated into 10 mL of HS broth, then the culture broths were incubated at 30 °C for 48 hours. Bacterial growth was determined by reading the turbidity in the culture broth with a spectrophotometer at 600 nm. 2.3.3. Influence of temperature on isolates growth 100 μL of a bacteria suspension of DO600 = 0.5 previously prepared in Tryptone Salt (TS) were inoculated into each tube. Then the tubes were incubated for 48 hours in an aerobic environment at different temperatures (30 °C, 35 °C, 40 °C and 45 °C). Bacterial growth was determined by reading turbidity in the culture broth using a spectrophotometer at 600 nm. 2.3.4. Influence of pH on isolates growth This method is based on the ability of isolates to resist pH variations. Evaluation of pH resistance allows the selection of isolates capable of resisting the influence of pH. Eight hundred (800) mL of the HS broth was prepared and divided World Journal of Advanced Research and Reviews, 2025, 27(01), 2672-2685 2676 into 8 vials due to 100 mL per vial. Sterilization to 121°C and cooling to 45°C were performed and ethanol was added at a final concentration of 4% in each 100 mL vial. This influence was achieved but the HS broth was adjusted to different pH: 2, 3, 4, 5, 7, 8, 10 and 12. 2.3.5. Influence of glucose on isolates growth The principle of this method is based on the ability of isolates to resist osmotic stress. The osmotic stress resistance evaluation allows to select isolates capable of growing under the influence of a glucose concentration. Six hundred (600) mL of the HS broth was prepared and divided into 5 vials at 100 mL per vial. Sterilization to 121°C and cooling to 45°C is performed. Each vial containing the medium is poured into test tubes at a rate of 10 mL per tube. This influence was performed as described above but the HS broth was adjusted to the following different glucose concentrations: 0%, 4%, 6%, 8%, 10% and 12%. 2.3.6. Acetic acid production, growth kinetics and pH evolution of isolates in liquid medium The acidity produced by selected strains was quantified. This assay was done by titration with sodium hydroxide solution, 0.1 N (NaOH 0.1 N) and phenolphthalein as a color indicator (Nanda et al. [24]). For this, the HS medium was used. A 24h bacterial culture of all selected isolate was previously carried out. Then, for each of them, 10 mL of culture medium was introduced into a test tube and inoculated with 100 μL of the different suspensions. Incubation was carried out under agitation at 160 tr/min. Every day, acetic acid production was quantified until constant values are observed while growth was determined by measuring the optical density (OD) at 600nm and pH evolution using pH meter. To determine the amount of acid produced, 3 drops of phenolphthalein were added to a beaker containing 2ml of culture medium. A volumetric dosage with NaOH 0.1 N contained in a burette, made it possible to quantify the production of acetic acid. Acetic acid production (AAP) per liter is given by the following relation: AAP = (N×Vb×1000×M) /Va Va: Test volume (mL); N = 0.1 : NAOH Normality ; Vb: NAOH Volume (mL); M: Acetic acid molar mass 2.4. Data processing The analysis of variances (ANOVA) followed by the Tukey test with a significance level of 5% was performed using the XLSTAT software version 2016. This software allowed to calculate the means, the distances of the microbiological parameters. 3. Results 3.1. Physico-chemical parameters The physico-chemical parameters studied are pH, moisture content, dry matter, alcohol content, vitamin C content and soluble dry extract. The results are recorded in table 1. Table 1 Physico-chemical parameters of Diospyros mespiliformis fruits Parameters Values Ph 2,85 ± 0,01 Moisture content (%) 74,97 ± 0,86 % Titratable acidity (meq/100 g) 3,38 ± 0 Dry matter (%) 25,03 ± 0,86 % Alcohol content (%) 2,5 ± 0 Vitamin C content (mg/100 g) 5,06 ± 2,16 Soluble dry extract (°B) 5 ± 0 World Journal of Advanced Research and Reviews, 2025, 27(01), 2672-2685 2677 3.2. Acetic acid bacteria isolated and identified 3.2.1. Isolated bacteria Macroscopic and microscopic observations allowed the selection of acetic bacteria. Acetic bacteria are small, flat colonies with smooth contours of beige color. They are also short bacilli. The biochemical tests carried out showed short Gram-negative bacilli, strict aerobic, positive catalase and negative oxidase (Figure 2). A total of thirty-two (32) strains were isolated and named with codes ranging from BAD1 to BAD32. Figure 2 Microscopic observation of acetic acid bacteria isolated 3.2.2. Acetic acid bacteria groups identified The group distinction test was to identify, among the 32 acetic bacteria isolates, those capable of using acetic acid as a carbon source. Thus, the culture media were prepared with this acetic acid as the sole carbon source. Results showed a yellow to green turn or no turn of strains (Figure 3). Of the 32 isolates tested, 23 (71.87%) showed a yellow to green turn indicating that these isolates have the ability to degrade acetic acid. However, 9 isolates (28.13%) could not degrade acetic acid. Isolates capable of degrading acetic acid belong to the Acetobacter group and those that do not have this ability belong to the Gluconobacter group. Figure 3 Tubes with or without a turn of the color indicator 3.3. Potential technological properties of isolated acetic bacteria 3.3.1. Acidification ability in solid medium of isolated acetic bacteria A total of thirty-two (32) isolates were screened. Results show that 24 isolates (75%) were able to acidify the medium and that 8 isolates (25%) were not able to acidify the medium. The isolates with the ability to acidify the medium show transparent halos around spots and those without the ability to acidify the medium did not do it (Figure 4). Depending on the diameter of measured halos, acidifying isolates can be classified into different groups: World Journal of Advanced Research and Reviews, 2025, 27(01), 2672-2685 2678 • High acidifiers with diameters ranging from 20 to 29 mm (39.14%); • Medium acidifiers with diameters ranging from 11 to 19 mm (30.43%); • Low acidifiers with a diameter ranging from 3 to 9 mm (30.43 %). Figure 4 Transparent halos of acidifying isolates 3.3.2. Ethanol resistance of isolates The ethanol resistance study assessed the ability of 9 isolates with best acidification to tolerate high alcohol concentrations (Figure 5). Thus, the culture media were prepared with varying proportions of ethanol (4%, 8%, 10%, 12%). In general, ethanol variation influenced the growth of individual isolates. All isolates had their best growth when the medium contained 4% ethanol. Above 4%, a decrease in growth is observed for all isolates studied. At 4% ethanol, isolate BAD5 had the best growth with OD600 = 0.987 while isolate BAD23 had the lowest growth with a OD600 = 0.658. At 12%, most isolates decrease until they cancel. Figure 5 Ethanol influence on isolates growth 3.3.3. Temperature influence on isolates growth Figure 6 shows the influence of temperature on bacteria growth. The selected bacteria had growth that varied at different temperatures. In general, the growth of all isolates studied decreases as temperature increases. It should be noted that all isolates had their best growth at 30°C. At this temperature, isolate BAD24 had the best growth (OD600 = World Journal of Advanced Research and Reviews, 2025, 27(01), 2672-2685 2679 0.983) and isolate BAD20 had the lowest growth (OD600 = 0.565). At 45°C, growth ranged from 0.055 to 0.524 and isolate BAD13 was the most resistant with OD600 = 0.524 and isolate BAD11 was the least resistant (OD600 = 0.055). Figure 6 Temperature influence on isolates growth 3.3.4. pH influence on isolates growth Growth of selected bacteria varies with the pH concentration in the medium (Figure 7). It should be noted that for the nine (9) isolates studied, growth increases to a peak above which it decreases. Thus, growth peaks varied from one isolate to another. Most isolates, eight in total had their growth peak at pH3 while isolate BAD20 had its growth peak at pH4. For growth values of all isolates tends to cancel. Figure 7 pH influence on isolates growth 3.3.5. Glucose influence on isolates growth The selected isolates showed good growth at different glucose concentrations (Figure 8). The growth of all isolates studied follows a Gaussian curve with a growth peak when the medium contains 4% glucose. Above 4% glucose in the medium, growth stabilizes slightly for eight (8) isolates (BAD5, BAD11, BAD13, BAD14, BAD19, BAD20, BAD23, BAD24) before dropping. Isolate BAD22, which had the best growth at 4% glucose (OD600 = 0.958), has a lower growth rate above this glucose concentration but still remains higher than the other eight (8) isolates. At a concentration of 8% glucose, the growth of isolate BAD22 is reversed while that of isolate BAD13 is reversed with 10% glucose in the medium. Growth of isolates BAD20 and BAD23 was nil at 12% glucose. Only isolate BAD14 had growth at 12% glucose (OD600 = 0.191). World Journal of Advanced Research and Reviews, 2025, 27(01), 2672-2685 2680 Figure 8 Glucose influence on isolates growth 3.3.6. Acetic acid influence on isolates growth Figure 9 shows the influence of acetic acid on bacteria growth. All isolates studied had their best growth when the medium did not contain acetic acid. However, given the pace of the different curves, acetic acid has little effect on growth in the isolates studied (OD600 greater than 0.5 for all isolates). With an acetic acid concentration of 4% in the medium, isolate BAD22 (OD600 = 0.547) had the best growth while the other isolates have similar growth. Figure 9 Acetic acid influence on isolates growth 3.3.7. Acetic acid production kinetics of selected isolates The curves obtained show a similar pace. From D1 to D2, acetic acid production increases regularly before reaching its peak at D2. From D2 to D3, production is constant. From D3 to D4, production drops. Beyond D4, acetic acid production is stable towards the end. BAD24 is the best producer (Figure 10).