Physicochemical characterization and microbiology quality of the Pentadesma butyracea fruit pulp collected from various parks in Benin

In Benin, P. butyracea fruit pulp undergoes significant post-harvest loss due to its low valorization. The collected fruits in five parklands from a survey of transformer's perception were characterized through their visual observations, the determination of their dimensions and pulp proportions. The nutritional value of pulp was evaluated through its chemical characterization. The results of this study revealed that four shapes (ellipsoid, globular, ovoid, ellipsoid twisted) identify the physical aspect of P. butyracea fruits in the P. butyracea parklands. The average length, diameter at the equator, mass and pulp proportion of fruits were 130.93 mm, 86.98 mm, 125.63 g and 35.79%, respectively. The physicochemical characterization showed that the P. butyracea fruit pulp contained 3.37–3.41 pH, 3.33–4.61% protein, 20.37–20.78% fiber, 2.78–3.57% ash, 6.31–6.51% crude lipid, 85.77–86.47% moisture, 21.54–23.22 mg/mL total phenolics, 0.73–1.03% titratable acidity, 65.10–66.94% carbohydrates, 335.75–338.99 kcal calorie and minerals (Ca, Fe, Zn, Mn). The antioxidant activity result showed that the P. butyracea fruit pulp is a valuable source of antioxidant pigments. Faecal coliforms are not detected in pulps and the aerobic mesophilic bacteria, yeasts and moulds, and Staphylococcus aureus were below detection level in P. butyracea fruit pulps.


Scientific Reports
| (2021) 11:17040 | https://doi.org/10.1038/s41598-021-96211-z www.nature.com/scientificreports/ as the glucides, the proteins, the fibres, minerals and the vitamins. It is then necessary to develop technologies of valorization of P. butyracea fruit pulp in Benin. For this reason, it is important to study the nutritional quality of the fruits pulp in Benin. The present study evaluated the nutritional value and microbiology quality of the P. butyracea fruit pulp collected from various parks in Benin and determined the various uses of fruit pulp using a quantitative survey approach and the Physical characterization of the fruit.

Materials and methods
Survey and data collection. The study was undertaken in five municipalities of nord Benin, namely Bassila, Tchaourou, Toucountouna, Natitingou and Kandi. An investigation was conducted between May and June 2020 in villages having a tradition of P. butyracea fruits processing. All participans were more than 18 years, selected from the study sites. The study interest and procedure were explained to the community by village leaders. Therefore, all participans approved the activity because they understood that it will allow them to valorize the P. butyracea fruit pulp. Each participant provided informed consent prior to responding to the survey. Individuals who did not provide informed consent could not proceed with the survey. Also, informed consent was obtained from a legal guardian on behalf of illiterate participants. This survey was approved by the Institution Ethics Board and the School of Nutrition and Food Sciences and Technologies (SNFST) of Abomey-Calavi University (UAC), approval N° F-025-2020-SNFST. To determine adequate number of respondents for our survey among the P. butyracea actors per parkland, the following formula was used: Ni = 4Pi(1 − Pi)/d2 where Ni is the total number of P. butyracea actors to be surveyed in parkland i, Pi is the proportion of P. butyracea actors found in previous studies in parkland i, and d is the expected error margin in the conclusion, which was fixed at 0.1 10 . Based on this method, a total of 260 subjects distributed in twelve villages of aforementionned municipalities was obtained. Data were collected by administering questionnaires to randomly selected and willing respondents. The questionnaires included the following aspects: P. butyracea actor's perception on fruit pulp, various uses, collection state and various methods of collection of the fruits of P. butyrace. In addition, the various fruit shapes available in each commune was determined. This research was performed in accordance with relevant guideline for humans and regulations.
Subjects. The socio-demographic characteristics of the participants in the quantitative survey were as fol- Physical charecterization of the P. butyracea fruits. The fruits evaluation has been done immediately after the sample collection. The characteristics analysed were: diameter, length, fruit mass and fruit pulp mass. Diameter and length (mm) of the fruits were measured with a precision caliper (0.01 mm) and mass of the fruits (fm) and pulps mass (pm) were determined using balance with precison is 1/10.000. Measurements were performed on 50 fruits chosen at random per sample and by shape (ellipsoid, globular, ovoid and ellipsoid twisted) from each park. The generated values allow calculating the proportion of pulp (P) with P(%) = pm/fm × 100.
Evaluation of physicochemical composition of P. butyracea fruit pulp. The proximate analysis of fruit pulp samples were analyzed using standard methods 11 . The moisture content was determined by drying about five grams of the fresh sample in an oven at 105 °C to constant weight. Crude ash was determined by incineration of 5 g of sample in a muffle furnace at 550 °C for 8 h. The nitrogen (N) content was estimated by micro-Kjeldahl method and crude protein content calculated as N% × 6.25. Crude lipid was exhaustively Soxhlet extracted from 5 g sample with nhexane for 4 h. Crude fibre content was determined by treating 2 g sample with 1.25% (W/V) H 2 SO 4 and 1.25% (W/V) NaOH. Minerals (calcium, magnesium, iron and zinc) of the samples were then analyzed with Atomic Absorption Spectrophotometer model Analysit-400. The pH was determined using a digital pHmeter (HI8418; Hanna instruments, Limena, Italy) calibrated with buffers at pH 4.0 and 7.0 (WTW, Weilheim, Germany). The titratable acidity, expressed as lactic acid, was carried out using the method described by Ref. 12 . The Brix values were carried out using a refractometer. The available carbohydrate (CHO) was calculated by difference. Calorific value (CV) was determined using the following equation: Preparation of extracts for the determination of total phenolics and antioxidant activity. Samples were extracted in methanol/HCl (99:1 (v/v) following the method described by Ref. 14 . Fifty milligrammes (50 mg) of each sample were extracted at room temperature with 1.5 mL of solvent under agitation using a magnetic stirrer for 30 min. The mixtures were centrifuged at 2500g for 10 min and the supernatants were collected. The residues were re-extracted twice under the same conditions, resulting in 3 mL crude extract. All extracts were used as they were after centrifugation for various analyses.
Total phenolics determination. Total phenolics were measured following the method of Ref. 15 modified by Ref. 14 as follows: to 300 μL of extract, 4.2 mL of distilled water, 0.75 mL of Folin-Ciocalteu's reagent (Merck, Germany) and 0.75 mL of sodium carbonate solution (200 g/L) were added. After incubation for 30 min, the optical density was measured at 760 nm against a blank. Gallic acid was used as standard and the results were expressed as gallic acid equivalent (GAE) per g of sample DM.
Antioxidant activity determination. The antioxidant activity was measured using DPPH (2,2-diphenyl-1-picrylhydrazyl). The DPPH method was conducted by adaptation as described by Scherer and Godoy 16 . Equal volumes (100 μL) of DPPH (50 μM) and plant extracts (12.5 mg/mL) were mixed and incubated for 20-30 min in the dark at room temperature. Then, the absorbance was read at 517 nm and the blank was a mixture of methanol and DPPH (v:v). This activity was given as % DPPH scavenging and calculated using Eq. (1): where A control is absorption of DPPH solution, and A sample is absorbance of the test sample. The half maximal effective concentration (EC 50 ) is the amount of sample necessary to decrease the absorbance of DPPH by 50%. It was determined graphically using a calibration curve in the linear range by plotting the extract concentration and the corresponding scavenging effect. Ascorbic and gallic acids were used as positive controls. All the analyses were carried out in triplicate.

Statistical analysis.
All data were expressed as mean ± standard deviation (n = 3 replicates). Data were analyzed using one-way ANOVA using SPSS 16.0. Duncan's multiple-range test was used to determine the difference between means. A significant difference was considered at the level of p ≤ 0.05.

Collection of P. butyracea fruits.
Fruits were collected in five muicipalities of nord Benin under permission from Department of the Environment of National Forestry Commission, approval N°2020-FOR-03-093-PENESSOULOU. Sometimes, there are the foresters who lead us in the parks to collect the P. butyracea fruits.

Experimental research and field studies on plants. All experimental protocol and methods
was approved by the Faculty of Agronomics Sciences (FSA) of Abomey-Calavi University (UAC), approval N°202040016. The fruits have been collected between May and June 2020 in five muicipalities of nord Benin, and transported to food laboratory of School of Nutrition and Food Sciences and Technologies of FSA for analyze. Moreover, an investigation was conducted between May and June 2020 in the villages retained as focus group. It was carried out through a guide of interview pre-established. This survey was approved by the School of Nutrition and Food Sciences and Technologies (SNFST) of FSA, approval N° F-025-2020-SNFST. Data were collected by administering questionnaires to randomly selected and willing respondents. To determine adequate number of respodents for survey, method according 10 used.

Results and discussion
Intercultural convergence uses of P. butyracea fruit pulp in Benin. The different uses of P. butyracea fruit pulp by rural households in the study zones are presented in Table 1. Among the different ethnic groups involved in the P. butyracea fruit pulp use, the Fulani reported to know about trees producing fruits with bitter pulp and their yellow color. The fruit pulp sometimes is consumed by the farmers in the fields. P. butyracea fruit pulp is used mainly in the therapeutic applications and less in cosmetic and food sector. All the socio-cultural groups interviewed use the pulp for therapeutic applications. More specifically, P. butyracea fruit pulp is used by Anii (100% of respondents), Kotocoli (96.80% of respondents), Boo (97.30% of respondents) and Waama sociocultural groups (94.30% of respondents) in the treatment of digestive system disorders such as bloated stomach and constipation. Reference 2 reported that the fruits are used to treat the digestives disorders, the genito-urinary infections and also in the human consumption. Apart from this traditional utilization, P. butyracea pulp is also Moreover, all socio-cultural groups interviewed use pulp to wash their pans, they also use it against rust. According the P. butyracea butter producers, the fruit is available from May to June. The period of abundance is the end of May in most of the parks. It are available at the beginning of the rainy season, a period characterized by general food scarcity in Sub-Saharan regions. Gathering of P. butyracea fruits is generally done by women and children early in the morning. They collect under the trees all fruits found without sorting. After gathering, the fruits are depulped in the field and kernels brought back home.
Physical characteristics of P. butyracea fruits. The dimensions of P. butyracea fruits from various parks in northern of Benin were presented in the Fig. 1 The highest length and diameter of fruits were recorded to the ellipsoid and globular shapes, while the lowest lengtth and diameter of fruits were found in the ellipsoid twisted shape. This result is in agreement with characteristics of fruits described by collectors during the survey. The collectors of P. butyracea fruits affirmed that the fruits having the ellipsoid and globular shapes are of big size. In addition, more than 70% of the fruits collected in the various parks are ovoid or globular forms.   Table 2. The mass of fruit from different parks ranged from 343.27 to 475.35 g, with a mean value of 403.30 g. In the same way, the mass and proportion of pulp ranged from 95.98 to 162.50 g, with an average value of 125.63 g, and 32.76 to 39.12%, with a mean value of 35.79%, respectively. There was a significant difference between the parks studied for fruits mass, pulp proportion and pulp mass (p ≤ 0.001). Thus, the highest fruit mass, pulp proportion and pulp mass were found on the fruits collected in Bassila and Tchaourou parks, while the lowest values of these parameters were observed on the fruits from Kandi and Toucountouna parks. The major factors that could affect these characteristics of fruits are rainfall. Thus, Tchaourou and Bassila are localised in the Sudano-Guinean transitional climatic zone with an average rainfall varying from 850 to 1850 mm and 1000 to 1300 mm, respectively. Kandi and Toucountouna are located in the Sudanian climatic zones with an average rainfall of 1200 and 1000 mm, respectively 5,17 . Moreover, fruit mass, pulp proportion and pulp mass varies significantly (p ≤ 0.001) between fruits shapes (Table 3) and ranged from 241.50 to 535.25 g, 81.75 to 204.50 g and 30.44 to 36.74%, respectively. The highest fruit mass, pulp proportion and pulp mass of fruits were recorded to the ellipsoid, ovoid and globular shapes respectively, while the lowest values were found in the ellipsoid twisted shape. The pulp proportion obtained was lower compared to the range of 57-70% for shea fruit pulp collected from various parks in Benin 18 . The ovoid shape has the proportion of pulp more significant (36.74%) which is definitely higher than those of the ellipsoid twisted (33.30%), ellipsoid (33.05%) and globular shape (30.44%).   Table 4. The moisture content of the P. butyracea fruit pulp ranged from 85.77 to 86.47%, with no significant different between the P. butyracea parks (p > 0.05). These values were similar to those of the pineapple (87%) and apple (86%) 19 and shea fruit pulp from various zones in Benin (77.56-80.89%) reported by Ref. 18 . The P. butyracea fruit pulp would not be microbiologically stable at this moisture content as it would allow the proliferation of microorganisms. The ash content of the pulps was comparable to that of the bush mango fruit pulp (02.10%) 20 but higher than the 0.33-0.71% reported for shea fruit pulp jam 21 .This is indicative of the high mineral content of P. butyracea fruit pulp. The pH of the pulps ranged from 3.27 to 3.41. The pH of the different pulps is not significantly different (p > 0.05). The low pH of the P. butyracea fruit pulp would not create favorable conditions for many organisms to sporulate and multiply 20 . The low pH and high acidity for these products was due to the organic acids in the fruit pulp 22 . These properties would confer long shelf life on the products by hindering proliferation of undesirable microorganisms 23 . The titratable acidity of the P. butyracea fruit pulp from different park in Benin ranged from 0.73 to 1.03%, with significant different between the parks. The highest value of titratable acidity was found in the samples from Kandi park while the lowest values of this parameter was observed in the samples from Bassila park. The value for the titratable acidity was similar to that reported by Ref. 19 in orange/pineapple juice blends (0.47-1.27%), but higher than 0.233-0.330% obtained by Ref. 21 in shea fruit pulp juice. The acids present in food not only improve its palatability but also influence its nutritive value 20 . The proteins value of fruit pulp from different parks ranged from 3.33 to 4.61% dw. There was a significant difference (p ≤ 0.05) between the parks studied for this parameter. These proteins values were lower than that of the shea fruit pulp (3.96-6.34%) reported by Ref. 18 , but is higher than 1.5-3.5% obtained by Ref. 9 in Uganda and 1.09% reported by Ref. 24 from the fresh shea pulp. In addition, Protein content of P. butyracea fruit pulp was similar to that found in mango pulp (4.8%) reported by Ref. 20 . The differences in the protein contents may be due to environmental differences in areas of production. The The value of crude fiber content for P. butyracea fruit pulp of 20.37-20.78% is within the crude fiber values of most wild and domesticated fruits and higher than in legumes with mean values ranging between 5 and 6% 25 . In addition, these values are high compared to the fiber value for bush mango pulp of 2.5% reported by Ref. 20 , and high than the shea fruit pulp collected from various parks in Benin with values ranging between 5.97 and 6.52% as reported by Ref. 18 . The highest crude fiber content of the P. butyracea fruit pulp makes it digestible beverage for children as reported by Ref. 26 for juices made from citrus fruits. It also implies that promoting consumption of P. butyracea fruit pulp is of great benefit to the human diet. The crude lipids content of P. butyracea fruit pulp ranged from 6.31 to 6.51%, with no significant different between the P. butyracea parks (p > 0.05). These values were higher than those of the shea fruit pulp from different shea zones in Uganda (1.5-3.5%) reported by Ref. 9 , and 1.4% obtained by Ref. 20 in bush mango fruit pulp. The values for carbohydrates ranged from 65.10 to 66.94% and showed no significant difference (p > 0.05) between the samples. This is in close relationship with the range 80.8% reported by Ref. 20 for bush mango fruit pulp. The carbohydrate value was higher when compared with values 7.3-9.9 g/100 g obtained by Ref. 27 in Butyrospermum paradoxum fruit pulp and 12.6-19.4 g/100 g reported by Ref. 9 for shea fruit pulp in different shea districts of Uganda. The P. butyracea fruit pulp was rich in carbohydrate when compared with other nutrients of the fresh pulp. The energy content of the pulp varies from 335.75 to 338.99 kcal, and shows no significant different (p > 0.05) between sites. The highest fat and carbohydrate contents of the P. butyracea pulp contributed to the higher energy content of the pulp. The energy obtained from P. butyracea pulp was higher than that of 1289.2 kj/100 g provided by the jams processed from shea fruit pulp reported by Ref. 21 . The minerals profile of the P. butyracea fruit pulp result was presented in Table 4. The most predominant mineral found in the P. butyracea fruit pulp was calcium with values ranged from 1793.71 to 1921.75 µg/g. The highest value of calcium was found in the samples from Toucountouna park while the lowest values of this mineral was observed in the samples from Tchaourou park. The calcium content was higher when compared with that of shea fruit pulp in Uganda (37.20-95.60 mg/100 mL) reported by Ref. 9 and mango fruit pulp (190 mg/100 mL) 20 . In addition, iron and manganese were also high with values ranged from 176.44 to 205.53 µg/g and 40.71 to 50.31 µg/g respectively. Iron and manganese were highest in the samples from Toucountouna park and the lowest value in the samples from Bassila and Tchaourou parks, respectively. The iron content was higher than 2.5 mg/100 g obtained by Ref. 20 in mango fruit pulp and 27.65 µg/kg reported by Ref. 24 from fresh shea fruit pulp in Ghana. Manganese content found in P. butyracea fruit pulp was higher than 12.50 µg/kg obtained by Ref. 24 from fresh shea fruit pulp in Ghana.
Microbial characteristics of P. butyracea fruit pulp. Hygiene quality of P. butyracea fruit pulp samples from various parks in Benin was presented in Table 5. Samples of P. butyracea fruit pulp had various microbial load with low count of aerobic mesophilic germs (1.70-2.75 log10 CFU/g), yeasts and moulds (1.85-2.97 log10 CFU/g), total coliforms (1.11-2.37 log10 CFU/g) and staphylococcus aureus (1.05-1.37 log10 CFU/g) ( Table 1). The number of aerobic mesophilic bacteria in all collected samples was close to the international standard of 4 log10 CFU/g 28,29 . However, the highest microbial count was found in sample collected in Tchaourou and Kandi parks, while the lowest microbial count was observed in fruit pulp from Bassila, Naitingou and Toucountouna. Difference between parks for the number of yeasts and moulds (p ≤ 0.001) and total coliforms (p ≤ 0.001) were observed, with samples collected from Tchaourou park giving significantly higher values of yeasts and moulds compared with the samples from other parks. Water and environment may play a major role in the yeasts and moulds contamination of P. butyracea fruit pulp especially during washing of fruits. The highest count of yeasts and moulds is also due to the acidic nature of P. butyracea fruit pulps which probably favours the growth of yeasts. Moreover, no faecal coliforms were detected in all fruit pulps. Furthermore, there is no significant difference in Staphylococcus aureus counts between the fruit pulp collected in various parks (p > 0.05). The presence of Staphylococcus aureus in the fruit pulp is attributed to its wide spread in the environment. The primary habitat of these organisms are the interior body and skin of man and animal from where this microbes are transferred to the fruits and subsequent transfer to the fruit pulp during treatment. However, the level of all microbial enumerated in P. butyracea fruit pulps was under the detection limit. The acidity of pulps could be a factor that limited the development of spoilage and pathogen microorganisms in pulps.  31 . In addition, the TPC of P. butyracea fruit pulp obtained in this study was higher than 226.25 mg/100 g reported by Ref. 24 from fresh shea fruit pulp in Ghana. The antioxidant capacity of fruits and vegetables is an important indicator of health promoters, and many methods have been developed to evaluate this particular capacity 32 . In this study the anti-radical properties of the fruit pulps was performed by DPPH radical scavenging assay. EC 50 , the effective concentration of the extracts (mg antioxidant/mg DPPH) required to scavenge 50% of DPPH radical are presented. Significant difference in EC 50 between the fruit pulp samples was observed (p ≤ 0.001). The pulp sample collected in Toucountouna park showed the highest EC 50 value, whereas the pulp sample from Kandi was found to have the lowest EC 50 value. A lower value would reflect greater antioxidant activity of sample. Moreover, the remaining DPPH in the fruit pulps ranged from 10.83 to 22.54%. This result suggests that P. butyracea fruit pulp plays an important role in the scavenging of free radical.

Conclusion
This study showed that P. butyracea fruit pulp has high total phenolic content. In addition, P. butyracea fruit pulp is a valuable source of antioxidant pigments. The proximate and the mineral composition values obtained showed that P. butyracea fruit pulp is rich in total carbohydrates, crude fiber, calcium and iron. This fruit pulp is a good source of the minerals which are useful for the proper functioning of the body and important in the diet. Thus, consumption of P. butyracea fruit pulp is highly recommended in Benin.

Data availability
All the data supporting the results are included within the article.  Table 6. Parameters of free radical scavenging activity by DPPH and total phenolics of P. butyracea fruit pulp by park. Mean ± Standard deviation; values with the same letter in the same column, are not significantly different at p < 0.05. Gallic acid -0.0020 ± 0.0001 9.83 ± 0.03