EVALUATION OF MICROBIOLOGICAL AND PHYSICOCHEMICAL Q UALITY OF BORASSUS AKEASSII FRESH SAP AND FERMENTED SAP (BANDJI) PRODUCED AT BURKINA FASO

Zongo Oumarou, Tapsoba François, Guira Flibert, Zongo Cheikna, Traore Yves and Savadogo Aly *. LABIA/CRSBAN/Département de Biochimie-Microbiologie , UFR-SVT, Université Ouaga 1 Pr Joseph KI-ZERBO, Burkina Faso, 03 BP 7021 Ouagadougou, Burkina Faso.  ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Palm wines are produced, consumed and appreciated by population of West Africa. Our previous study deal with impact of technological diagram on biochemical and microbiological quality of Borassus akeassii wine produced traditionally in Burkina Faso. This study aims to evaluate the microbiological and physicochemical quality of the fresh and fermented sap from Borassus akeassii. Thirty (30) samples of fresh and fermented sap were collected from traditional producers. The microbiological analysis was carried out using standard microbiology methods and physicochemical parameters were determined by AOAC methods. The analyzes of different samples of sap showed that total count of mesophilic bacteria was between 2.0x10 6 and 1.7x10 9 CFU/ml ; yeasts between 2.2x10 5 and 2.5x10 8 CFU/ml ; lactic acid bacteria (LAB) flora between 1.9x10 4 and 1.8x10 7 CFU/ml and acetic acid bacteria (AAB) between 1.3x10 5 and 3.1x10 7 CFU/ml. Coliforms, Staphylococcus aureus and Salmonella sp strains were found in few samples of fresh sap but absent in the fermented palm sap. Total sugars and Ascorbic acid content of saps ranged from 0.55 ± 0.05 to 12.5 ±0.1% (w/v) and 0.78 ± 0.06 to11.01 ±0.22 % (w/v) respectively. The application of good hygiene practices during the collection, selling or packaging of the sap is needed and could improve the quality of palm wine.

Introduction:-
The palm trees (family of Arecaceae or Palmeae) are largely widespread in the intertropical areas of Asia, America and Africa (Miège, 1985). They gave innumerable products to the local populations of the developing countries. The sap of the palm trees (one of products) is collected in the whole world by the local populations of the tropical and subtropical areas (Essiamah, 1983;Swing and De Ley, 1977). In Africa, the sap is extracted from the various species of palm trees such as Elaeis guineensis, Raphia hookeri, Phoenix dactylifera, Borassus aethiopum, Cocos nucifera and Borassus akeassii (Ouoba et Mollet et al., 2000). After the extraction, the crude sap of the palm trees is subjected to a spontaneous fermentation to give an alcoholic drink called palm wine. According to country, the palm wine is known under various names: toddy in India, emu or ogoro in Nigeria, lambanog in Phillipines, taberna in Mexico (Santiago-Urbina et al., 2013;Noll, 2008). In Burkina Faso, the palm wine usually called "bandji" is obtained by natural fermentation of the sap of Borassus akeassii, a new palm tree species identified in south-west 1490 (Bayton and Ouédraogo, 2009 ;Bayton et al., 2006). The extraction of the sap is practiced twice per days (morning and evening) indifferently of the sex of palm tree following a seasonal mode. The sap is a white liquid milky, flocculent, and characterized by a gas effervescence resulting from the spontaneous fermentation, which testifies to the presence of fermentative micro-organisms (Combet-Blanc, 1997). The methods of tapping palm trees are various but in general, tapping involves perforation of the trunk, insertion of a tube in the hole and collection of the sap in a container (gourd, clay pot, plastic container, glass bottle or calabash) (Dalibard, 1999). According to Santiago-Urbina and Ruíz-Terán (2014), the methods of tapping palm trees depend on the locality but in general, two methods are practiced. In the first method, the sap is obtained from a live standing tree and the second, the tree is felled or cut down before tapping. The sap of the palm tree contains essentially sugar (10-12% of saccharose), soluble proteins, amino acids, amides, minerals and vitamins (Tiépma et al., 2013;Heller, 1981;Bassir, 1968). Half of the total sugars are fermented during first 24 hours and ethanol content of the fermented palm sap reaches maximum of 5.0 -5.28 % (v/v) after 48 hours (Sekar and Mariappan, 2005).
Presence of micro-organisms such as yeasts, lactic acid bacteria (LAB), acetic acid bacteria (AAB), enterobacteria, Bacillus spp., Micrococcus spp. and Staphylococcus spp. has been reported (Malonga et al., 1995 ;Atputharajah et al., 1986 ;Okafor, 1975Okafor, , 1972. Several studies showed that the palm wine results from an alcoholic, lactic and acetic fermentation (Tapsoba et Atputharajah et al., 1986 ;Okafor, 1978). However, the most important role are played by yeasts, LAB and AAB. Generally, palm wine is good for health (Olawale et al., 2010). A balanced administration of fresh and fermented date sap was found to improvise the treatment of hemoglobin deficient anaemic patients and to supplement vitamin-B12 level in the vitamin deficient patients (Debmalya and Mazumdar, 2008). Nutritionally, this drink is a source of sugars and vitamins interesting that complements the daily food intake of consumers (Okafor, 1978;Van Pee and Swings, 1971). It is also involved in traditional ceremonies such as weddings, christenings, funerals, self-help work and as a source of income for rural populations. However, palm wine is traditionally produced by local people. It is diluted frequently with untreated water and directly consumed without any treatment. It is thus necessary to evaluate the quality of this local drink.

Sampling:-
The biological material consisted of ten (10) samples of fresh sap, ten (10) samples of fermented sap after 24 hours of collection and ten (10) samples of fermented sap after 48 hours of collection. Fresh sap is the sap which has been accumulated during the night and collected early the morning. Fermented samples are samples which have been stored and fermenting for additional 1 or 2 days at ambient temperature (25-30°C) spontaneously. Samples (500ml) was collected from traditional palm wine tapper in the village of Tiékouna (6 km from Banfora towards Sindou) and Bounouna (located 4 km the entry of Banfora) in sterile containers. Samples were stored immediately on ice and transported to the laboratory for physicochemical and microbiological analyses.
Physicochemical Analysis:-Physicochemical analysis includes pH, total acidity, sugar, Ascorbic acid (Vitamin C) and alcohol content. The pH and total titrable acidity was determined using methods described by Amoa-Awua et al. (2007). The percentage of sugar expressed as degree Brix was measured by refractometry method using a refractometer (METTLER TOLEDO, B/0311) (AOAC, 2000). Vitamin C content was determined by titration method using DIP (2,6-Dichlorophénolindophénol) as described by AOAC (1990). Alcoholic content of the samples was determined by densimetry method using hydrostatic balance according to AOAC (2000). The samples were initially degassed with the Ultrasound (BRANSON, 1510E) during 15 minutes then filtered using filter papers (Whatman).Then 30 ml of distilled water are added to 100 ml of filtrate sample. The mixture obtained was distilled with electrochemical distiller (GIBERTINI, B/0303) after addition of 5 ml of the catalyst solution (solution of calcium hydroxide 2M) and of some drops of antisolution foams (silicone solution 30%). After distillation, 100 ml of distillate were collected and the percentage of alcohol (v/v) is read directly using alcohol meter (Super Alcomat).

Microbiological Analysis:-
The microorganisms were counted according to ISO 7218 (2007): Microbiology of food and animal feed -General rules for microbiological analyzes. The amount of micro-organisms was determined by serial dilutions and spread plate technique was used. Ten milliliter (10 ml) of each sample was diluted with 90 ml of sterile buffered peptone water and well mixed. Successive dilutions of the sap were prepared in screw test tubes and appropriate dilutions were poured into plates on appropriate selective media then enumerated. Aerobic mesophilic flora were counted on 1491 the PCA agar (Plate Count Agar) incubated at 30 ° C after 24 to 72 hours under aerobic conditions. Sabouraud agar containing Chloramphenicol was used for enumeration of yeasts, the plates were incubated aerobically at 30 ° C for 72 hours. Lactic acid bacteria (LAB) were enumerated on MRS agar (de Man, Rogosa and Sharpe) containing Nystatin (antifungal) which were aseptically added to inhibit the growth of yeasts (100 mg / l) and plates incubated at 30 ° C for 4 days. For Acetic acid bacteria (AAB) enumeration, GYC agar (glucose yeast extract and calcium carbonate) were used. Penicillin (12.5 mg / l) and Nystatin (100 mg / l) were added to GYC medium to inhibit LAB and yeast respectively, and plates were incubated at 30 ° C for 5 to 6 days. The total and faecal coliforms were counted on Violet Red Bile Lactose Agar (VRBL) and the plates were incubated for 24 to 48 hours at 37 ° C for total coliforms and 44 ° C for faecal or thermotolerant coliforms. Chapman's agar was used for the enumeration and isolation of Staphylococcus aureus.The plates were incubated aerobically at 37 ° C for 24 to 48 hours. Gram stain, the catalase test and oxidase were performed to confirm the presence of Staphylococcus aureus. The number of colonies counted were expressed as colony forming units (CFU) per ml. Finally, Salmonella and Shigella were tested on Salmonella-Shigella Agar after a pre-enrichment using buffered peptone water and enrichment with Rappaport Vassiliadis Soja buffered. The suspect colonies (uncolourless colonies with or without black center) were selected and purified on Mueller Hinton agar then identify by biochemical tests such as Gram stain, gaz and H2S production, lactose, glucose, urease production, indole, citrate, mannitol , motility.

Statistical Analysis:-
The data were seized on Excel 2010 and analyzed with software XL STAT 7.5.2. One-way analysis of variance (ANOVA) were used to determine whether there are any significant differences between the various averages of the different parameters. The difference between the averages is significant when p< 0,05.

Results of physicochemical Analyses:
The physical and chemical quality of Borassus akeassii palm sap was evaluated by the determination of some parameters such as pH, total acidity, total sugars, vitamin C and the alcohol content.

Analyse of fresh sap:
The pH of fresh sap samples ranged from 4.05 ± 0.15 to 4.86 ± 0.04; total acidity from 0.2± 0.01 to 0.48 ± 0.03 % (table 5). The total sugar content was between 8.00± 0.00 and ± 12.5 ± 0.10% and vitamin C content between 1.57 ± 0.22 and 11.01 ± 0.22%. The alcohol content ranged from 0.30 ± 0.02 to 2.39 ± 0.04%. The differences between the averages are meaningful to the different physico-chemical parameters (p <0.05). The fresh sap is the sap that has been accumulated throughout the night. This sap still has high levels of sugar and vitamin C despite spontaneous fermentation held. Maintaining these parameters is due to the continuous accumulation of the sweet sap of the palmyra in the fermented or fermenting sap  Analyse of fermented sap:-Analysis of fermented sap for 24 hours (Table 6) showed a pH between 3.50 ± 0.00 and 3.83 ± 0.03 and a total acidity between 0.35 ± 0.02 and 0.59 ± 0.02%. Total sugars varied from 2.40 ± 0.00 to 7.1 ± 0.20%, Vitamin C from 2.58 ± 0.11 to 7.19 ± 0.22% and the alcohol content from 1.82 ± 0.01 to 4.70 ± 0.12%. The differences between the averages are meaningful to the different physico-chemical parameters (p <0.05). The pH of the fermented sap for 48 hours (Table 7) was between 3.25 ± 0.01 and 3.51 ± 0.01; total acidity between 0.43 ± 0.01 and 0.76 ± 0.01% ; the total sugar content ranged from 0.55 ± 0.05 to 2.4 ± 0.1%; the rate of Vitamin C varies from 0.78 ± 0.06 to 4.38 ± 0.11% and the alcohol level between 3.53 ± 0.03 and 6.13 ± 0.01%. The differences between the averages are meaningful to the different physico-chemical parameters (p <0.05). Compared to fresh sap, the fermented sap for 24 hours and 48 hours contain low levels of sugars and vitamin C. The fermented sap has higher acidity and alcohol content than fresh sap. These results could be explained by the fermentation activity of the microflora of the sap. Indeed, the natural microflora (bacteria and yeasts) uses vitamins during their growth and continuously converts the sugars into alcohol and / or organic acids. The low pH of some samples could be due to the intense activity of acidproducing bacteria (LAB and AAB) (Tapsoba et al., 2011).
1496  2014) showed an alcohol content of 5.80 ± 2.13 and 4.7 ± 1.47% (v / v) and a total acidity of 0.64 ± 0.08 and 0.82 ± 0.29 % (w/v). These results could be due to the initial physico-chemical composition of the sap and the nature of fermentation. In view of the physical and chemical characteristics, the fresh sap has good nutritional quality while fermented sap has a fairly good nutritional quality because of its acidity.

Conclusion:-
This study allowed to evaluate the microbiological and physico-chemical quality of the sap collected from Borassus akeassii. The microbiological analysis results show contamination of the fresh sap by coliforms, strains of Staphylococcus aureus and Salmonella sp. The presence of these pathogens responsible for foodborne illness can be a risk to consumer health. However, no pathogen was found in the fermented palmyra sap for 24 hours and 48 hours' time. The physicochemical analyzes revealed that the fresh sap contain large amounts of sugars and vitamin C than fermented sap. Fermentation appears as a means of improving the quality of the boxwood wine through the inhibition of pathogens. However, the acidity of the fermented sap causes loss of its organoleptic quality, making it undesirable to consumers. Fermented sap for 24 hours would be recommended for consumption. Good practices of production and hygiene are needed to be implemented during collection for quality improvement.