The description of the processes was carried out twice at two production sites Loul-séssène and Boyard in the Fatick region (Senegal) in May 2019. Each unit operation was identified and described. Moreover, material flows and physical parameters were identified and recorded. This allowed establishing the synthetic diagram to obtain the “Mbite” drink which is based on the fruits of Sclerocarya birrea.

The various samples collected during the description of the processes were analyzed to assess both the effect of the involved factors on the quality of the “Mbite” drink and the overall performance of the production process. Two samples per site are sent to the laboratory for the purposes of biochemical and microbiological analyses.

Classical physicochemical analysis methods were used to characterize the fruits of Sclerocarya birrea and the “Mbite” drinks. These are pH, titratable acidity, volatile acidity, ethanol content, polyphenols, tannins, antioxidant activity, color indices, reducing and total sugars according to standards AFNOR [8] . All analyses were done in duplicate.

Lactic acid bacteria, yeasts and moulds were counted in accordance with French standards [9] . The standards and culture media used are presented in Table 1 .

The analysis of variance (ANOVA) makes it possible to define the interdependence relationships, for example between the analyzed samples and the physicochemical parameters. The statistical differences are then compared with a threshold of 5%.

The “Mbite” drinks collected at the production sites were analyzed during storage at 25˚C (ambient temperature). The physicochemical and biochemical parameters had been studied every day for one month.

The Optimization of fermentation was carried out with two strains of yeast: Saccharomyces cerevisiae and Saccharomyces boulardii. The biological conditions were linked to the optimal parameters of the strains’ activity while taking into account the physicochemical characteristics of the raw (unfermented) “Mbite” drinks. The latter were thus incubated at 20˚C in a thermostatically controlled refrigerator. Monitoring with a frequency of 6 hours made it possible to describe the evolution of the fermentation by the strains.

The alcoholic drink “Mbite” is traditionally prepared, and the diagram is composed of two main stages: extraction of the marula juice and fermentation ( Figure 1 ).

The manufacturing of the “Mbite” begins with manual extraction of the juice. This is a difficult step which involves using stems of Guiera senegalensis or Combretum glutinosum to facilitate the separation of the pericarp from the almond. As a result, two types of extracts are obtained: the pure juice called “O gone” and the pericarp macerate called “A thiamba” in the local language (Seerer, Senegal). The extracts are then filtered and mixed according to the producers. Finally, a 2-day natural fermentation produces the alcoholic fermented drink made from Sclerocarya birrea ( Figure 2 ).

Marula juice has traditionally been used to make two types of drinks. The tangy and nutritious juice is reserved for children and the alcoholic drink is exclusively for adults [10] . In Zimbabwe, the local drink “Umkumbi” is prepared after extracting marula juice using tapered goat horns. The must undergoes a first natural fermentation of 24 hours and is then clarified using a basket of woven palm leaves [11] . The manufacturing process of “Buganu” drink from Swaziland has the particularity of extracting the marula juice by grinding in a wooden mortar [6] . In the case of “Mbite” drink, clarification by decantation takes place prior to the fermentation to eliminate as much mucilage as possible. Filtration of the must is then done using millet straws (Pennisetum glaucum L.) as support.

In the “Mbite” manufacturing, no differentiated step of adding leaven has been detected. In fact, it is a spontaneous fermentation linked to the endogenous yeasts in S. birrea fruits [5] [11] . This is well confirmed by the microbiological analyses on the fermented extracts. The yeasts and lactic acid bacteria were counted at respective values of 10⁶ and 10⁴ CFU/ml ( Table 2 ).

The results of the analysis of S. birrea drinks correspond to samples from the three manufacturing sites ( Table 3 ). “Mbite” are not very acidic with a pH of 3.82 - 3.97. They are also very rich in polyphenols at respective concentrations of 158.25; 151.10 and 124.92 mg GA/100 ml. However, there is a significant difference in the obtained ethanol contents (4.09; 4.04 and 1.85 ml/100 ml) in “Mbite”.

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The ethanol content was monitored in the beverages prepared during the processes’ descriptions ( Figure 3 ). These samples stored at 25˚C (ambient temperature) reach a limit alcohol concentration of 5% (v/v) after 3 days. Furthermore, the pH and titratable acidity are 3.57 - 3.80 and 12.65 - 15.43 mEq/100 ml, respectively.

The concentrations of tannins (8.04 - 9.28 mg/100 ml) and polyphenols (124.92 - 158.25 mg/100 ml) as well as the color indices reflect good stability of the beverages during storage.

The maturation of fermented drinks is the phase corresponding to the peak of the ethanol concentration, but mostly to the development of aromatic and taste compounds. It depends on the duration of the various fermentations (main or secondary) which condition the sanitary and market qualities. This maturation period is 2 days for the Zimbabwean drink “Umkumbi” [11] ; and 3 days for “Buganu” from Swaziland at ambient temperature (25 - 30˚C) [6] .

The volatile acidities found in “Mbite” drinks respectively 1.6; 2.4 and 2.6 g of acetic acid/l are well above the tolerated limit (1.2 g/l) in wines [12] . It would be tempting to deduce that secondary fermentation leads to the production of organic acids, diacetyls and volatile esters [11] [13] . The same conclusions have been obtained in several traditional African drinks made from Marula [5] [6] [11] . “Mbite” has an alcoholic strength of 2 to 4% (v/v) which is quite similar to those of “Umkumbi” and “Buganu” varying from 2 to 5% (v/v) [5] [6] .

Various genomic studies have shown the preponderance of strains of Lactobacillus, Leuconostoc, Saccharomyces, Salmonella and Shigella in local marula-based drinks [5] [11] .

These germs naturally present in the marula fruit are involved in the fermentation process and in many cases of food poisoning [14] . However, the high acidity of fermented drinks made from Sclerocarya birrea participate in the inhibition of pathogenic microorganisms such as Escherichia coli [15] . Nevertheless, it would be advisable to test controlled fermentations for better control of sanitary and hygienic quality.

Traditional marula-based drinks are considered to be fairly stable given their acidic pH (3.5 - 4.0) and alcoholic strength of 2 to 5% (v/v) [5] [6] . The fact remains that they present high health risks due to the spontaneous nature of the fermentation involving the native flora of the juice [11] [16] . In this sense, controlled fermentation trials will make it possible to control the process, which will condition both the hygienic and organoleptic qualities.

The samples analyzed were the S. birrea juice obtained by manual extraction and filtered with a filter bag of 0.45 µm porosity. The extracts were then autoclaved at the scale of 120˚C/15 minutes. Table 4 presents the physicochemical and biochemical characteristics of both the sterilized and unsterilized juices.

Unsterilized and sterilized S. birrea juices are not acidic with pH of 4.82 and 4.90, respectively. This translates into titratable acidities of 4.60 and 4.75 mEq/100 ml, respectively. These results are quite different from the samples from the Ferlo zone (Senegal) with a pH of 3.88 - 3.95 and are highly acidic (150 - 199 mEq/l) [17] . These parameters are closely related to the maturity state of the plant and to pedoclimatic conditions. However, the juices of the fruit of Sclerocarya birrea have high contents of antioxidant compounds (polyphenols: 221 - 236 mg/100 ml and tannins: 18 - 19 mg/100 ml), of reducing sugars (5.28 - 5.88 g/100 ml). and total sugars (8.86 - 11.69 g/100 ml).

The heat treatment in an autoclave did not induce any major changes in the physicochemical and biochemical characteristics. Only the color indices have undergone a significant modification. Unsterilized and sterilized juices are statistically similar for both nutritional and energetic compounds. Indeed, the objective of sterilization is to eliminate all pathogenic and spoilage microorganisms while preserving the nutritional and sensory qualities of the product. Concerning the marula juice, sterilization had to eliminate the native flora composed of pathogens such as Salmonella and Shigella [11] and Lactobacillus which are responsible for secondary fermentation. Also, the noted color indices prove that there were no major browning reactions.

The sterilized juices of S. birrea were inoculated with Saccharomyces cerevisae strain: MC1 and MC2; and Saccharomyces boularii strain: MB1 and MB2. Monitoring the ethanol content established that the fermentation was optimal at 24 hours for MC1 and MC2 drinks and at 30 hours for MB1 and MB2 drinks. Alcohol production is slow in the early hours of fermentation and tends to speed up after 12 hours of storage ( Figure 4 ).

Alcoholic fermentation corresponds to the breakdown of sugars into ethanol and carbon dioxide by yeasts. These fungi are responsible for producing the energy necessary for their growth and development. In the case of industrial beer such as wine, Saccharomyces cerevisae remains the used species par excellence. Regarding Saccharomyces boulardii, it is a probiotic used as an anti-diarrhea. It participates in the maintenance and restoration of the intestinal flora of both the large and small intestines. Traditional fermented drinks are characterized by the fact that multiple fermentations can occur in them [5] [18] . In previous works, fermentation germs belonging to the genus Saccharomyces and Lactobacillus as well as some pathogens were discovered [5] [16] .

Sterilization ensured better control of both fermentation and hygienic quality. Contrary to the traditional method, the unique alcoholic fermentation triggered by the Saccharomyces strains resulted in a substantial reduction in the fermentation time from 3 days to 24 hours. Furthermore, the incubation temperature is beneficial for the preservation of antioxidant molecules. Studies have shown that fermentation of S. birrea juice at a temperature below 30˚C allows more than 90% of the polyphenols to be retained [16] . This is a clear advantage for industrial production.