In the Republic of the Congo, there is a wide variety of traditional fermented drinks, including sugar cane wine (“loungouila”), palm wine (“nsamba”), honey wine (“duma”). Other local wines basing on banana, orange, pineapple and grapefruit are not yet documented. The plantain wine commonly known as “banana wine” is known as “Mbamvu” in some Republic of Congo areas [1]. This fermented local beverage is produced according to traditional methods generally in areas where raw materials are grown. The rather crude fermentation technique consists in letting the juice extracted from the fruit turn to produce alcohol. Spontaneous fermentation is achieved by the presence of microorganisms from the plant material Physicochemical parameters are generally poorly controlled [1].

Also known as “Urwagwa” in Rwanda, banana wine is one of the oldest and most important beverages found in most East African countries. It is mainly consumed in three quarters of the Rwandan territory, where banana cultivation is a major agricultural activity [2].

Traditional practices show that Mbamvu is prepared from mature plantain slices suspended in water for one (1) to three (3) weeks. The microorganisms responsible for alcoholic fermentation are usually present on the surface of the raw materials that serve as their substrate [3]. In addition, the hygienic quality is based on the transformation conditions and the material used [4].

The choice of the fruit is justified by the fact that the plantain is the most consumed fresh fruit in the world [5] but beverage produced from plantains is little known.

So far, the techniques of making our local beverages such as nsamba “loungouila”, orange wine and “Mbamvu” are poorly controlled and pose many difficulties from the hygienic point of view, in terms of preservation, rate of fermentation, alcohol, taste variation, which can lead to different health problems.

Some fermented foods and beverages have been studied in Republic of Congo and molecular identification of lactic acid bacteria, Bacillus spp., Sacharomyces spp. have been done as well [6] - [11]. Multiplex PCR techniques have been used to identify lactic acid bacteria [12] [13]. The concept “multiplex PCR” refers to a development of the PCR technique allowing the amplification, in a single reaction, of several distinct DNA segments. The pairs of primers corresponding to the different loci to be analyzed are introduced into the same reaction tube [12] [13]. PCR multiplexe amplicon using 16S RNA and 23S RNA genes were estimated on Lactobacillus mali, Pediococcus pentosaceus, Leuconostoc mesenteroides and Oenococcus oeni [14]. Virulence genes including fsr, efm, esp, cylA, cad1, ace, gelE, and asa1 have been used to identify Enterococcus spp. in multiplex technique [15]. Several lactic acid bacteria have been shown to be secreted biosurfactants which are amphiphilic compounds produced by either on the cell surface or secreted extracellularly [16] [17] [18]. Biosurfactant plays an important role in antimicrobial activities [16] [18] [19].

To our knowledge, few studies from the microbiological and biomolecular point of view have been carried out to be secreted directly biosurfactant in banana wine during fermentation. In the context of this work, this beverage seems to be interesting for the identification of biomolecules secreted during fermentation. To this end, in order to valorize that banana wine, some questions could be issued: What diversity of microorganisms can we encounter in banana wine? What are the secreted biomolecules in plantain wine? What is the role in preservation?

In this work the main goal was to appreciate banana wine by its richness Biomolecules secreted during fermentation. Banana has been shown to widely contain bioactive molecules like polyphenol, flavonoids and carotenoids playing important roles in the well-being of humans [5]. The role of bacteriocins and biosurfactants should be considered again. Indeed, in many departments of the Republic of Congo, Congolese are consumers of fermented beverages and sometimes seek for new tastes, novel sensations and good protective beverage. Some molecule contained in banana including vitamin B, protein, amino acids and calories are also important. Plantain is mainly characterized by its sugar content and its density, a factor that conditions subsequent fermentation and ethanol production [5].

We have clearly shown that biosurfactants are highly secreted during fermentation in the extracellular medium. 15 isolates were found as part of this job. They are all capable of producing biosurfactants. 66.66% produce extractible biosurfactants. Until the writing of this paper, no study on fermented foods and

drinks has shown the secretion of biosurfactants directly in the ferment. The biosurfactants extracted with chloroform and ammonium sulphate are able to inhibit the growth of pathogenic bacteria including E. coli, S. flexneri M90T, Salmonella spp, P. aeruginosa and S. aureus.

The tendency of lactic acid bactreria is to acidify the medium [4] [11] [26]. The capacity of microorganisms to secrete and biosurfactants in the extracellular medium could explain the absence of the pathogenic bacteria of the genus Shigella, Salmonella, Staphylococcus and although Shigella is able to live 2 hours inside of the stomach and cause bacillary dissentery [27] [28] [29]. Since the fermentation of the plantain wine lasts 96 hours, and the pH goes down very quickly, it is almost difficult for Shigella spp. and Salmonella spp. to resist in this condition. Lactobacillus spp. are also able to secrete other biomolecules such as bacteriocins [26] [30] which can also inhibit the growth of the bacterial pathogens mentioned above [31]. Biosurfactants have already been proposed as a preservative in the food industry [17] [32] [33]. The evaluation and the functional characterization of a biosurfactant produced by L. plantarum CFR 2194 have been done. Fourier transform infrared spectroscopy (FTIR) spectra demonstrated that biosurfactants were constituted by protein and polysaccharide fractions by possessing a glycoprotein like structure [34]. We can postulate that our Biosurfactant could be a glycoprotein because this can be easily extracted from supernatent by using ammonium sulfate ((NH₄)₂SO₄) favourizing by the salting out mecanisms [35]. The probiotic effect and ability of bacteria of the Lactobacillus spp. to inhibit the growth of pathogenic bacteria has already been demonstrated [31] [36] [37] [38]. Many studies have focused on the inhibition of pathogens by targeting bacteriocins. A previous study on banana wine in Rwanda also showed the absence of these pathogenic bacteria [39].

By using specific culture media we have shown that the bacteria like Shigella flexneri, Salmonella spp., E. coli, Pseudomonas aeruginosa, Staphylococcus, Clostridium spp. are totally absent on the tested samples. MRS allow us to isolate 15 isolates from plantain wine. One step PCR multiplex have been used to identify isolates among five lactic acid bacteria species including Enterococcus faecium, Pediococcus acidilactici, Lactobacillus sakei, Lactobacillus plantarum, Lactobacillus curvatus.

The most representative being L. plantarum (33%) associated to Is2, Is9, Is12 and Is13. It should be interesting notified that no study has been focused on genes encoding for bacteriocins to discriminate bacilli and cocci from lactic acid bacteria in a one step multiplex-PCR. By the way several methods of screening, purification and antimicrobial potentialities of bacteriocin in Health Care for human being have been mentioned [40] [41] [42]. The development and validation of a one step Multiplex-PCR assay for the detection of ten Lactobacillus species, has been recently documented [12] [43]. In addition random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis was used to identify strains of L. plantarum [13] [44] [45].

This work allowed showing that biosurfactant can be secreted directly in the extracellular medium. Lactobacillus isolated from plantain wine is a part of this job. A novel one step multiplex PCR that targets genes encoding for bacterioscins allowed to identify L. plantarum (Is2, Is9, Is12 and Is13) that are able to secrete biosurfactants playing an important role in the preservation of banana wine. The biosurfactant extracts with chloroform and ammonium sulphate are able to inhibit the growth of pathogenic bacteria such as Shigella flexneri, Salmonella spp., Pseudomonas aeruginosa and Staphilococcusn aureus. This work also constitutes a scientific support, a reference tool and a source of information that may be of interest to future scientists and researchers who would like to deepen the analysis. The processes and methods we used, could serve as a basis and example for the manufacture of all other local wines under better conditions and standards for the health of the population. The control of local beverages production could be the very important input of nutritious beverages for the benefit of the people because contains very important bioactive compounds including polyphenol and flavonoïds and carotenoids.

We are grateful Prof. Clobite Bouka Biona (Université Marien Ngouabi), and Dr. Bienvenu Dinga (Université Marien Ngouabi) for their continuous encouragements and material support. This work was supported in part by Institut National de Recherche en Sciences Exactes et Naturelles (IRSEN).

The authors declare that the research was conducted in the absence of any intellectual commercial or financial relationships that could be construed as potential conflicts of interest.

Moukala, M.B., Kayath, C.A., Ahombo, G., Dangui, N.P.M., Kinavouidi, D.J.K., Mouélé, E.C.N. and Diatewa, M. (2019) Giving More Benefits to Biosurfactants Secreted by Lactic Acid Bacteria Isolated from Plantain Wine by Using Multiplex PCR Identification. Advances in Microbiology, 9, 917-930. https://doi.org/10.4236/aim.2019.911058