First Development of a Biotechnological Ferment Based on a Consorsium of the Genus Bacillus for the Optimization of the Fermentation Process of Cassava Tubers

Due to its nutritional values, cassava has become an unavailable food and is one of the essential foods in the Republic of Congo. Fermentation of tubers is still traditional. Fiftyrod-shaped spore-forming bacteria were screened and carried out in batch mode for the fermentation abilities of cassava tubers in order to develop biotechnological starter. The Penetrometry Index (PI) has been used to screen bacteria and 16SrRNA as well as fibEone step multiplex PCR which were used to molecularly identify isolates. Emulsification Index, Proteolytic as well as amylolytic, and cellulolytic activities of some strains were quantitatively evaluated for prooving orgaleptic characterics. As results Bacillus subtilis (MT994787), Bacillus subtillis (MT994789), Bacillus tequilensis (MT994788), Bacillus safensis, and Bacillus subtilis have been identified. Single isolates were able to ferment tubers in 48 h and 72 hours meanwhile Bacillus consortia were able to shift fermentation of tubers from 48 hours to 24 hours. The consortium could be used as the major bacterial starters. Strains were associated with the ability to secrete biomolecules including biosurfactants, protease, amylase and cellulase.


Introduction
The traditional fermentations of the cassava retting process are a major concern in rural areas in the Republic of Congo. Fermentation processes are based on natural ferments which create an infernal expectation of the fermentation time. The expected products generally have a non-standardized quality [1]. Fermentation does not present greater acceptance in the sensorial analysis for taste, aroma and general acceptance. Nowadays the work of microorganisms has been well documented to considered like an indisputable input of fermentation including the genera Bacillus [2], Lactobacillus [3] [4], Lactoccoccus [4], Saccharomyces [5] and Candida [6] and molds as well. The Saccharomyces cerevisiae is the only commercially available probiotic yeast used in controlled fermentations of bread and other dairy products from pastries [6] even if Bacillus GRAS species are on its way to be accepted like a probiotic and prebiotic bacteria [7].
Manihot esculenta, Cranzt, commonly called cassava [8], manioc, yuca, macaxeira, mandioca, aipim, and agbeli, is a woody shrub of the spurge family, Euphorbiaceae is extensively cultivated as an annual crop in Republic of Congo for its edible starchy tuberous root and all other derived products [9]. This plant represents nearly 80% of local consumption [1].
Bacteria of the genus Lactobacillus have already and intensively been used as a starter culture [10]. It is often difficult to accept that during a traditional fermentation there is only one genus of bacteria. Strains are usually in the form of anecosystem consortium [11]. Without citing the other genera, bacteria of the genus Bacillus are ubiquitous in acid or alkaline fermentations [2] and they should probably play several roles still unclear since the fermentative capacity seems to be associated only with yeasts and lactic acid bacteria. In addition Bacillus species have been used as a starter [12] for improving acetoin and tetramethylpyrazine in Baoning bran vinegar [13] and for bettering the production of okpehe, a traditional African fermented condiment in Nigeria [14]. It has been demonstrated that Bacillus species possess important characteristics for further development of starter cultures and its organoleptic characteristics [15].
By the time of drafting this paper no study has separately and specifically shown the genus Bacillus roles in the direct fermentation of cassava tubers and retting as well. The processing and production of cassava are rudimentary in the Republic of Congo. Developing a biotechnological ferment could be of great added value among the responses to be given to achieve food self-sufficiency. In this way, the objective of this work is to isolate and to evaluate the fermentation potential of Bacillus species from cassava tubers to select those favorable to be used as starter cultures for the elaboration of a cassava fermented food and derived products.

Isolation of Strains with the Ability to Ferment Tubers
Samples from fermented tubers have been collected. Dilutions were done and microorganism suspension was streaked on Mossel supplemented with polymixin B. The Petri dishes were incubated at 37˚C for 24 h to 48 h. Each colony of different appearance was separately isolated. Purification of the isolates was rigorously done by successive cultures. Purity was estimated by using a microscope for morphological characterization. Gram status was determined by using 3% KOH. Sporulation, hydrogen peroxide (H 2 O 2 ), and oxidases tests were used for biochemical characterization.
Three ( Tuber penetrometer resistance is an effective and reliable method for evaluating cassava tubers strength. A mecanic penetrometer has been used to introduce in fermented tubers. During the fermentation process the penetrometry Index (PI) have been assessed. The values were established according to the texture of the fermented cassava tubers. A score of ten (10) was associated with the tuber whose the penetrometer completely has been penetrated and broke the tubers.
An index of seven (7) to eight (8) was associated with the tuber with penetration of the stem creating cracks. An index of five (5) was associated with the tuber that the penetrometer entered but did not break. Zero (0) was associated with the tuber whose peak strength and maximum stress were not allowable.

The Production of Enzymes and Biosurfactant
The Proteolytic, amylolitic and cellulolytic activities of some Bacillus strains were assessed for the ability to secrete proteases, amylase and cellulase in the extracellular environment as described and modified [16]. The E24 was investigated by adding crude oil with LB medium in 1:1 ratio (v/v). The solution was vortexed for 5 min and incubated for 24 h. Emulsification percentage was calculated through the height of emulsion layer. In addition, E24 was determined for gasoline, diesel fuel or SAE 140. All the experiments were performed in triplicates. E24 = Height of emulsion layer/Total height of solution ×100.

Molecular Identification of Microorganisms
The recent molecular identification using the fibE gene encoding for the fibrinolytic enzyme has been used for targeting strains like Bacillus amyloliquefaciens, B. subtilis, B. pumilus, B. licheniformis, B. altitudinis, B. mojavensis, B. safensis, and B. atrophaeus. Extraction and purification of isolate genomic DNA were performed according to the NucleoSpin Microbial DNA (Macherey-NAGEL) kit. Briefly, isolates were grown in 5 mL LB broth for 24 h at 37˚C with stirring. The DNA purity was assessed by electrophoresis on agarose gel and by the ratio of optical densities 260/280 nm. 5 μL of each amplification product was mixed with 2 μL of loading buffer (BIOKE). Mixtures were subjected to electrophoresis on 1% agarose gel (w/v). The 10 kb 2-Log (BIOKE) was used as a molecular weight marker. The housekeeping 16S rRNA gene has been amplified by PCR (Thermal Cycler, Bio-Rad) by using universal primers fD1 (5'-AGACTTTGATCCTGGCTCAG-3' and rP2 (5'-ACGGCTACCTTGTTACGACTT-3'). 5 μL of each amplification product was mixed with 2 μL of loading buffer (BIOKÉ). Mixtures were subjected to electrophoresis on 1% agarose gel (w/v). The 10 kb 2-Log (BIOKÉ) was used as a molecular weight marker. The PCR products were purified using the solution of Gel Extraction kit (Omega Bio-tek), and the purified products were subjected to sequencing by the Sanger technique (3130 × l Genetic Analyser (Applied Biosystems)). The sequences obtained were aligned with the software BioNumerics 7.5 (Applied Maths, Belgium) and corrected manually to resolve discrepancies between the sense and antisense strands. Sequences were compared with homologous sequences contained in the sequence databanks through NCBI (National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the BLASTn program based on the identification criterion. All sequences have been stored in NCBI GenBank data.

The Abilities of Isolates to Secrete Biomolecules
Exploring bacterial communities with biomolecule production was purposed in this study. The isolates with a good fermentation capacity were subjected to be assessed for secretion of Biomolecules such as biosurfactants, cellulases, amylases, pectinases and proteases. By exploiting proteolytic activity we showed that isolates were able to secrete proteases in the extracellular medium. The diameter of clear zone around the wells indicates proteolytic digestion of the skimmed has been calculated (Figure 3(a)). Amylolytic and Cellulolytic activities have been carried out at 37˚C for enzymatic production and rate have  been done (Figure 3(b)). To highlight if identified strains could involve in the production of with more successful, a qualitative and quantitavie test called emulsion index in 24 hours (E24) has been conducted by inoculating precultures in flasks containing the nutrient broth. Incubation has been done overnight at 37˚C. As results the so-called starters were able to secrete biosurfactants by mixing with either gasoline. The average emulsion index (EI24) for gasoline are illustrated in Figure 3(b).

Discussion
In this in vitro work we showed that Bacillus species could be used to ferment tubers from cassava crop. We found for the first time that Bacillus subtilis (  (Gen bank: MT994788), Bacillus Safensis and Bacillus subtillis that were isolated by using 16 rRNA gene and fibE one step multiplex PCR [22]. When Bacteria have been mixed in pairs, they systematically reduce the duration of fermentation for only 24 hours, testifying various interactions including mutualism and symbiosis between the strains. It's worthy to recognize that in traditional fermentation bacterial ecosystem as well as yeast are co-cultivated [23] [24]. Solid-state Co-cultivation of Bacillus subtilis and Bacillus mucilaginosus have been showed to stimulate cell growth [24].
In this work we showed that Bacillus subtilis (Gen bank: MT994787), Bacillus subtillis (Gen bank: MT994789), Bacillus tequilensis (Gen bank: MT994788) and Bacillus safensis were able to produce enzymes (cellulase, protease and amylase) and biosurfactants as well. The production of enzymes and biosurfactants by the isolated bacteria could give great added value. A couple of organoleptic characteristics are the work of these biomolecules. Incorporation of lipopeptide MSA31 biosurfactant in muffin showed improved organoleptic qualities compared to positive and negative control [25]. Then the genus Bacillus is known for its ability to produce extracellular enzymes such as amylases [26], pectinases [27] [28] [29], cellulases [30] [31] [32], proteases [33] [34] and other biomolecules as well [35].

Conclusion
The use of bacteria of the genus Bacillus as a starter for Fermentation of cassava tubers improves and shortens the fermentation time to 24 h when bacteria are placed in consortium. This study opens the way for an important characterization of a local ferment which will make fermentation possible in short time to compensate the lack of cassava tubers in the Republic of Congo. The stater cultures are being inverstigated.