Introduction

Introduced to Africa by the Portuguese around the middle of the 15th century, cassava, its scientific name Manihot esculenta Crantz, is a plant of the Euphorbiaceae family with tuberous roots. It is a plant that is an integral part of the diet of more than half a billion human beings around the world with an estimated global production of 302.7 million tonnes during the year 2020 where Africa produces on average half of this production¹.

In Côte d’Ivoire, cassava occupies second place among food crops with 6.4 million tonnes after yam. It is a commodity used for the preparation of several local dishes, most of which require the use of a traditional leaven cassava. The preparation of most of these dishes requires the use of traditional sourdough or cassava ferment.  The preparation of most of these dishes requires the use of traditional sourdough or cassava ferment. For thousands of years, humans have used the fermentation process for food processing and preservation techniques. It is a process that is generally implemented to diversify different types of foods, make otherwise inedible foods edible, improve nutritional value and energy requirements, decrease toxicity, preserve foods, and reduce cooking times². The African continent has a wide variety of fermented foods that generally have a significant impact on the nutritional quality and socio-economic status of the population³. In Côte d’Ivoire, local food fermentation processes are spontaneous and the microorganisms involved are lactic acid bacteria, Bacillus, yeasts and molds⁴. These microorganisms not only reduce the growth of pathogens through the production of various organic compounds but also sometimes have probiotic potential.

Nowadays, studies are turning to the search for probiotics of microbial origin. These are mainly bacteria and yeasts present in certain fermented foods⁵. Their use, initially based on empirical observations, is currently more rational and supported by numerous studies⁶. Bacteria of the genera Lactobacillus and Bifidobacterium, qualified as « beneficial bacteria » and residents of the intestinal tract have been mainly studied and used⁷. Numerous studies show that these probiotic microorganisms are capable of interacting with the intestinal immune system leading to a regulatory immune response.

The concept of probiotic has been extended to the use of other strains absent from the endogenous flora of the intestinal tract. Among them, bacteria of the Bacillus genus capable of resisting in the form of spores to extreme pH conditions during passage through the stomach and possess antimicrobial activities against pathogens and immunomodulatory properties. The ingestion of probiotic Bacillus spores does not lead to the definitive establishment of the strain within the microbiota. Indeed, probiotics of the Bacillus genus are considered transient residents of the intestinal flora and their potential development within this environment depends on the nutritional conditions prevailing there. Among these bacteria of the Bacillus genus, those isolated during cassava fermentation have shown their ability to play a role in tissue degradation and detoxification of cassava⁸ and to produce a diversity of enzymes^9,10. However, in Côte d’Ivoire, their role has not yet been elucidated in terms of their probiotic activity.

In this context, many discussions have suggested the use of microbial starters as the best approach to improve not only the quality of cassava ferment and the resulting dishes but also the health of the consumer. It is in this context that this study was initiated in order to evaluate some probiotic activities of Bacillus strains involved in cassava fermentation.

Materials and Methods

Study material

The study material consists of six strains of Bacillus each with its code, namely Bacillus subtilis (E7-B4), B. toyonensis (AB2-3), B. pumilus (AB3-5), B. methylotrophicus (AB4-6), B. vallismortis (E3-B7) and B. amyloliquefaciens (E8-B2), isolated from traditional cassava ferments, identified genetically and coming from microbial heritage of the Laboratory of Biotechnology and Microbiology of Foods of NANGUI ABROGOUA University, Côte d’Ivoire.

Conservation and purification of Bacillus strains

Bacillus strains were stored at -80°C in Plate Count Agar (PCA) medium supplemented with 1% starch. The cultures were revived in brain heart broth then incubated at 37°C for 24 hours. After incubation, the strains were subcultured on Moselle agar then incubated at 37°C for 24 hours.

Growth of Bacillus strains at different acidic pH concentrations

The study of the growth of Bacillus strains at different pHs was carried out according to the method¹¹. Heart-brain broths were prepared at different acid pH (2; 3) and control pH (6.8) by adjustment with 1M HCl. The simple prepared medium served as a control. The medium was divided into different test tubes at the rate of 3 mL per tube. The sterile medium contained in each tube was inoculated at 1% v/v with a pre-culture of Bacillus seeded in brain heart broth at 37°C for 24 hours, the load of which was adjusted to 3.10⁸ CFU/mL. The seeded media were incubated at 37°C for 24 hours. At each time including 0 hour, 4 hours and 24 hours, a tube is removed and the optical density was read at 600 nm using a spectrophotometer (BK-UV1000).

Growth of Bacillus strains at different concentrations of bile salts

The growth of Bacillus strains at different concentrations of bile salts was carried out according to the method¹¹. Different quantities of Ox-bile were added to the different heart-brain broths, namely 3 g, 6 g, and 9 g to give the concentrations of 0.3%, 0.6% and 0.9% of bile salts respectively. Broth without Ox-bile served as a control. The medium was divided into different test tubes at the rate of 3 mL per tube and autoclaved at 121°C for 15 min. Each medium contained in a tube was inoculated at 1% v/v with a Bacillus pre-culture seeded in brain heart broth at 37°C for 24 hours and the load was adjusted to 3.10⁸ CFU/mL. Then, the media were incubated at 37°C for 24 hours. A tube is removed at each time (0 hour, 4 hours and 24 hours) and the optical density is read at 600 nm.

Resistance of Bacillus strains to antibiotics

Resistance of Bacillus strains to antibiotics was carried out using the disk diffusion method described by¹². First, a Bacillus pre-culture was carried out in brain heart broth and incubated at 37°C for 24 hours. For each strain, the optical density was read at 600 nm and adjusted to 0.2. Then, 100 µL of each of the strains were placed in empty Petri dishes. The Mueller Hinton medium, sterilized in an autoclave at 121°C for 15 min, was poured into the petri dishes containing the inoculum then homogenized. After solidification of the medium, discs of eight antibiotics from seven families of antibiotics in particular Beta-lactams (Penicillin : 10 µg and Amoxicillin : 30 µg), Carbapenems (Imipenem : 10 µg), Aminosides (Gentamycin : 10 µg), Quinolones (Ciprofloxacin : 5 µg), Rifamycin (Rifampicin : 30 µg) Glycopeptides (Vancomycin : 30 µg) and Phenicolates (Chloramphenicol : 30 µg) were placed on the previously prepared Petri dishes and then incubated at 37°C for 24 hours. After incubation, zones of inhibition observed around the discs were measured and the results were expressed as sensitive (S) where the inhibition diameter > 15 mm or as resistant (r) where the inhibition diameter ≤ 15 mm¹³. Bacterial strains of E. coli, S. aureus and Salmonella sp. have been used for antibiotic control.

Antimicrobial activities of Bacillus strains

This study was carried out according to the method described by¹⁴. Bacillus strains were cultured in brain heart broths and incubated at 37°C for 24 hours. After incubation, the Bacillus cultures were centrifuged at 6000 rpm at 4°C for 20 minutes. Then, 100 µL of the supernatant of each of the strains were deposited in 6 mm diameter wells dug under aseptic conditions with the tip of a Pasteur pipette on the nutrient agar. Then, the boxes were placed at 4°C for 2 hours to allow proper diffusion of the antimicrobial substance then incubated at 37°C for 24 hours. The results were read by measuring the diameter of the inhibition zones formed around the wells¹⁴. At this level, the result is positive when the inhibition diameter > 15 mm and lower when the inhibition diameter ≤ 15 mm.

Growth of Bacillus strains at different temperatures

The growth of Bacillus strains at different temperatures was carried out following the method¹¹. Different brain heart broths were prepared and then distributed in different test tubes at a rate of 3 mL per tube. After sterilization by autoclaving at 121°C for 15 minutes, the media contained in the test tubes were then inoculated at 1% (v/v) by a pre-culture of Bacillus strains seeded in brain heart broth at 37 °C for 24 hours and whose load was adjusted to 3.108 CFU/mL. The seeded media were incubated at different temperatures including 37°C and 45°C for 24 h. A tube is removed at each time (4 and 24 h) and the optical density is read at 600 nm using the spectrophotometer (BK-UV1000).

Determination of hemolytic activities of Bacillus strains

The Bacillus strains revivified on YPDA agar were tested for their hemolytic activity using blood agar supplemented after autoclaving at 121°C for 15 min with sterile sheep blood (7%, v/v) following the protocol described by¹⁵. Ten (10) μL of each Bacillus strains suspension were inoculated onto the surface of the culture medium per spot and then the medium was incubated at 37°C for 48 hours in an oven (BJPX-H64II, China). Positive activity is reflected by a zone of lysis around Bacillus strains colonies. The non-hemolytic reaction was recorded as one producing no effect on blood agar.

Ability of Bacillus strains to form a biofilm

The Bacillus strains were cultured in brain heart broths at 37°C for 24 hours and then the optical density was adjusted to 0.5. A volume of 1 mL of each culture was aseptically introduced into Eppendorfs tubes and then incubated at 37°C for 24 hours. Subsequently, the tubes were centrifuged at 4000 rpm for 10 minutes and the pellets were washed 3 times with NaCl solution (0.9%) then dried at 50°C for 30 minutes. Bacillus biofilms were stained with 1 mL of 0.1% crystal violet for 20 min and rinsed with NaCl solution (0.9%). The dye was then eluted with ethanol (96%) and the contents of the Eppendorfs tubes were transferred into reading tubes and quantified by measuring the absorbance at a wavelength of 595 nm. Thus, the capacity of a Bacillus strain to form a biofilm is considered positive for any the optical density ≥ 0.5¹⁶.

Statistical analysis

Statistical processing of the results was carried out using R software version 4.1.1. The means of the different parameters studied were compared using a one-way analysis of variance (ANOVA 1) associated with the Tukey’s Test at the threshold of 5%.

Results

 Growth capacity of Bacillus strains at different acidic pH

Table 1 presents the growth capacity of Bacillus strains at different acidic pH. The growth of Bacillus strains in an acidic environment was observed after 0 hours, 4 hours and 24 hours of incubation at pH 2, pH 3 and pH 6.8. Some strains of Bacillus show good growth. At 0 hour of incubation, all strains have practically the same growth at pH 2, pH 3 and pH 6.8. After 4 hours of incubation, strains AB2-3 and E7-B4 showed the strongest growth respectively (9±1.06) × 10⁷ CFU/mL and (7.29±1.40) × 10⁸ CFU/mL at pH 2. At pH 3, strain AB2-3 showed the strongest growth at 4 hours and 24 hours respectively (5.14±0.15) × 10⁸ CFU/mL and (1.14±0.22) × 10⁹ CFU/mL. On the other hand, at pH 2 and pH 3, strain E8-B2 showed the lowest growth at 4 hours and 24 hours of incubation. All strains have practically the same growth at pH 6.8 apart from the E8-B2 strain whose growth amounts to (2.20±0.2) × 10⁹ CFU/mL.

Growth capacity of Bacillus strains at different concentrations of bile salts

Bacillus strains showed good growth ability in the presence of different bile salt concentrations from 0.3% to 0.9% during all incubation hours. After 4 hours of incubation with the concentration of 0.3%, the results obtained showed significant differences (P<0.05) between all strains. Strain E8-B2 showed the highest growth at 0.3% (1.54±0.21) × 10⁹ CFU/mL bile salts. Also, strain AB2-3 had the strongest growth (3.15±0.70) × 10⁹ CFU/mL at 24 hours of incubation. As for strains E3-B7 and E7-B4, they showed stronger growth (1.69±0.70) × 10⁹ CFU/mL and (3.11± 0.63) × 10⁹ CFU/mL respectively at 4 hours and 24 hours of incubation at a concentration of 0.6%. A significant difference (P<0.05) was observed between the strains at the concentration of 0.9% bile salts. However, strains E8-B2 and E3-B7 showed similar growth (1.24±0.45) × 10⁹ CFU/mL at 4 hours. At 24 hours, strains E8-B2 and E3-B7 similarly showed the highest growth (2.11±0.9) × 10⁹ CFU/mL. However, no significant difference (P˃0.05) was observed between the strains at 0 hour, 4 hours and 24 hours at the control concentration (0%) in bile salts where all the strains had the same growth (Table 2).

Table 1: Growth of Bacillus strains at acidic pH 2, pH 3 and pH 6.8 at 0 hour, 4 hours and 24 hours of incubation

AB2-3 : Bacillus toyonensis ; AB3-5 : B. pumilus ; E7-B4 : B. subtilis ; AB4-6 : B. methylotrophicus ; E3-B7 : B. vallismortis ; E8-B2 : B. amyloliquefaciens ; Average values ​​of 3 repetitions ; In the same line, values ​​followed by the same alphabetical letter are not statistically different (P >0.05) (Tukey, HSD).

Table 2: Growth of Bacillus strains at different concentrations of bile salts at 0 hour, 4 hours and 24 hours

AB2-3 : Bacillus toyonensis ; AB3-5 : B. pumilus ; E7-B4 : B. subtilis ; AB4-6 : B. methylotrophicus ; E3-B7 : B. vallismortis ; E8-B2 : B. amyloliquefaciens ; Average values ​​of 3 repetitions ; In the same line, values ​​followed by the same alphabetical letter are not statistically different (P >0.05) (Tukey, HSD).

Resistance of Bacillus strains to antibiotics

The antibiotics that were used in this part of the study were Amoxicillin, Ciprofaxin, Imipenem, Gentamycin, Penicillin, Oxacillin and Rifampicin. All 6 Bacillus strains tested (B. toyonensis, B. pumilus, B. subtilis, B. methylotrophicus, B. vallismortis and B. amyloliquefaciens) demonstrated multi-resistance with regard to Ciprofaxin (100%). All strains showed their multi-sensitivity (100%) towards 7 antibiotics (Amoxicillin, Imipenem, Vancomycin, Gentamycin, Penicillin, Chloramphenicol and Rifampicin) except B. pumilus and B. methylotrophicus which were sensitive to Rifampicin (77%) and B. subtilis which was multi-resistant to Rifampicin (100%) (Table 3). The sensitive (A) and resistant (B) inhibition diameters of antibiotic disks against the Bacillus strains tested are presented in Fig. 1.

Table 3: Resistance of Bacillus strains to antibiotics

AB2-3 : Bacillus toyonensis ; AB3-5 : B. pumilus ; E7-B4 : B. subtilis ; AB4-6 : B. methylotrophicus ; E3-B7 : B. vallismortis  ; E8-B2 : B. amyloliquefaciens ; R : resistant ; S : sensitive ; Ciproflox : Ciprofaxin ; Rifampi : Rifampicin ; Amoxic : Amoxicillin ; Imipen : Imipenem ; Vanco : Vancomycin ; Chloram : Chloramphenicol ; Penicil : Penicillin ; Gentamy : Gentamycin.   

Figure 1: Petri dish photograph showing the sensitive (A) and resistant (B) inhibition diameters of antibiotic disks against Bacillus strains. Click here to view Figure

Antibacterial activities of Bacillus strains

The antibacterial activity of Bacillus species was evaluated on three pathogenic bacteria in particular E. coli, S. aureus and Salmonella sp. The vast majority of Bacillus species inhibited more than half of the pathogenic bacteria. The highest inhibition rate (96.7%) was obtained with B. amyloliquefaciens on S. aureus while the lowest rate (50.15%) was observed with B. subtilis on E. coli (Table 4). Pathogen inhibition rates are between 65.09% and 80.27%. The most inhibited pathogen was S. aureus (80.27%) and the least inhibited was E. coli (65.09%) (Fig. 2).

Table 4: Inhibition rate (%) of different Bacillus species of traditional cassava ferments

Figure 2: Rate of pathogens inhibited Click here to view Figure

Growth capacity of Bacillus strains at different temperatures

The growth capacity of Bacillus strains at different temperatures is presented in Table 5. Generally, all Bacillus strains showed growth at 37°C and at 44°C after 4 hours and 24 hours. However, growth at 44°C is lower than that at 37°C. After 4 hours of incubation time at a temperature of 37°C and 44°C, a significant difference (P<0.05) exists between the growth of the strains studied. At 37°C, the E8-B2 strain exhibited the highest growth (2.27±0.10) × 10⁸ CFU/mL at 4 hours while the E3-B7 strain also showed the highest growth (4.51±0.2) × 10⁸ CFU/mL at 24 hours. At 44°C, strain AB2-3 showed the highest growth (1.54±0.15) × 10⁸ CFU/mL at 4 hours while strain E3-B7 showed high growth (2.23±0.20) × 10⁸ CFU/mL at 24 hours.

Table 5: Growth of Bacillus strains at different temperatures (37°c and 44°c) for 4 hours and 24 hours

AB2-3 : Bacillus toyonensis ; AB3-5 : B. pumilus ; E7B4 : B. subtilis ; AB4-6 : B. methylotrophicus ; E3B7 : B. vallismortis ; E8B2 : B. Amyloliquefaciens ; Average values ​​of 3 repetitions ; In the same line, values ​​followed by the same alphabetical letter are not statistically different (P >0.05) (Tukey, HSD).

Hemolytic activities of Bacillus strains

Hemolytic activities of Bacillus strains were highlighted. Of all the six Bacillus strains tested, none of them secrete enzymes with hemolytic activities into their production environments (Table 6). The illustration reflecting the absence of hemolytic activity is shown in Fig. 3.

Table 6: Production of hemolytic enzymes by Bacillus strains

Figure 3: Photograph reflecting the Absence of Hemolytic Activity Click here to view Figure

AHA: No Lysis Zone around colonies

Ability of Bacillus strains to form a biofilm

The ability of Bacillus strains to form a biofilm is presented in Fig. 4. In general, all Bacillus strains studied showed a good ability to form a biofilm through the optical density values. The different optical density values ​​being between 0.663±00 nm for the strain AB4-6 (B. methylotrophicus) and 3.15±02 nm for the strain E3-B7 (B. vallismortis) greater than 0.5, which thus shows the ability of all Bacillus strains tested to form a biofilm.

Figure 4: Ability of Bacillus species to form biofilm Click here to view Figure

Discussion 

The observed growth of Bacillus strains tested at acidic pH (pH 2 and pH 3) could allow them to pass the gastrointestinal barrier governed by an acidic pH of between 2 and 3. Probiotic strains should therefore survive and grow at these pH. The ability to survive stomach acid varies greatly between genera and species.

All strains tested showed good growth at pH 2 and pH 3 during the different times studied. But at pH 2, B. toyonensis and B. subtilis showed the highest growth. Our results are in agreement with those¹⁷ who reported good resistance of Bacillus strains to acidic. Furthermore^18,19 have reported the tolerance of vegetative Bacillus cells to acidic conditions. These results could be explained by the fact that bacterial spores are dormant multilayer cellular structures resulting from sporulation which occurs when certain bacteria are placed in an unfavorable environment. Furthermore, these results could also be explained by the production of bacterial enzymes and metabolites produced by Bacillus strains. Indeed, according²⁰, the production of metabolites including organic acids, sideerophores and enzymes such as ACC deaminase by Bacillus allows them to tolerate acidic and stressful conditions.

The tolerance of bacteria to bile salt concentrations is a criterion for which condition their survival in the conditions of the Gastro-Intestinal tract and to colonize the intestinal environment. All Bacillus strains studied were resistant to bile salt concentrations. Our results corroborate those of^21,22 who reported the survival of Bacillus strains at bile concentrations and to adapt to them in a stable manner.

This study also showed that most of the Bacillus strains tested are multi-resistant to the antibiotic Ciprofloxacin and the same as B. subtilis which is multi-resistant to Rifampicin. These results are consistent with those of the work of^23,24 which reported the resistance of Bacillus strains to the antibiotics ciprofloxacin and rifampicin. These results could be due to certain proteins, notably bacitracin, synthesized by Bacillus strains. Indeed²¹, reported that Bacillus strains produce the substance bacitracin for their natural resistance to antibiotics. According to these same authors, this resistance of Bacillus strains to antibiotics occurs either through the specific transport protein which takes bacitracin out of the cell or through an undecaprenol kinase. The resistance of Bacillus strains to antibiotics could also be explained by intrinsic or acquired factors. Indeed, according to^25,26, the resistance of Bacillus strains to antibiotics occurs either by the transfer of a gene from a plasmid or by the mutation of a bacterial gene. Furthermore, the majority of Bacillus strains studied were sensitive to most of the antibiotics studied. These results corroborate those of^27,28,29 who reported that several strains of Bacillus expressed no resistance to several antibiotics including Vancomycin, Gentamicin, Chloramphenicol, Imipenem and Penicillin. This sensitivity of different Bacillus strains to antibiotics could be due to their intrinsic gene as reported by²⁹.

The Bacillus strains tested had an inhibitory power on E. coli, S. aureus and Salmonella. Our results corroborate those of^30,31 who reported the inhibition of E. coli, S. aureus and Salmonella bacteria by several Bacillus species. Similar observations on the antibacterial activity of Bacillus species have been reported on the inhibition of E. coli and S. aureus by B. vallismortis³². These results could be due to various antibacterial agents produced by the Bacillus strains. Indeed, according to³³, several strains of Bacillus produce a wide range of antibacterial agents including peptide and lipopeptide compounds which have an inhibitory effect on Gram-positive and Gram-negative pathogenic bacteria. These results could also be explained by the competitive exclusion mechanism. Indeed, according to^34,35, when probiotics compete with pathogenic bacteria on adhesive and nutrient receptors, they can effectively destroy them with various antimicrobial agents including bacteriocins, hydrogen peroxides, organic acids, peptide and lipopeptide compounds that they secrete. Furthermore³⁶, indicated that B. subtilis produces extracellular compounds including proteases and bacteriocins involved in antagonistic activities against pathogenic bacteria. The inhibition of several pathogenic bacteria of food origin including E. coli, S. aureus and Salmonella by bacteriocins has been reported^37,38.

In this study, none of the six Bacillus strains studied showed hemolytic activity in the blood after 48 hours of incubation, although all were able to grow. Non-hemolytic strains are considered safe for their hosts while hemolytic strains are considered pathogenic. Our results corroborate those of^39,40 who reported the non-hemolytic activity of several Bacillus species including B. amyloliquefaciens, B. pumilus, B. subtilis and B. amyloliquefaciens. These results could be explained by the absence of hemolytic agents which are responsible for the secretion and release of hemoglobin as indicated by^41,42. According to these same authors, the hemolytic activity is due to the positive regulation of the transcription of the zrt1 gene which codes for a zinc/iron permease, which suggests that iron can be acquired from binding to a host molecule such heme which is used to transport blood gases to cause hemolysis. These results could also be explained by the fact that the expression of hemolysin, which is an inherent factor, is triggered under specific conditions⁴³. Microorganisms exhibiting hemolytic activity can disrupt red blood cells resulting in the release of hemoglobin. This activity is often associated with the production of hemolysins which can have cytolytic effects on host cells and with the reduction in the hemoglobin content available as a source of Iron⁴⁴. In Bacillus, hemolytic activity is known to be a virulence factor contributing to pathogenicity and therefore poses a risk to the health of the host.

All Bacillus strains tested during this work showed good growth at temperatures of 37°C and 44°C, knowing that 30°C is their optimal growth temperature. The results corroborate those of⁴⁵ who reported the growth of Bacillus strains at 37°C. Studies carried out by⁴⁶ showed that probiotic Bacillus spores could germinate in the intestine, particularly under the extremely high conditions of temperature, pH and bile salts favoring their development in the tract in order to exert its beneficial effects. Furthermore⁴⁷, reported the growth of Bacillus strains at 45°C very close to the temperature studied (44°C).

The ability of potentially probiotic Bacillus species to form biofilm remains an important property given, which provides an ability rapid assimilation and metabolism. In our study, all Bacillus species tested showed an ability to form a biofilm. The highest biofilm aggregates were seen with B. toyonensis, B. subtilis and B. vallismortis thus confirming the work of^48,49 who reported biofilm formation by several Bacillus species. These results would be due to the synthesis of extracellular matrices of Bacillus species which maintain all the constituent cells. Indeed, according to^50,51, the EPS and TasA operon genes which are the two essential components of the B. subtilis biofilm matrix contribute to the establishment of a stable biofilm. Furthermore, these same authors also reported that in B. subtilis, the extracellular matrix includes components including exopolysaccharide synthesized by the products of the epsA-O operon, amyloid-type fibers encoded by tasA and γ-poly-DL-glutamic acid which can promote the formation of submerged biofilms.

Conclusion

At the end of this study, we can conclude that the different strains of Bacillus, namely B. amyloliquefaciens, B. subtilis, B. methylotrophicus, B. toyonensis, B. pumilus and B. vallismortis are resistant to acidic pH (pH 2 and pH 3) with higher growth observed in B. toyonensis and B. subtilis. In addition, all strains showed good growth at 44°C and 37°C, knowing that their optimal growth temperature is 30°C. All these strains also showed good growth in the presence of bile salts during all hours of incubation (0 hour, 4 hours and 24 hours). All Bacillus strains studied demonstrated multi-resistance to Ciprofaxine and were sensitive to Amoxicillin, Imipenem, Gentamycin, Penicillin, Oxacillin and Rifampicin. The vast majority of Bacillus strains inhibited more than half inhibited Salmonella and E. coli. No strain of Bacillus studied showed hemolytic activity and these strains showed a good capacity to form a biofilm.

Acknowledgment

All authors would like to thank the producers of traditional cassava ferments and without forgetting the managers of the Biotechnology and Microbiology Laboratory at Nangui Abrogoua University

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest 

Data Availability

Data used to support the findings of this study are included in the article 

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

This work was carried out with the collaboration of all the following authors.

Authors Zamblé Bi Irié Abel Boli and Yao Serge Junior N’goran carried out the literature searches, drafted the experimental protocol and participated in writing the final manuscript.

Author Abodjo Celah Kakou designed and validated the study.

Author Kouassi Roselin Cyrille Goly processed the formal analysis and software.

Authors Rose Koffi-Nevry and Marina Koussemon supervised the study and corrected the final manuscript. All authors read and approved the final manuscript.

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