Probiotic profiling of Leuconostoc species isolated from a traditional fermented cassava product

The various properties of lactic acid bacteria have made them good auxiliary in the manufacturing process in agro food industries and farms. They are widely used as probiotics which can be defined as living microorganisms that have beneficial effects on human health probiotics could replace antibiotic growth promoters in livestock without creating new threats such as that observed with antibiotics. Before their use as probiotics lactic acid bacteria require a perfect knowledge in view to their biochemical and genetic characteristics because it is difficult to differentiate morphologically some Leuconostoc and Lactobacillus strains using morphological characteristics. This study was undertaken in order to evaluate the probiotics potential of Leuconostoc strains isolated from traditional fermented cassava. The results showed that 5 strains of Leuconostoc have antibacterial activity against Staphylococcus aureus (MetiR), Klebsiella pneumoniae (BLSE), Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. The molecular identification of species using the conserved region of the 16S rRNA helped distinguish the species Leuconostoc mesenteroides. All these results showed that the studied Leuconostoc strains could be used as potential probiotics for the biopreservation of various foods.


INTRODUCTION
Probiotics are defined as living microorganisms that have beneficial effects on human health (FAO/ WHO, 2002).Indeed, several studies have demonstrated that probiotics may enhance growth performance, immunity and disease resistance (Saxelin et al., 2005;Ezendam and van Loveren, 2006;Ström-Bestor and Wiklund, 2011).Despite the numerous definitions, the criteria to select probiotic strains are total safety for the host, resistance to gastric acidity and pancreatic secretions, adhesion to epithelial cells, antimicrobial activity, inhibition of adhesion of pathogenic bacteria, evaluation of resistance to antibiotics, tolerance to food additives and stability in the food matrix (Soccol et al., 2010).In addition, the functional properties of probiotics include hypocholesterolemic activity by lowering plasma cholesterol, preventing and treating diarrhoea (Liong and Shah, 2005).The mechanisms by which probiotics exert their beneficial effects on the host include the reduction of luminal pH, competition with pathogens for adhesion sites and nutritional sources, secretion of antimicrobial substances, toxin inactivation, and immune stimulation (Salminen et al., 2004).The most commonly used probiotics are the strains of lactic acid bacteria such as Lactobacillus, Bifidobacterium and Streptococcus (Streptococcus thermophilus); the first two are known to resist gastric acid, bile salts and pancreatic enzymes, to adhere to colonic mucosa and readily colonize the intestinal tract (Fioramonti et al., 2003).
The use of selected probiotics from alternative sources known as "unconventional sources" is likely to increase.These unconventional sources include non-intestinal sources and non-dairy fermented food products, such as traditional fermented foods, traditional fermented drinks, vegetables, and fruit juices (Ramirez-Chavarin et al., 2013;Siddiqee et al., 2013).In Côte d'Ivoire, cassava (Manihot esculenta) root is traditionally fermented into a traditional microbial starter called "mangnan" that is used to prepare a really appreciated food called "attiéké" defined as fermented and steamed semolina cassava (Assanvo et al., 2006;Dje et al., 2008).Studies on this traditional microbial starter showed that the dominant microflora consists of lactic acid bacteria (LAB) (Assanvo et al., 2006).LAB, a group of Gram-positive, non-spore forming, non-motile microorganisms can produce inhibitory compounds such as lactic acid, bacteriocin and hydrogen peroxide preventing the growth of harmful microorganisms.The role of LAB in improving the shelf life and nutritional quality of fermented foods and beverages, controlling diarrhea, as well as their antimicrobial properties have also been established.However, despite an increasing interest in LAB, there is a paucity of literature regarding novel and emerging uses of LAB as probiotics, especially from traditional African fermented foods.Thus, the objective of the current study was to characterize the potential probiotic properties of Leuconostoc species isolated in previous studies (Coulibaly et al., 2016) from traditional fermented cassava.

LAB and indicator strains
The microbial strains used in this study are shown in Table 1.Five (05) LAB belonging to Leuconostoc genus, previously isolated from traditional fermented cassava (Coulibaly et al., 2016)

Antimicrobial activity determination
The method of agar spots as described by Larpent-Gourgaud et al. (1997) was used to evaluate the antimicrobial activity of the selected LAB strains.For this Petri dish, MRS agar was spotted with a 24-h colony of the LAB strain.The plates were seeded at 37°C for 24 h.At the same time, the indicator strains were subcultured in BCC broth for 3 h and then isolated on selective agar and incubated at their optimum growth temperature for 18 h.Each indicator strain was suspended in 2 ml of 0.85% NaCl and then vortexed.The OD was adjusted to 2.5 Mc Farland.Then, the inoculum was obtained by mixing 1 ml of inoculum of each strain in 9 ml of physiological water.The boxes with the spots were inoculated by flooding and the dishes were observed after 24 h of incubation at 37°C.The size of the zones of inhibition produced was measured.

Morphological, physiological and biochemical identification
The Gram characteristics of the isolates were determined using light microscope (Leica DM 1000, France) following staining.LAB are known to be Gram-positive.Cultures were grown in appropriate MRS media at 37°C for 48 h under anaerobic conditions.Cells from fresh cultures were used for Gram staining.
The determination of fermentation profiles (heterofermentative or homofermentative) was performed by inoculating 10 ml MRS broth containing bell Durhams at 28°C for 24 h (Harir et al., 2009).
For the fermentation of sugars, MRS media broth without glucose and supplemented with bromocresol purple as indicator was used (Mannu et al., 2000).For this, 9 ml of the medium is left in test tubes and sterilized for15 min.One milliliter of the sugar solution (10%, p/v) was aseptically added after filtration on a membrane with 0.45 µm porosity.Incubation was performed at 28°C for 48 h.Tested sugars were arabinose, sucrose, fructose, trehalose and esculin (Garvie, 1983).
The method described by Leveau et al. (1991) was used to determine the dihydrolase arginine (ADH), while the ability to grow at 10, 37 and 44°C was determined after incubation of inoculated Petri dishes at these temperatures.

DNA extraction and 16s rDNA amplification
A colony from culture was resuspended in 300 µl pure water in 1.5 ml Eppendorf tubes.DNA isolation and purification were realized using Instagen Matrix kit (Bio-Rad, USA) according to the manufacturer's instructions.PCR targeting the 16s rDNA of LAB were done as described by Bayane et al. (2006).The amplification reactions of 16S rDNA region were realized in a final volume of 50 µl containing 1X Master Mix (5 PRIME HotMasterMix, 5PRIME), 1 µl of DNA template (approximately 50 ng), 1 µM of each primer (Table 2) and PCR grade water.
The amplification was carried out in a 2720 ABI thermalcycler (Applied Biosystems, Syngapore).PCR conditions started with an initial denaturation at 94°C for 2 min; 36 cycles consisting of 1 min denaturation at 94°C for 30 s, annealing at 58°C and elongation at 65°C for 2 min.Then, a final extension for 7 min at 65°C ended the PCR reaction.PCR products were revealed in 1% agarose gel electrophoresis containing ethidium bromide (1 µg/ml).

Sequencing of the amplified DNA and data analysis
PCR products were sent to a Company (GATC BIOTECH, GERMANY) for sequencing.Sequences obtained were compared to those listed in Genbank (National Center for Biotechnology Information) using the nucleotid BLAST 2.5.0 tool (Zheng et al., 2000;Aleksandr et al., 2008).Similarity percentages were determined between the isolated sequences in this study and the closest sequences listed in GenBank.Sequences were considered similar when they have at least 99% percentage of similarity.Phylogenetic constructions were done after re-alignment of the sequences using MEGA 7.0.14(Kumar et al., 2016).The maximum likelihood and UPGMA algorithms (USA) were chosen for trees construction.

Antibacterial activity
Among the nine LAB strains tested for antibacterial activity in solid medium, only 5 strains (BL1, BL7, BL39, BL44, and BL61) showed good activity against all the indicator strains.The inhibition diameters measured are superior or equals to those of reference strains (L.plantarum, E. faecium, and W. confusa) (Table 3).
Inhibition diameter is denoted positive when greater than 8 mm (Schillinger et al., 2001).BL39, BL44 and BL61 strains inhibited the growth of all indicators Gram positive strains Staphylococcus hemolyticus MetiR and Staphylococcus aureus ATCC 25923.
Bacteria of the genus Leuconostoc, in association with the mesophilic LAB are capable of inhibiting the growth of pathogenic microorganisms such as S. aureus, Klebsiella pneumoniae BLSE, Escherichia coli, Salmonella Typhimurium and reference test strains, E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.Indeed, Todorov and Dicks (2005) have shown the growth inhibitory of E. coli and P. aerugionosa by a product of Leuconostoc mesenteroides subsp.mesenteroides.The antagonistic action of LAB against pathogens such as Salmonella, S. aureus, and E. coli was also confirmed by studies of Makras et al. (2006).
The diameters of inhibition of the indicator strains S. aureus ATCC 25923, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were higher than those of the three reference strains.The strain BL44 showed a strong  et al. (2005).
The antagonist effect as shown is this study is related to the biosynthesis of inhibitor compounds observed in strains of lactic bacteria (Servin, 2004).Indeed, organic acids are able to acidify the cytoplasm after dissociation and inhibit the cellular enzymatic activity of acid-sensitive pathogens (Tou et al., 2006;Djéni et al., 2008).This decrease in pH can therefore affect the viability of pathogenic bacteria (Bruno et al., 2002;Servin, 2004).This inhibition effect may be related to a competition with nutrients.An increase in the number of LAB obtained during a probiotic treatment would make it possible to reduce the substrates available for the implantation of pathogenic microorganisms (Fooks and Gibson, 2002).This justify the fact that all bacteriocins produced by LAB antimicrobial activity against Gram + (Dortu and Philippe, 2009).
Leuconostoc producing bacteriocin can be found in different food products including meat, cereals and milk (Hastings et al., 1994;Wulijideligen et al., 2012).

Morphological, biochemical and physiological characteristics
Morphological, biochemical and physiological examinations of the strains with antibacterial activity are shown in Table 4. Bacterial cells were spherical grouped into short chains and diplococcic.All strains were heterofermentative after 24 h.None of strains possessed hydrolase arginine.

Phylogenetic analysis and identification
The PCR targeting the 16 rDNA showed that all the strains that have demonstrated antibacterial activity belong to the lactic bacteria family.Positive samples detected highlighted a 1500 bp PCR product on agarose gel (Figure 1).

Sequences analysis and phylogeny
The 5 Leuconostoc PCR 16 S rDNA products were sequenced.The BLASTn's results confirmed that all the 5 isolates BL1, BL7, BL39, BL44 and BL 61 were genetically related to L. mesenteroides with 99% identity.The multiple sequence alignment of the Ivoirian Leuconostoc strains with other isolated strains from other region of the world show that all the 5 Ivoirian strains presented identical sequence.The genetic tree analysis showed clearly that those strains form a new genetic group related to L. mesenteroides on the basis of Blast results but are genetically distant (Figure 2).The 16S rRNA gene sequences of BL1, BL7, Bl39, BL44, and BL61 where deposited in GenBank nucleic acid sequence database under accession number KM518656 to KM518660.
The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Zheng et al., 2000).The tree with the highest log likelihood (-2249.2146) is as shown in Figure 2. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.The tree is drawn to scale, with branch lengths measured in the number of substitutions  per site (next to the branches).The analysis involved 22 nucleotide sequences.Codon positions included were 1st+2nd+3rd+Noncoding.There were a total of 515 positions in the final dataset.Evolutionary analyses were conducted in MEGA7 (Aleksandr et al., 2008).

Conclusion
In conclusion, it can be stated that strains of LAB that have antibacterial properties belong to L. mesenteroides.In view to their strong inhibitory properties on P. aeruginosa and on methicillin-resistant S. aureus, these strains could be used as biopreservatives of various foods.Furthermore, a better knowledge of other criteria for selecting the probiotics associated with technological properties would make them potential candidates for the formulation of ready to use probiotics

CONFLICT OF INTERESTS
The author(s) have not declared any conflict of interest.

Figure 2 .
Figure 2. Molecular phylogenetic analysis by Maximum Likelihood method of Leuconostoc strains isolated from traditional fermented Cassava.

Table 1 .
LAB and indicators strains.

Table 2 .
Features of primers used for PCR.

Table 3 .
Diameter of inhibition (mm) of indicators strains.

Table 4 .
Biochemical characteristics of the selected Leuconostoc strains.
inhibition (60 mm) of growth of the P. aeruginosa ATCC 27853.All strains diameters inhibition of E. coli and Staphylococcus were similar to those obtained by Labiou