Molecular Characterization of Lactic Acid Bacteria Isolated from Starter Dough of Sudanese Sorghum Fermented Flat Bread (Kissra)

MATERIALS AND METHODS

Preparation of the fermented kissra dough: The fermented dough was obtained from the famous women of kissra makers in locality of Bahri, Khartoum State, Sudan. About 100 g of sorghum flour were mixed with 200 mL water in a round plastic container (Khumara). 25 g of previously fermented dough were added to the mixture to act as a starter culture. The dough was allowed to ferment for 3 h at room temperature.

Isolation of LAB: The fermented dough was prepared from sorghum as mentioned above. Ten mL fermented dough was sterilized diluted with saline, plated on De Man Rogosa Sharpe (MRS) agar (Himedia, Mumbai, India) and incubated anaerobically at 37°C for 24-48 h. The dominant colonies on MRS agar which were milky white, circular, convex, elevated and non-pigmented were chosen and further sub cultured. The colonies were streaked on MRS agar to check for purity. The pure cultures were overlaid with glycerol and preserved for further study Pal et al.¹⁸.

Classical characterization of LAB isolates: Growth was assayed in MRS broth at room temperature and gram staining test, catalase production, endospore staining test, motility test, oxidase test, oxidative and fermentative test (O∕F) and glucose (acid) test were carried out for the isolates as described by Harrigan¹⁹.

DNA isolation: Genomic DNA was isolated by the procedure as described by Moore et al.²⁰.

(RAPD) analysis: RAPD analysis was carried out using the primers R2 5’-GGCGACCACTAG 3’ and M13 5’ GAGGGTGGCGGTTCT-3’²¹. Maxime PCR PreMix Kit (i-Taq) for 20 μL rxn was used to achieve the PCR process with little modification of external addition of 2.5 μL of MgCl₂.

The PCR cocktails (20 μL) consisted of 1 μL of the primers, 2 μL of DNA, 2.5U i-Taq DNA polymerase, 2.5 mM of each dNTP, 1 X reaction buffer, 1X gel loading buffer and 2.5 μL of MgCl₂. Amplification conditions were initial denaturation at 94°C for 5 min, 40 cycles of 94°C for 1 min, annealing at 38°C for R2 and 40°C for M13 for 45 sec and elongation at 72°C for 1 min, followed by a final elongation at 72°C for 10 min²¹. The PCR products were visualized by running in 1.5% agarose gel electrophoresis with 100 bp DNA ladder (Sigma, Saint Louis, USA). The electrophoresis conditions were 100 V, 60 mA, for 20 min with 1X TBE as the running buffer.

Molecular characterization by 16S rRNA gene analysis: Amplification of 16S rRNA gene was performed from genomic DNA of the isolates using universal primers fD1 (5’-GAGTTTGATCCTGGCTCA-3’) and rP2 (5’-ACGGCTACC TTGTTACGACTT-3’)²², with some modification. PCR cocktails (20 μL) consisted of 1 μL of the primers, 2 μL of DNA, 2.5 U i-Taq DNA polymerase, 2.5 mM of each dNTP, 1X reaction buffer, 1X gel loading buffer and 2.5 μL of MgCl₂. Amplification conditions were initial denaturation at 94°C for 3 min, 20 cycles of 94°C for 30 sec, annealing at 48°C for 45 sec and elongation at 72°C for 1 min, followed by a final elongation at 72°C for 10 min.

Purified PCR products were sequenced with automated DNA sequencer with specific primers using the facility at Macrogen Inc (Macrogen Inc., Seoul, Korea). Phylogenetic analysis was performed for the isolates using MEGA 6.0 software²³.

Accession number: These sequence data have been submitted to the GenBank database (http://www.ncbi.nlm. nih.gov/gen bank/) under accession number KX430760 to KX430768.

RESULTS AND DISCUSSION

The present study deals with classical and molecular characterization of LAB isolates from fermented kissra dough. This study forms a broader objective to obtain a uniform consortium of strains having many beneficial properties as starter culture for commercial purposes.

Phenotypic characterization of LAB isolates: There were 75 lactobacilli isolates. The results of phenotypic characterization of LAB isolates tested are illustrated in Table 1. The results showed that, all isolates were Gram-positive, catalase negative, non-endospore-forming, non-motile and produced acid without production of gas from glucose. Among them, 89.3% (67 isolates) were rods, which occurred either singly or in pairs, when tested under microscope. These isolates were assigned to the genus Lactobacillus. While, 10.7% (8 isolates) were cocci which occurred in pairs or tetrads, 4% (3 isolates) of these cocci were identified as pediococcal strains. Also 6.7% (3 isolates) of these cocci exhibited a well-rounded cell morphology typical of the lactococcus when tested under microscope.

RAPD-PCR result: RAPD analysis was performed initially to cluster the isolates using two different primers R2 and M13. Group (1L): Isolates 3, 4 and 7 having similar pattern in the RAPD analysis belonged to a single group. Group (2L) and Group (4L) included isolates 5 and 9, respectively and having different patterns clustered into different groups, while Group (3L): Included isolates 8, 22, 27, 6, 48, 56, 58, 70, 72, 73, 26, 44, 51 and 36. Group (5L): Isolates 17 and 14 having similar patterns. Group (6L): The biggest one included isolates 20, 21, 24, 11, 12, 15, 55, 35, 10, 60, 18, 13, 61, 62, 37, 40, 38, 41, 32, 39, 63, 65, 68, 71, 50, 45, 74, 64, 1, 2, 19, 47, 67, 46, 53, 54, 57, 16, 49, 52, 23, 31 and 75 showed similarity. Group (7L): Isolates 25, 59, 33 and 69 having the same cluster. Group (8L): Isolates 30 and 34 belonged to same group and finally Group (9L): Isolates 42, 66, 43, 28 and 29 having the same cluster (Fig. 1a and b). Thus, nine different clusters were clearly observed based on the RAPD analysis.

16S rRNA result: The 16S rRNA was analyzed for the nine different clusters of isolates.

Table 1:	Phenotypic characterization of the isolated LAB from Sudanese kissra fermented dough

Fig. 1(a-b):	RAPD analysis using the primer R2 and M13, (a) Group 1L to 5L and (b) Group 6L to 9L
	M = 100 bp marker

The PCR products were sequenced and were subjected to nucleotide BLAST. 4% (Group 1L) showed 100% homology towards Pediococcus acidilactici and 6.7% (Group 9L) showed 100 homology towards Lactococcus lactis subsp. lactis strain SFL. Among the rest of the 67 lactobacillus isolates, 1.6% (Group 2L) showed 100% homology towards L. murinus, also same percentage (1.6%) (Group 4L) reported as L. casei strain IMAU70007. 2.9% (Group 5L) showed 100 homology towards L. plantarum strain KLAB4. The same percentage (2.9%) (Group 8L) showed 100% similarity towards L. fermentum. 5.9% (Group 7L) showed 100% homology towards L. casei strain SWU30436., 20.9% (Group 3L) showed 100% similarity towards L. plantarum strain 1.0557CGMCC, while the majority of the isolates 64.2% (Group 6L) showed 100% homology towards L. plantarum strain CSI7.

Multiple sequence alignment was carried out by BioEdit software and later phylogenetic analysis was performed using software MEGA 6.0. All the isolates were phylogenetically closely related to Lactobacillus plantarum and Lactococcus lactis (Fig. 2).

The population of indigenous LAB tend to dominate sour dough fermentations by the production of acid in the fermented dough²⁴. Majority of LAB isolated in this study were homofermenters. This is in agreement with other research workers who reported the predominance of obligately homofermentative LAB in fermented maize meal for the production of sour bread and homofermentative lactobacilli and Pediococcus spp. from the final sour dough for production of Swedish rye bread^25-27.

Lactobacillus fermentum, Lactobacillus plantarum and Lactococcus lactis subsp. lactis were isolated from household and laboratory prepared bushera (Ugandan traditional non-alcoholic fermented beverage)²⁸. Similarly, in Nigeria, Olasupo et al.²⁹, showed that lactic acid bacteria, notably L. lactis was isolated from a dairy product called “wara”.

The lactic acid bacteria identified in kissra have been reported in other fermented foods. L. plantarum has been isolated from the raw material, sorghum powder and also from corresponding fermented and cooked fermented samples³⁰. L. plantarum has been shown to be the dominant organism at the end of several natural cereal fermentations^31-33, as for instance in maize-derived products like ogi^34-37. L. plantarum has also been identified as the predominant species in most vegetable fermentations³⁸. The microbial composition of some African traditional fermented cereals such as “poto poto” (a maize dough from the Republic of Congo) and “de’gue” (a millet dough from Burkina Faso) has been shown by molecular techniques to include the presence of L. plantarum³⁹. In some West African countries, the production of “fufu” (fermented cassava product), “ogi” (fermented maize, sorghum, or millet gruel), “fura da nunu” (fresh cow’s milk with fermented millet gruel) and “pito” and “burukutu” (cereal-based alcoholic beverages) are largely brought about by lactic acid bacteria and yeast, with L. plantarum predominating³⁵. The dominant species L. plantarum frequently occurs (spontaneously) in high numbers in most lactic acid fermented foods, especially when the food is based on plant material, for example, in brined olives, makdous and fermented vegetables.

Fig. 2:	Phylogenetic tree with the 16S rRNA gene using the MEGA 6.0 program by neighbor-joining (NJ) method
	Out rooting done by E. coli

Thus, individuals consuming these products also consume a large numbers of L. plantarum together with L. casei, L. fermentum and pediococci, which were found in these products⁴⁰.

In another study, L. plantarum was the dominant lactic acid bacteria isolated in different batches of “pito” and “burukutu” collected from local producers in Nigeria⁴¹. The dominance of L. plantarum at the late stages of fermentation has been attributed to its high acid tolerance^34,32,38, Hounhouigan et al.⁴², El Mardi⁴³, found the bacteria in rob (Sudanese fermented milk) from the suburbs of Khartoum to be Lactobacillus fermentum and Lactococcus lactis. Abdelgadir et al.⁴⁴ found in a traditional Sudanese fermented camel's milk product, gariss, L. fermentum was the dominant spices. Among LAB, Lactobacillus plantarum are the species most widely described in acid-fermented meat products⁴⁵.

L. fermentum have been suggested to be the predominating microorganisms during the fermentation of fufu and ogi, two Nigerian foods⁴⁶, kenkey, a Ghananian fermented maize dough⁴⁷, mawe, a Benin fermented maize dough⁴² and agbelima, a Ghananian cassava dough⁴⁸. These species have also been reported to occur in fermented plant materials and sour dough^49,50, in sorghum beer⁵¹ and in togwa⁵².

CONCLUSION

The results of this study have indicated that several different species of lactic acid bacteria can be implicated in the fermentation of kissra. Therefore, there is a need for investigation into the selection of the most suitable strains for controlled fermentation of kissra. The starch fermenting strains might be important in the development of the starter cultures and for use in the development of small-scale commercial production of kissra.

ACKNOWLEDGMENTS

The authors are grateful to the Sudan University of Science and Technology (SUST) for providing technical facilities. The authors would also like to thank Mr. Taj eldein Mustafa, technician at the Department of Food Science and Technology, Faculty of Agriculture, University of Khartoum. Thanks are extended to technical staff at The Research Laboratory, College of Medical Laboratory Science, (SUST).