Numeration and identification of thermotolerant endospore-forming Bacillus from two fermented condiments Bikalga and Soumbala.
Bikalga and Soumbala are condiment products of traditional uncontrolled alkaline fermentation of Hibiscus sabdariffa and Parkia biglobosa seeds respectively. These food additives are used and produced as major condiments in many African countries including Burkina Faso, Cameroon, Mali, Niger, Senegal, Sierra Leone and Sudan among others. Bikalga and Soumbala are produced by women and constitute economical resource. Soumbala is known under different appellations depending of the country: dawadawa or iru in Nigeria and North of Ghana [26,37,11], netetou in Senegal , afitin in Benin , kinda (Sierra Leone), natto in Japan and kinema in Nepal [5,38]. Bikalga is also called Dawadawa botso in Niger , Datou in Mali , Furundu in Sudan , Mbuja in Cameroon . They are excellent sources of proteins with essential amino acids also containing lipids, carbohydrates, essential fatty acids and vitamins [6,33,40]. Many families in west Africa often used Soumbala and Bikalga as low-cost meat substitute. These condiments improve nutritional values of foods as well as sensory properties as taste enhancer; contain antioxidant and neutraceuticals that provide health.
Several works were dedicated to the characterization of the microflora of these fermented products [11,4,32,17,21,29]. These studies demonstrated the prevalence of Bacillus species (B. subtilis, B. coagulans, B. amyloliquefaciens, B. pumilus, B. cereus, B. thuringiensis, B. brevis and B. licheniformis) in the fermentation process and their role in the bioconversion of the products. Ouoba et al.  in their recent studies sowhed that Bacillus subtilis, Bacillus licheniformis, B. pumilus, B. cereus, B. badius, B.sphaericus and B. fusiformis are microorganisms present and involved in Bikalga fermentation. Parkouda et al.  were showed that these condiments contained several Bacillus strains which play a key role during fermentation for the manufacture of the product. Micro-organims involves or responsible for the fermentation of Soumbala have been identified as Bacillus spp. with B. subtilis as the predominant species [26,2,25,12,24].
The initial boiling of Parkii biglobosa and Hibiscus sabdariffa seeds during their traditional preparation process of these two Condiments may be way of thermotorant microorganisms selection and destroyed non-Bacillus species; this could explain the dominance of Bacillus species in Soumbala and Bikalga. However there is no information or few data on numeration of the thermotorant flora of these two condiments well consumed in Burkina Faso. This work deals with numeration and identification of thermotorant bacteria from Soumbala and Bikalga.
Materials and methods
Collection of Samples:
Bikalga and Soumbala samples were bought from various small markets (Table1) of Ouagadougou and Gaoua. Samples were transported at the microbiology laboratory immediately after collection and analysed or stored under refrigeration until analysis.
10 g of each sample was placed in a sterile stomacher bag containing 90 ml of peptone saline water and subsequent serial dilution up to [10.sup.-10] of each sample was made. Before analysis each sample (10 g plus 90ml peptone saline water) was heated at 90[degrees]C, 100[degrees]C, 105[degrees]C for 15 minutes. 0.2 ml of each appropriate dilution was inoculated triplicate in Plate Count Agar and incubated at 37[degrees]C for 24 H for viable colonies count. After incubation developed colonies on agar were counted for each sample.
Micro-organisms Primary Characterization:
To confirm that the isolated colonies are belonging to Bacillus species different tests were made. Sixten bacteria colonies from Soumbala and Bikalga were selected (5 from Soumbala and 11 from Bikalga), isolated and characterized according to methods described by Harrigan and McCance , ICSMF ; Collins and Lyne . After colony counting different colonies were picked at random from petri dish and identified by cell morphology and motility, gram reaction, glucose metabolism , lecithinase, VP (Voges-Proskauer test), urease, catalase, starch hydrolysis, casein hydrolysis, sporulation, NaCl.
Selected Strains Characterization by PCR using Bacillus genus Specific Primer:
DNA Extraction and Preparation:
5ml of an overnight culture were used, cells were harvested by centrifugation and washed twice with Nacl 0.9%. Isolation of DNA was conducted with PROMEGA kit and protocol technics (Promega Corporation, Madison, WI 53711-5399 USA).
PCR Reaction and Electrophoresis:
Primers (B--K1/R: 5'-TCACCAAGGCRACGATGCG-3' and B-K1/F: 5'-CGTATTCACCGCGGCATG-3') were used for the identification of Bacillus strains . PCR mixture consisted of 1.5 [micro]l of each primer (20 [micro]M), 12.5 [micro]l (1.2 [micro]M) of Master Mix (Fermentas GMBH, St-Leon, Rot, Germany), 8 [micro]l of [H.sub.2]O, 1.5 [micro]l of DNA (10-30 ng. [micro][l.sup.-1]) in a final volume of 25 [micro]l. Thermal cycling was carried out using a Eppendorf AG (Hamburg, German) Mastercycler gradient as follows: initial denaturation at 94[degrees]C for 3 min followed by 30 cycles of denaturation at 94[degrees]C for 1min, primer annealing at 43[degrees]C for 30 s primer extension at 72[degrees]C for 45 s and final extension at 72[degrees]C for 10min. The expected fragment size is 1114bp. 10[micro]l of the amplified products of PCR were analysed by electrophoresis in 1% (w/v) agarose gels stained with ethidium bromide (0.5 mg [m.sup.-1]). The gels were visualised with an Ultraviolet Illuminator and Digitally recorder (GelDOC Bio-Rad, Hercule, USA).
Dermination of Proteolytic Activity:
Proteolytic activity of the isolates was determined using skim milk agar. The culture supernatant were spotted on the surface of the skim milk agar plates and incubated at 37[degrees]C for 24h. The development of a clear zone was considered as proteolytic activity .
For determination of amylolytic activity starch agar was used and incubated at 37[degrees]C for 48h. Enzymatic activity was indicated as clearing zones on the plates. For observation of amylolytic activity the agar plates were flooded with iodine solution.
Results and Discussion
The count of thermotolerant bacteria in Soumbala (Table 2) at 90[degrees]C, 95[degrees]C, 100[degrees]C and 105[degrees]C ranged from 1.35 x [10.sup.9] to 3 x [10.sup.9] cfu/g, 1.24 x [10.sup.9] to 2.78 x [10.sup.9] cfu/g, 0.54 x [10.sup.9] to 2.27 x [10.sup.9] cfu/g, 3.5 x [10.sup.7] to 1.54 x [10.sup.9] cfu/g respectively. Thermotolerant bacteria in Bikalga (Table 3) at 90[degrees]C, 95[degrees]C, 100[degrees]C and 105[degrees]C ranged from 1.9x[10.sup.7] to 12.8x [10.sup.7] cfu/g, 2x[10.sup.6] to 11.2 x [10.sup.7] cfu/g, 0 to 9.6 x [10.sup.7] cfu/g, 0 to 7.6 x [10.sup.7] cfu/g. These results indicated that Soumaba contained more themotolerant bacteria than Bikalaga. In all samples samples of Bikalga and Soumabala we numbered thermotolerant bacteria at 90[degrees]C, 95[degrees]C, 100[degrees]C and 105[degrees]C, except in of Bikalga samples B2 at 10[degrees]5C and B3 at 100[degrees]C and 105[degrees]C we didn't numbered no themotolerant . This analysis of heat treatment microbial flora in Bikalga and Soumbala revealed the occurring of both Bacillus genera. Colonies morphology on nutrient agar clearly indicates the presence of more than one type of isolate, confirming thus the diversity of flora.
-Primary Characterization, Proteolytic and Amylolytic Activities:
Five (5) representative strains from Soumbala and eleven (11) representative strains from Bikalga were selected for morphological and biochemical characterization (Table 4). All selected strains are Gram-positive, endospore-forming catalase positive; these selected strains are identified according to Biochemical, morphology and PCR characteristics (Table 4): five (5) are Bacillus coagulans, four (4) are Bacillus cereus, three (3) are Bacillus pumilis, two (2) are Bacillus firmus, one (1) is Bacillus subtilis and one (1) is Bacillus licheniformis. Bacillus subtilis, Bacillus cereus, Bacillus pumilus were identified in Soumbala and Bacillus licheniformis, Bacillus cereus, Bacillus pumilus, Bacillus coagulans, Bacillus firmus were identified in Bikalga (Table 4). Further examination (Table 4) showed that the selected strains hydrolysed casein (15 strains) and starch (6 strains), grew in medium with 5 % to 8% NaCl (10 strains for 5% NaCl, 6 strains for 6% NaCl, 5 strains for 7% NaCl and 3 strains for 8% NaCl). The heating has destroyed the non-Bacillus species and others microorganisms in volved in Soumbala and Bikalga process. Ouoba et al.  reported extra and intracellular protease enzymes pronounced activity in Bacillus species. Bacillus counts in soybean dawadawa by Ogbadu and Okagbue , Opai-Tetteh  and Omafuvbe et al. (2000) were estimated in order of [10.sup.10] to [10.sup.11] cfu/g.
The amplification with PCR using Bacillus genus primers gave expected fragments 1114 bp for the sixteen selected strains (Figure 1). These results confirm that the selected strains are effectively belonging to the Bacillus genus.
The count of bacteria was dominated by Grampositive, catalase-positive, rods, endospore-forming all these characteristics are those of Bacillus group , this fact is due to heating during the numeration at 90[degrees]C ,95[degrees]C, 100[degrees]C and 105[degrees]C for 15 minutes. The initial boiling of Parkii biglobosa and Hibiscus sabdariffa seeds during their traditional preparation process of these two condiments and the heating during the numeration of microorganisms in our samples may be way of thermotorant microorganism selection and destroyed non-Bacillus species; this could explain the dominance of Bacillus species in Soumbala and Bikalga. Also The pH of these two condiments are alkaline this fact can explain why we get otptimum spore heat resistance and high number of Bacillus counted.
Among Sixteen (16) selected strains 15 have proteolytic activity this result indicate that Bikalga and Soumbala are rich in proteolytic Bacillus spp.
[FIGURE 1 OMITTED]
Bacillus spp. was found to be dominant in iru and sonru  in dawadawa  in Soumbala [12,5,33,31] in Meju and others fermented soybean foods [19,9,20,18]. Lipolysis and proteolysis are important processus observed in fermented condiments [1,33]. Bacillus subtilis was encountered and used as starter culture for production of locust bean daddawa [16,27].
B. subtilis was encountered and used as starter culture for production of locust bean daddawa [16,27].
Proteins degradation in fermented condiment contributes to the development of the texture, flour and organoleptic quality. Lipolysis and proteolysis are important processus observed in fermented condiments [1,39,33]. Proteolytic activity of Bacillus can lead to the liberation of bioactive compounds which could give somes interesting role and characteristics of fermented condiments. The production of several proteolytic enzymes enhance the growth of Bacillus species in fermented food . The hydrolysis of protein during Bikalga and Soumbala fermentation leads to the alkalinity due to the protease and deaminase enzymes produced by the Bacillus group. Amylase activity has been reported to be the second most important activity during condiment production. Strain with protease activity will be relevant for flavor development and release of amino acids during condiment process. Strains with good protease and amylase will be more relevant as starter culture.
The Bacillus spp play important role in Maari fermentation  such as development of aroma and flavor. Bacillus subtilis was also present in others similar alkaline fermented condiments such as afitin  dawadawa  iru , Soumbala  kinema  mbuja . Bacillus subtilis and Bacillus licheniformis members of Bacillus group have same ability to degrade locust bean proteins. Proteins degradation is likely to produce essential amino acids and bioactive peptides useful for human nutrition.
Fermented soybean foods have several functional properties like antioxidant, antimutagenesis, immunomodulatory, antithrombosis and fibrinolytic [7,9].
This work indicates that Bacillus group are endospore-forming thermotolerant bacteria involved in Soumbala and Bikalaga fermentation and contribute to their sensor and nutritional characteristics. Enumeration result shows that the thermoteroleant bacteria are important and dominant in these two condiments. These result can be explain by the fact that Bikalga and Soumbala are condiments product of traditional uncontrolled alkaline fermentation of Hibiscus sabdariffa and Parkii biglobosa seeds respectively after long time cooking process.
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(1) Savadogo Aly, (1) Ilboudo A. Jules, (2) Gnankine Olivier, (1) Traore Alfred S
(1) Laboratoire de Microbiologie et de Biotechnologie Centre de Recherche en Sciences Biologiques, Alimentaires et Nutritionnelles(CRSBAN). Departement de Biochimie-Microbiologie (DBM) ; Unite de Formation et de Recherche en Sciences de la Vie et de la Terre (UFR/SVT) ; UNIVERSITE DE OUAGADOUGOU, Tel/Fax (226) 50 33 73 73 03 BP 7'3', Ouagadougou, BURKINA FASO
(2) Laboratoire d'Entomologie Fondamentale et Applique/UFR-SVT
Savadogo Aly, Departement de Biochimie-Microbiologie /CRSBAN /03 BP 7131 Ouagadougou, Burkina Faso
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Table 1: Sampling of Bikalga and Soumbala Samples Codes samples Number of sample S1, S3, S4, S6, S9, S10 Soumbala 6 B1,B2, B5, B7, B10 Bikalga 5 S2 Soumbala 1 S5 Soumbala 1 S7 Soumbala 1 B9 Bikalga 1 S8 Soumbala 1 B6 Bikalga 1 B3, B4 Bikalga 1 B8 Bikalga 1 Samples Codes Place of sampling S1, S3, S4, S6, S9, S10 Zogona market B1,B2, B5, B7, B10 (Ouagadougou) S2 Somgande market (Ouagadougou) S5 Goua market (Gaoua) S7 Nabigyaar market B9 (Ouagadougou) S8 Zone I market B6 (Ouagadougou) B3, B4 Karpala Market(Ouagadougou) B8 Goughin (Ouagadougou) S=Soumbala, B= Bikalga Table 2: Microbial analysis of Soumbala after treatment at different temperature. Soumbala samples 90[degrees]C 95[degrees]C CFU [g.sup.-1] S1 2.76 x [10.sup.9] 2 x [10.sup.9] S2 2.81 x [10.sup.9] 1.8 x [10.sup.9] S3 2.96 x [10.sup.9] 2.56 x [10.sup.9] S4 2.89 x [10.sup.9] 2.69 x [10.sup.9] S5 3 x [10.sup.9] 2.78 x [10.sup.9] S6 2.45 x [10.sup.9] 2.37 x [10.sup.9] S7 1.84 x [10.sup.9] 1.24 x [10.sup.9] S8 2.84 x [10.sup.9] 1.65 x [10.sup.9] S9 1.4 x [10.sup.9] 1.28 x [10.sup.9] S10 1.35 x [10.sup.9] 1.3 x [10.sup.9] Soumbala samples 100[degrees]C 105[degrees]C CFU [g.sup.-1] S1 1.51 x [10.sup.9] 3.5 x [10.sup.7] S2 1.56 x [10.sup.9] 1.10 x [10.sup.9] S3 2.02 x [10.sup.9] 1.54 x [10.sup.9] S4 2.27 x [10.sup.9] 5.8 x [10.sup.8] S5 2.19 x [10.sup.9] 1.21 x [10.sup.9] S6 1.98 x [10.sup.9] 6.7 x [10.sup.8] S7 6.2 x [10.sup.8] 3.8 x [10.sup.7] S8 7.1 x [10.sup.8] 5 x [10.sup.8] S9 8.9 x [10.sup.8] 6.2 x [10.sup.8] S10 5.4 x [10.sup.8] 1.12 x [10.sup.8] Values are weighted means of three replicates Table 3: Microbial analysis of Bikalga after treatment at different temperature. Bikalga samples 90[degrees]C 95[degrees]C CFU [g.sup.-1] B1 2.3 x [10.sup.7] 2.2 x [10.sup.7] B2 3.6 x [10.sup.7] 4.5 x [10.sup.7] B3 1.9 x [10.sup.7] 2. [10.sup.6] B4 4.5 x [10.sup.7] 7.1 x [10.sup.7] B5 5 x [10.sup.7] 6 x [10.sup.7] B6 3.5 x [10.sup.7] 2.2 x [10.sup.7] B7 6 x [10.sup.7] 5.3 x [10.sup.7] B8 10 x [10.sup.7] 6.9 x [10.sup.7] B9 12.5 x [10.sup.7] 11.2 x [10.sup.7] B10 12.8 x [10.sup.7] 10 x [10.sup.7] Bikalga samples 100[degrees]C 105[degrees]C CFU [g.sup.-1] B1 2.1 x [10.sup.7] 1 x [10.sup.7] B2 3.2 x [10.sup.7] 0 B3 0 0 B4 5.6 x [10.sup.7] 4.4 x [10.sup.7] B5 5.5 x [10.sup.7] 7 x [10.sup.6] B6 1.3 x [10.sup.7] 1.2 x [10.sup.7] B7 4.2 x [10.sup.7] 2.7 x [10.sup.7] B8 6.8 x [10.sup.7] 2 x [10.sup.7] B9 3.2 x [10.sup.7] 2.9 x [10.sup.7] B10 9.6 x [10.sup.7] 7.6 x [10.sup.7] Values are weighted means of three replicates Table 4: Biochemical and morphology characters of isolates of Soumbala and Bikalga. Isolated Isolated at 90[degrees]C Characters S1 B1 B2 Gram + + + Catalase + + + Sporulation + + + Voges Proskauer + - + Lecihinase - - + Starch hydrolysis - - - Casein hydrolysis +14mm +10mm +18mm Urease - - - NaCl (5%) + - - NaCl (6%) + - - NaCl (7%) + - - NaCl (8%) - - - Presumptive B. B. B. Identification pumilus coagulans cereus Isolated Isolated at 95[degrees]C Characters B3 S2 S3 B4 Gram + + + + Catalase + + + + Sporulation + + + + Voges Proskauer + - - + Lecihinase - - + - Starch hydrolysis - +13mm +13mm +16mm Casein hydrolysis +14mm +10mm +8mm +10mm Urease + - + - NaCl (5%) + + + + NaCl (6%) + + - + NaCl (7%) + + - + NaCl (8%) + - - + Presumptive B. B. B. B. Identification pumilus subtilis cereus licheniformis Isolated Isolated at 100[degrees]C Characters B5 S4 B6 B7 Gram + + + + Catalase + + + + Sporulation + + + + Voges Proskauer + + - + Lecihinase - - - - Starch hydrolysis +16mm - +17mm +15mm Casein hydrolysis +10mm +9mm -0 mm +12mm Urease - - + - NaCl (5%) - + + - NaCl (6%) - - + - NaCl (7%) - - - - NaCl (8%) - - - - Presumptive B. B. B. B. Identification coagulans pumilus firmus coagulans Isolated Isolated at 105[degrees]C Characters B8 S5 B9 B10 B11 Gram + + + + 11 Catalase + + + + 0 Sporulation + + + + 0 Voges Proskauer - + + + - Lecihinase - + - + - Starch hydrolysis +16mm - - - - Casein hydrolysis +4mm +11mm +14mm +6mm +6mm Urease - + - - - NaCl (5%) + - - + 6 NaCl (6%) - - - + - NaCl (7%) - - - + - NaCl (8%) - - - + - Presumptive B. B. B. B. B. Identification firmus cereus coagulans cereus coagulans + = positive reaction; -= negative reaction; B = strains from Bikalga (B1, B2,B3, B4, B5, B6, B7, B8, B9, B10); S = strains from Soumbala (S1, S2, S3, S4, S5)
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|Title Annotation:||Original Article|
|Author:||Aly, Savadogo; Jules, Ilboudo A.; Olivier, Gnankine; Traore, Alfred S.|
|Publication:||Advances in Environmental Biology|
|Date:||Aug 1, 2011|
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