Technological properties of indigenous Lactococcus lactis strains isolated from Lait caillé, a spontaneous fermented milk from Burkina Faso.

The experiments reported in this research paper aimed to determine the technological properties of indigenous Lactococcus lactis strains isolated from Lait caillé, a spontaneous fermented milk, from the perspective of starter culture development. Fermentations were conducted to determine the acidification patterns. The ropy character, growth in 0.04 g/ml NaCl and citrate metabolism were additionally tested. Furthermore, the rheological properties of samples from selected strains and the impact of cold storage were evaluated. Based on the rate of acidification, the indigenous strains were divided into 2 groups depending on their fermentation time, i.e. 10-13 h (fast acidifier), and up to 72 h (slow acidifier), respectively. The physiological tests suggested that most of these strains produced exopolysaccharides but none could ferment citrate. The flow properties of the samples inoculated by the fast acidifier strains showed a time-dependent shear thinning behaviour, while their viscoelastic properties corresponded structurally to those of weak gels. Cold storage decreased the viscosity and CFU counts for most of the indigenous strains tested. This study is a step towards the definition of starter cultures for African spontaneous fermented milks such as Lait caillé.

Traditional African Dishes Prepared From Local Biofortified Varieties of Pearl Millet: Acceptability and Potential Contribution to Iron and Zinc Intakes of Burkinabe Young Children.

Biofortification is among the food-based strategies, recently implemented and still in development, to fight micronutrient deficiencies. Three cereal-based traditional dishes of Sub-Saharan Africa (tô paste, pancakes, and gruel) prepared from one local (Gampela), or two biofortified (GB 8735 and Tabi) varieties of millet were assessed for their (i) acceptability by local consumers, (ii) iron and zinc absorption predicted by phytate-to-mineral molar ratios and (iii) contribution to the iron and zinc requirements of young children. Tasters preferred the color, texture, and taste of dishes prepared with the local variety, whether or not the grains were decorticated. Hedonic and preference tests showed no significant difference between the two biofortified varieties, but the cooks reported different behaviors during processing. Biofortified millet contained up to two times more iron than the local variety, reaching 6.5 mg iron/100 g dry matter. Iron and zinc contents remained higher in biofortified varieties even after decortication. Iron content in the dishes was highly variable, depending on iron loss and potential contamination during processing. The phytate-to-mineral molar ratios of all dishes indicated low iron absorption, independent of the millet variety, but improved zinc absorption in dishes prepared with biofortified varieties. The contribution of a dish prepared with one of the two biofortified millet varieties to the recommended iron and zinc intakes for 6-11-month-old children was estimated to be about 5 and 7%, respectively, compared to 2 and 4% for the same dish prepared with local millet. For 12-23-month-old children, the contribution to the recommended intakes was estimated to be about 14 and 12% with biofortified millet, respectively, and about 6 and 7% with local millet. The use of biofortified millet varieties could be complementary to food diversification strategies to increase iron and zinc intakes. As in Ouagadougou, cereals are eaten in different forms by young children several times per day, iron and zinc intakes could be improved in the long term by using the biofortified varieties of pearl millet.

Identification of the predominant microbiota during production of lait caillé, a spontaneously fermented milk product made in Burkina Faso.

The spontaneously fermented curdled milk product from Burkina Faso, lait caillé is prepared by traditional processing from raw unpasteurised milk. The fermentation lasts 1-3 days. This study aims to identify the predominant microbiota involved in lait caillé fermentation from cow milk. A survey on lait caillé end-products from local markets showed pH ranges of 3.5 to 4.2. Counts of total lactic acid bacteria (LAB) were 7.8 ± 0.06 to 10.0 ± 0.03 log CFU/g and yeast counts were 5.3 ± 0.06 to 8.7 ± 0.01 log CFU/g, together with considerate amounts of Enterobacteriaceae < 3.00 to 8.4 ± 0.14 log CFU/g. Sampling throughout the entire fermentation of lait caillé was performed at a traditional house-hold production site. A drop in pH from 6.7 ± 0.01 at 0 h to 4.3 ± 0.08 in the end-product (59 h) was found. Total LAB counts increased to 8.6 ± 0.02 log CFU/g in the end-product, while yeast and Enterobacteriaceae counts reached 6.4 ± 0.11 and 6.7 ± 0.00 log CFU/g, respectively. LAB and yeasts isolated during the fermentation were clustered by (GTG)5 repetitive-PCR fingerprinting followed by 16S and 26S rRNA gene sequencing, respectively. Microbial successions were observed with Leuconostoc mesenteroides being the predominant LAB followed by Pediococcus pentosaceus and Weissella paramesenteroides at the onset, while Lactococcus lactis and Enterococcus spp. where the predominant LAB after 7 h of fermentation. During the first 18 h Candida parapsilosis was the dominant yeast species, while from 35 h to the end-product, Saccharomyces cerevisiae predominated. The microbial safety risk pointed out in this study, showed the need for implementation of good manufacturing practices including pasteurisation and use of well-defined starter cultures.

Rapid screening of starter cultures for maari based on antifungal properties.

Forty Bacillus isolates obtained from maari (used as condiment in Burkina Faso) including 17 B. subtilis, 4 B. circulans, 7 B. pumilus and 6 B. licheniformis were investigated for use as starter cultures in maari production. The isolates were screened by PCR for the sfp gene responsible for the production of the lipopeptide biosurfactant, surfactin. The sfp gene was detected in all of the seventeen B. subtilis isolates, in 2 out of 7 B. pumilus, in 4 out of 6 B. licheniformis whereas no B. circulans was positive for the sfp gene by PCR screening. Furthermore, all the 40 Bacillus spp. were screened for biosurfactant production and inhibitory activity against Aspergillus flavus, A. niger, A. versicolor and Rhizopus oryzae. Results demonstrated a relationship between the presence of the sfp gene and the antifungal activity and biosurfactant production of Bacillus isolates. In addition, molecular typing of the 17 B. subtilis isolates by Multilocus Sequence Typing (MLST) resulted in 15 Sequence Types, one of them included three strains. Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR), used for B. licheniformis, B. megaterium, B. circulans and B. pumilus revealed that the inhibitory activity and biosurfactant production were strain-dependent. Finally, the detection of chitinase (chi) and ß-glucanase (glu) biosynthesis genes was found to be associated with the antifungal activity for 16 B. subtilis isolates. The present work provides a greater understanding of the antifungal activity and biosurfactant production ability within the Bacillus spp. isolated from maari and contributes to the selection of Bacillus isolates to be used as starter cultures for controlled production of maari.

Hanseniaspora jakobsenii sp. nov., a yeast isolated from Bandji, a traditional palm wine of Borassus akeassii.

Investigation of the microbial diversity of Bandji, a traditional palm wine from Burkina Faso (West Africa) revealed the presence of two yeast isolates (YAV16 and YAV17T) with unusual phenotypic and genotypic characteristics. The isolates divide by bipolar budding with no production of ascospores. Phylogenetic analysis of concatenated sequences of the 26S rRNA gene D1/D2 and internal transcribed spacer (ITS) regions indicated that the novel species was most closely related to Kloeckera lindneri and Hanseniaspora valbyensis. The new isolates differed from K. lindneri NRRL Y-17531T and H. valbyensis CBS 479T by substitutions in the D1/D2 region of 12 and 16 nt respectively. The divergence in the ITS region from the closely related species was characterized by substitutions of 45-46 nt. Repetitive palindromic PCR (rep-PCR) profiles of YAV16 and YAV17T were also significantly different from those of K. lindneri MUCL 31146T ( = NRRL Y-17531T), H. valbyensis NCYC 17T ( = CBS 479T) and other species of the genus Hanseniaspora. Based on the results of the phenotypic and genotypic characterizations, it was concluded that the new isolates represent a novel species for which the name Hanseniaspora jakobsenii sp. nov. is proposed with YAV17T ( = CBS 12942T = DSM 26339T = NCYC 3828T; MycoBank number MB 805785) as the type strain.

Variability of vitamins B1, B2 and minerals content in baobab (Adansonia digitata) leaves in East and West Africa.

The regional variability and age-age correlation on vitamin B1, vitamin B2 and minerals (Ca, Mg, P, K, Cu, Fe, Mn, Na, and Zn) concentration in baobab leaves were investigated. Baobab was cultivated from seeds from 11 countries including Benin, Burkina Faso, Kenya, Malawi, Mali, Mozambique, Niger, Tanzania, Togo, Senegal, and Sudan. Vitamins B1 and B2 content were assessed using microbiological VitaFast kits methods and minerals by atomic absorption and flame spectrometry methods. Overall, the results showed a higher content of vitamin B2 compared to vitamin B1 with the highest vitamin B2 content (1.04 ± 0.05 mg/100 g DM) from Senegal. The highest iron (Fe) content of 26.39 mg/100 g was found in baobab leaves from Mali. For age-age correlation, adult baobab leaves of Nankoun in Burkina Faso provided the highest calcium (Ca) content of 3373 mg/100 g. However, for provenance trial, young plants from three communities of Burkina Faso showed the highest calcium (Ca) and potassium (K) content. The study demonstrated that vitamins B1 and B2 and mineral contents in baobab leaves vary with the country and the age of the tree. Vitamin B1 content was higher in baobab leaves from ascendants compared to those from descendants, while in contrast vitamin B2 content was higher in the leaves from the descendants compared to their ascendants (mother tree).

Characteristics and phylogeny of Bacillus cereus strains isolated from Maari, a traditional West African food condiment.

Maari is a spontaneously fermented food condiment made from baobab tree seeds in West African countries. This type of product is considered to be safe, being consumed by millions of people on a daily basis. However, due to the spontaneous nature of the fermentation the human pathogen Bacillus cereus occasionally occurs in Maari. This study characterizes succession patterns and pathogenic potential of B. cereus isolated from the raw materials (ash, water from a drilled well (DW) and potash), seed mash throughout fermentation (0-96h), after steam cooking and sun drying (final product) from two production sites of Maari. Aerobic mesophilic bacterial (AMB) counts in raw materials were of 10(5)cfu/ml in DW, and ranged between 6.5×10(3) and 1.2×10(4)cfu/g in potash, 10(9)-10(10)cfu/g in seed mash during fermentation and 10(7) - 10(9) after sun drying. Fifty three out of total 290 AMB isolates were identified as B. cereus sensu lato by use of ITS-PCR and grouped into 3 groups using PCR fingerprinting based on Escherichia coli phage-M13 primer (M13-PCR). As determined by panC gene sequencing, the isolates of B. cereus belonged to PanC types III and IV with potential for high cytotoxicity. Phylogenetic analysis of concatenated sequences of glpF, gmk, ilvD, pta, pur, pycA and tpi revealed that the M13-PCR group 1 isolates were related to B. cereus biovar anthracis CI, while the M13-PCR group 2 isolates were identical to cereulide (emetic toxin) producing B. cereus strains. The M13-PCR group 1 isolates harboured poly-γ-D-glutamic acid capsule biosynthesis genes capA, capB and capC showing 99-100% identity with the environmental B. cereus isolate 03BB108. Presence of cesB of the cereulide synthetase gene cluster was confirmed by PCR in M13-PCR group 2 isolates. The B. cereus harbouring the cap genes were found in potash, DW, cooking water and at 8h fermentation. The "emetic" type B. cereus were present in DW, the seed mash at 48-72h of fermentation and in the final product, while the remaining isolates (PanC type IV) were detected in ash, at 48-72h fermentation and in the final product. This work sheds light on the succession and pathogenic potential of B. cereus species in traditional West African food condiment and clarifies their phylogenetic relatedness to B. cereus biovar anthracis. Future implementation of GMP and HACCP and development of starter cultures for controlled Maari fermentations will help to ensure a safe product.

Iron contamination during in-field milling of millet and sorghum.

Nutritionally, contaminant iron in foods may lead to overestimation of the satisfaction of iron requirement while iron deficiencies remain a widespread health problem. Iron contamination was measured in millet and sorghum grains after decortication and in-field milling using different equipments in Burkina Faso. Total iron content did not change significantly after decortication, probably due to a balance between losses resulting from the removal of iron-rich peripheral parts and contamination. Total iron contents increased significantly after mechanical milling irrespective of whether iron or corundum grindstones were used. Contamination was highly variable, ranging from 3 to 6 mg iron/100 g DM, and was mainly due to wear of the milling equipment. After in vitro digestion of traditional cereal dishes prepared with iron-contaminated or uncontaminated flours, the contaminant iron was found mainly in the insoluble fraction. Only in sorghum was a small proportion (4%) bioaccessible, showing that contaminant iron has poor nutritional interest.

Co-production of surfactin and a novel bacteriocin by Bacillus subtilis subsp. subtilis H4 isolated from Bikalga, an African alkaline Hibiscus sabdariffa seed fermented condiment.

Bikalga is a Hibiscus sabdariffa seed fermented condiment widely consumed in Burkina Faso and neighboring countries. The fermentation is dominated by Bacillus subtilis group species. Ten B. subtilis subsp. subtilis (six isolates) and Bacillus licheniformis (four isolates) isolated from traditional Bikalga were examined for their antimicrobial activity against a panel of 36 indicator organisms including Gram-positive and Gram-negative bacteria and yeasts. The Bacillus spp. isolates showed variable inhibitory abilities depending on the method used. Both Gram-positive and Gram-negative bacteria were inhibited in the agar spot assay while only Gram-positive pathogens were inhibited in the agar well diffusion assay. Cell free supernatants (CFS) of pure cultures of 3 B. subtilis subsp. subtilis (G2, H4 and F1) strains inhibited growth of Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus and Bacillus cereus, while CFS of 2 B. licheniformis (E3 and F9) strains only inhibited M. luteus. The antimicrobial substance(s) produced by B. subtilis subsp. subtilis H4 was further characterized. The antimicrobial substance(s) produced by H4 was detected from mid-exponential growth phase. The activity was sensitive to protease and trypsin, but resistant to the proteolytic action of proteinase K and papain. Treatment with α-amylase and lipase II resulted in a complete loss of antimicrobial effect, indicating that a sugar moiety and lipid moiety are necessary for the activity. Treatment with mercapto-ethanol resulted in a significant loss, indicative of the presence of disulfide bridges. The antimicrobial activity of H4 was heat resistant and active at pH3-10. PCR detection of yiwB, sboA, spoX, albA and spaS, etnS genes and genes coding for surfactins and plipastatins (fengycins) indicated a potential for subtilosin, subtilin and lipopeptide production, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out and a single band of approximately 4kDa had antimicrobial activity. Ultra high performance liquid chromatography-time of flight mass spectrometry (UHPLC-TOFMS) analysis of the 4kDa band allowed identification of surfactin and a protein with a monoisotopic mass of 3346.59Da, which is dissimilar in size to subtilosin and subtilin. Surfactin is a cyclic lipoheptapeptide, which contains a ß-hydroxy fatty acid, but no di-sulfide bridges or sugar residues. The complete loss of activity upon amylase treatment indicates that surfactin was not responsible for the observed antimicrobial effect. However, it cannot completely be ruled out that surfactin acts synergistically with the detected protein, though further investigations are needed to confirm this.

Inhibition of Bacillus cereus growth by bacteriocin producing Bacillus subtilis isolated from fermented baobab seeds (maari) is substrate dependent.

Maari is a spontaneously alkaline fermented food condiment made from baobab tree seeds. Due to the spontaneous nature of maari fermentations growth of the opportunistic human pathogen Bacillus cereus is occasionally observed. Bacillus subtilis strains are important for alkaline seed fermentations because of their enzymatic activities contributing to desirable texture, flavor and pH development. Some B. subtilis strains have antimicrobial properties against B. cereus. In the present work, three bacteriocin producing B. subtilis strains (B3, B122 and B222) isolated from maari were tested. The production of antimicrobial activity by the three strains was found to be greatly influenced by the substrate. All three B. subtilis strains produced antimicrobial activity against B. cereus NVH391-98 in BHI broth as determined by the agar well diffusion assay, whereas no antimicrobial activity was detected in whole cooked baobab seeds and in 10% (w/v) grinded baobab seeds. Incorporation of BHI with up to 5% (w/w) grinded baobab seeds enhanced the antimicrobial activity of B. subtilis compared with pure BHI in a strain dependent manner. Incorporation of BHI with 50% (w/w) baobab grinded seeds decreased the antimicrobial activity. Addition of the inorganic salts FeCl3, MgSO4 and MnSO4 has previously been reported to increase bacteriocin production of B. subtilis, but the addition of these salts to 10% (w/v) grinded baobab seed broth did not cause antimicrobial activity. Survival of B. cereus NVH391-98 in co-culture with B. subtilis was tested in BHI broth, 10% (w/v) grinded baobab seed based broth and during baobab seed fermentation to produce maari. B. cereus NVH391-98 grew well in all three substrates in mono-culture. All the 3 B. subtilis strains were able to decrease B. cereus NVH391-98 to levels below the detection limit (<10 CFU/ml) in BHI, but not in baobab seed based substrates, even though the outgrowth of B. cereus NVH391-98 was delayed by up to 40 h. In conclusion, production of antimicrobial activity by the investigated B. subtilis strains is highly substrate-specific and strain-specific. The three B. subtilis strains delayed but did not prevent B. cereus outgrowth during baobab seed fermentations. Maari is produced through mixed microbial population fermentations. B. subtilis co-starter culture candidates originating from maari which are able to prevent pathogen outgrowth remain to be identified.

Bacteriocin formation by dominant aerobic sporeformers isolated from traditional maari.

The antimicrobial activity of 8 Bacillus spp. and 2 Lysinibacillus spp. representing the predominant aerobic sporeformers during traditional maari fermentations, a traditional fermented baobab seeds product from Burkina Faso, was investigated. The antimicrobial activity was assessed against a total of 31 indicator organisms representing various Gram-negative and positive pathogens. The screening showed that 3 Bacillus subtilis strains (B3, B122 and B222) in particular had antimicrobial activity against some Gram-positive organisms and were selected for further studies. It was found that the antimicrobial substances produced were heat stable, in-sensitive to catalase, sensitive to protease and trypsin but resistant to the proteolytic action of papain and proteinase K and equally active at pH values ranging from 3 to 11. Bacteriocin secretion started in late exponential growth phase and maximum activity was detected during the stationary growth phase. The production of bacteriocin by B. subtilis B3, B122 and B222 was dependent on the aeration conditions. Maximum production of bacteriocin was observed under reduced aeration. Specific primers were used to screen isolates B3, B122 and B222 for genes involved in the synthesis of the bacteriocins subtilosin A, subtilin, sublancin and ericin. Amplicons of the expected sizes were detected for iywB, sboA, sboX, albA and spaS involved in the biosynthesis of subtilosin and subtilin, respectively. The translated nucleotide sequences had 100% identity to the YiwB, SboX and SboA amino acid sequences of the subtilosin A producing B. subtilis subsp. subtilis strain 168. Interestingly there was a 3 amino acid deletion at the N-terminal part of AlbA in B3, B122 and B222 that probably alters the activity of this enzyme. Analysis of the spaS gene sequences of B3, B122 and B222, encoding a subtilin precursor peptide, showed that the translated nucleotide sequence had 98% identity with the corresponding SpaS amino acid sequence of subtilin producing B. subtilis subsp. spizizenii strain ATCC6633.

Microorganisms associated with Maari, a Baobab seed fermented product.

A microbiological study was carried out on Baobab fermented seeds (Maari) obtained from 4 different production sites in Burkina Faso (Mansila, Toulfé, Ouagadougou and Gorgadji). A total of 390 representative isolates comprising 251 aerobic mesophilic bacteria (AMB) and 139 lactic acid bacteria (LAB) were isolated and identified to species level using a combination of pheno- and genotypic methods including conventional morphological analysis, carbohydrate fermentation profiling, rep-PCR ((GTG)(5)-fingerprinting) and 16S rRNA gene sequencing. The fermentation of Baobab seeds was initiated by the AMB identified as Bacillus subtilis (82% of AMB isolates) and Staphylococcus sciuri (18% of AMB isolates). No lactic acid bacteria were isolated at the beginning of the process. After 24h fermentation time, Enterococcus faecium appeared in the fermenting seeds and remained until the end of the fermentation, as the predominant LAB. In Maari collected from retail outlets the AMB count ranged from 6.7log10CFU/g to 10log10CFU/g while the LAB load ranged from 4.4log10CFU/g to 9.9log10CFU/g. The AMB were identified as belonging to genus Bacillus (12 species), Staphylococcus (3 species) and one species of Aerococcus, Macrococcus, Leifsonia, Kurthia, Proteus, Acinetobacter and Globicatella, respectively. A putatively novel, previously undescribed Corynebacterium sp. was also found. E. faecium was the dominant LAB in all investigated retail samples except one sample dominated by Pediococcus acidilactici.

Ability of selected lactic acid bacteria to ferment a pearl millet-soybean slurry to produce gruels for complementary foods for young children.

To assess the ability of lactic acid bacteria to improve some nutritional characteristics of the pearl millet-soybean slurry to prepare complementary foods for young children in African countries, inoculation was performed using strains previously selected for their ability to hydrolyse starch, phytate, or alpha-galactooligosaccharides (alpha-GOS). For the sake of comparison with the action of a natural microflora, fermentation was also performed by back slopping inoculation, that is, with a sample obtained from spontaneously fermented traditional pearl millet slurry obtained from a small scale processing unit in Burkina Faso (Ouagadougou). Starter cultures thrived on the slurry as shown by counts on MRS agar, TTGE fingerprints, and fermentation patterns. The fermentation of precooked slurries inoculated by back slopping or with mixed cultures containing the amylolytic strain Lb. plantarum A6 enabled partial starch hydrolysis. Corresponding gruels had a suitable consistency for young child feeding at high dry matter content, and a high energy density: 88.7 +/- 4.2 and 75.8 +/- 5.1 kcal/100 g of sweetened gruel, for the gruels inoculated by back slopping or with Lb. plantarum A6, respectively. Unexpectedly, no decrease in phytates was observed in any of the experiments, suggesting the presence of one or more inhibitory compounds in soybean. Furthermore, preprocessing conditions before fermentation affect the carbohydrate composition of slurry and have a more profound effect than fermentation on the reduction of the alpha-GOS content.

The microbiology of alkaline-fermentation of indigenous seeds used as food condiments in Africa and Asia.

Alkaline-fermented food condiments play an important role in the diets of many people in developing and a few developed countries. The rise in pH during production of these foods is due to the ability of the dominant microorganisms, Bacillus spp., to hydrolyze proteins into amino acids and ammonia. Studies have been undertaken which have investigated a number of these products like dawadawa, ugba, bikalga, kinema, natto, and thua-nao. In this review, current knowledge about the principal microbiological activities and biochemical modifications which occur during the processing of the alkaline condiments including nutritional, antimicrobial, and probiotic aspects are discussed. The current use of molecular biology methods in microbiological research has allowed unambiguous and more reliable identification of microorganisms involved in these fermentations generating sufficient knowledge for the selection of potential starter cultures for controlled and better production procedures for alkaline-fermented seeds condiments.

Genotyping of starter cultures of Bacillus subtilis and Bacillus pumilus for fermentation of African locust bean (Parkia biglobosa) to produce Soumbala.

Bacillus spp. are the predominant microorganisms in fermented African locust bean called Soumbala in Burkina Faso. Ten strains selected as potential starter cultures were characterised by PCR amplification of the16S-23S rDNA intergenic transcribed spacer (ITS-PCR), restriction fragment length polymorphism of the ITS-PCR (ITS-PCR RFLP), pulsed field gel electrophoresis (PFGE) and sequencing of the 968-1401 region of the 16S rDNA. In previous studies, the isolates were identified by phenotyping as Bacillus subtilis and Bacillus pumilus. The phenotyping was repeated as a reference in the present study. The ITS-PCR and ITS-PCR RLFP allowed a typing at species level. The PFGE was more discriminative and allowed a typing at strain level. Full agreement with the phenotyping was observed in all cases. The sequencing of the 16S rDNA allowed the identification at species level with an identity from 97% to 100% comparing the sequences to those from the GenBank databases. The desired cultures of B. subtilis and B. pumilus from African locust bean fermentation were distinguished by ITS-PCR and ITS-PCR RLFP from Bacillus cereus and Bacillus sphaericus which sometimes occur in the beginning of the fermentation.