A review of microbes and chemical contaminants in dairy products in sub-Saharan Africa.

Animal milk types in sub-Saharan Africa (SSA) are processed into varieties of products using different traditional methods and are widely consumed by households to support nutritional intake and diet. Dairy products contain several microorganisms, their metabolites, and other chemical compounds, some with health benefits and many others considered as potential health hazards. Consumption of contaminated milk products could have serious health implications for consumers. To access the safety of milk products across SSA, studies in the region investigating the occurrences of pathogens as well as chemical compounds such as heat stable toxins and veterinary drug residues in animal milk and its products were reviewed. This is done with a holistic view in light of the emerging exposome paradigm for improving food safety and consumer health in the region. Herein, we showed that several published studies in SSA applied conventional and/or less sensitive methods in detecting microbial species and chemical contaminants. This has serious implications in food safety because the correct identity of a microbial species and accurate screening for chemical contaminants is crucial for predicting the potential human health effects that undermine the benefits from consumption of these foods. Furthermore, we highlighted gaps in determining the extent of viral and parasitic contamination of milk products across SSA as well as investigating multiple classes of chemical contaminants. Consequently, robust studies should be conducted in this regard. Also, efforts such as development cooperation projects should be initiated by all stakeholders including scientists, regulatory agencies, and policy makers to improve the dairy product chain in SSA in view of safeguarding consumer health.

Halomonas nigrificans sp. nov., isolated from cheese.

A Gram-stain-negative, rod-shaped Proteobacteria isolate, MBT G8648T, was obtained from an acid curd cheese called Quargel. The isolate was moderately salt tolerant and motile, with numerous peritrichous flagella. The 16S rRNA gene sequence analysis indicated that the strain belongs to the genus Halomonas, with 98.42 % 16S rRNA gene sequence similarity with Halomonas titanicae BH1T as nearest related neighbour. Further comparative sequence analysis of secA and gyrB genes, as well as physiological and biochemical tests, revealed that this bacterium formed a taxon well-separated from its nearest neighbours and other established Halomonas species. Thus, the strain represents a new species, for which the name Halomonas nigrificans sp. nov. is proposed, with strain MBT G8648T (=LMG 29097T =DSM 105749T) as type strain.

Novel Siphoviridae phage PMBT4 belonging to the group b Lactobacillus delbrueckii subsp. bulgaricus phages.

A novel Lactobacillus delbrueckii bacteriophage PMBT4 was isolated from the Nigerian fermented milk product nono. The phage possesses a long and thin, non-contractile tail and an isometric head, indicating that it belongs to the Siphoviridae family. A neck passage structure (`collar`), previously hypothesized to be encoded by two genes located in the Lactobacillus delbrueckii phage LL-K insertion sequence (KIS) element, as well as in two additional Lb. delbrueckii phages Ld17 and Ld25A, could also be observed on an estimated 1-5% of phage particles by transmission electron microscopy. However, neither mapping of high throughput sequencing data to KIS element genes from Lb. delbrueckii phages LL-K, Ld17 and Ld25A nor PCR amplification of the KIS element genes could corroborate the presence of these genes in the PMBT4 genome. The PMBT4 genome consists of 31,399 bp with a mol% GC content of 41.6 and exhibits high (95-96%) sequence homologies to Lb. delbrueckii phages c5, Ld3, Ld25A and Ld17, which assigned PMBT4 as a new member of this genus, i.e. the Cequinquevirus genus.

Isolation and Characterization of Potential Starter Cultures from the Nigerian Fermented Milk Product nono.

Nono, an important traditional fermented dairy food produced from cow's milk in Nigeria, was studied for microbial diversity and for starter culture development for industrial production. On the basis of a polyphasic approach, including phenotypic and genotypic methods such as 16S rRNA gene sequencing, repetitive element PCR (rep-PCR) fingerprinting metagenomics, and whole genome sequencing, we identified Lactobacillus (Lb.) helveticus, Limosilactobacillus (L.) fermentum, Lb. delbrueckii, and Streptococcus (S.) thermophilus as predominant bacterial species involved with milk fermentation during traditional nono production in Nigeria, while the predominant yeast species in nono was identified as Saccharomyces cerevisiae. Using metagenomics, Shigella and potential pathogens such as enterobacteria were detected at low levels of abundance. Strains of the predominant lactic acid bacteria (LAB) were selected for starter cultures combination on the basis of their capacities for rapid growth in milk and reduction of pH below 4.5 and their gelling characteristic, which was demonstrated noticeably only by the S. thermophilus strains. Whole genome sequence analysis of selected bacterial strains showed the largest assembled genome size to be 2,169,635 bp in Lb. helveticus 314, while the smallest genome size was 1,785,639 bp in Lb. delbrueckii 328M. Genes encoding bacteriocins were not detected in all the strains, but all the LAB possessed genes potentially involved in diacetyl production and citrate metabolism. These bacteria isolated from nono can thus be used to improve the microbial safety quality of nono in Nigeria, in addition to improving technological parameters such as gelling viscosity, palatability, and product consistency.

Diversity of bacillus species isolated from okpehe, a traditional fermented soup condiment from Nigeria.

The diversity of Bacillus species isolated from the fermented soup condiment okpehe in Nigeria was studied using a combination of phenotypic and genotypic methods. Fifty strains presumptively characterized as Bacillus spp. using the API 50 CHB test were further identified by PCR of randomly amplified polymorphic DNA (RAPD) and by amplified ribosomal DNA restriction analysis (ARDRA) genotyping methods. ARDRA fingerprinting with HhaI, HinfI, and Sau3AI restriction enzymes did not allow successful differentiation between the Bacillus species, except for distinguishing B. cereus from other Bacillus species. This problem was overcome with the combination of RAPD PCR and ARDRA genotypic fingerprinting techniques. Sequencing of 16S rRNA genes of selected strains representative of the major clusters revealed that the Bacillus strains associated with this fermentation were B. subtilis, B. amyloliquefaciens, B. cereus, and B. licheniformis (in decreasing order of incidence). The presence of enterotoxin genes in all B. cereus strains was demonstrated by multiplex PCR. The high incidence of detection (20%) of possibly pathogenic B. cereus strains that contained enterotoxin genes indicated that these fermented foods may constitute a potential health risk.

Draft Genome Sequences of Lactobacillus helveticus, Lactobacillus fermentum, and Lactobacillus delbrueckii Strains from African Fermented Nono.

The genomes of predominant Lactobacillus helveticus, Lactobacillus fermentum, and Lactobacillus delbrueckii strains from fermented nono were sequenced. The genome sizes were 2.1, 1.9, and 1.7 Mbp, respectively, and the GC contents were 36.5%, 51.5%, and 49.7%, respectively. Annotation revealed some genes for bacteriocin and for the potential production of aroma compounds.

Comparison of the microbial composition of African fermented foods using amplicon sequencing.

Fermented foods play a major role in the diet of people in Africa, where a wide variety of raw materials are fermented. Understanding the microbial populations of these products would help in the design of specific starter cultures to produce standardized and safer foods. In this study, the bacterial diversity of African fermented foods produced from several raw materials (cereals, milk, cassava, honey, palm sap, and locust beans) under different conditions (household, small commercial producers or laboratory) in 8 African countries was analysed by 16S rRNA gene amplicon sequencing during the Workshop "Analysis of the Microbiomes of Naturally Fermented Foods Training Course". Results show that lactobacilli were less abundant in fermentations performed under laboratory conditions compared to artisanal or commercial fermentations. Excluding the samples produced under laboratory conditions, lactobacilli is one of the dominant groups in all the remaining samples. Genera within the order Lactobacillales dominated dairy, cereal and cassava fermentations. Genera within the order Lactobacillales, and genera Zymomonas and Bacillus were predominant in alcoholic beverages, whereas Bacillus and Lactobacillus were the dominant genera in the locust bean sample. The genus Zymomonas was reported for the first time in dairy, cereal, cassava and locust bean fermentations.

Complete Genome Sequence of Ochrobactrum haematophilum FI11154, Isolated from Kunu-Zaki, a Nigerian Millet-Based Fermented Food.

Ochrobactrum haematophilum FI11154 was isolated from kunu-zaki, a Nigerian traditional fermented millet-based food. Here, we present the first complete genome sequence of this species. The genome consists of five replicons and contains genes related to iron uptake and phosphatase activities.

Isolation and Characterization of Lactic Acid Bacteria from Fermented Goat Milk in Tajikistan.

The lactobacilli associated with a fermented goat milk product from Tajikistan were isolated to characterize their technological properties and antibiotic resistances in order to assess their suitability for development as starter cultures. In this study, twenty three strains were identified by 16S rRNA sequencing as typical dairy-associated lactic acid bacterial strains, i.e. L. plantarum, L. pentosus, L. delbrueckii, L. helveticus and L. paracasei. These strains were generally susceptible to most antibiotics tested in this study and this allowed a selection of strains as safe starters. The draft genomes of four representative strains were sequenced and the number of contigs of the four assembled genomes ranged from 51 to 245 and the genome sizes ranged from 1.75 to 3.24 Mbp. These representative strains showed differences in their growth behavior and pH-reducing abilities in in vitro studies. The co-inoculation of these Lactobacillus spp. strains together with a yeast Kluyveromyces marxianus MBT-5698, or together with the yeast and an additional Streptococcus thermophilus MBT-2, led to a pH reduction to 3.4 after 48 h. Only in the case of fermentation inoculated with the co-culture, the viscosity of the milk increased noticeably. In contrast, fermentations with single strains did not lead to gelation of the milk or to a decrease in the pH after 24h. The results of this study provide a comprehensive understanding of the predominant lactobacilli related to Tajikistani fermented milk products.

In vitro fermentation studies for selection and evaluation of Bacillus strains as starter cultures for the production of okpehe, a traditional African fermented condiment.

Selected Bacillus and Enterococcus strains, isolated from traditional okpehe fermentations, were studied for their suitability as starter cultures in laboratory-scale fermentations of Prosopis africana seeds for the production of okpehe, a traditional fermented vegetable product of Nigeria. The strains were selected on the basis of highest proteolytic activity, as determined with the APIZYM (BioMerieux) test. The choice of starter strains was narrowed to Bacillus subtilis strains BFE 5301 and BFE 5372. These were determined as the best starter combination because of rapid growth, high amylolytic and proteolytic activities, high levels of polyglutamic acid production by strain BFE 5372, as well as bacteriocin production by strain BFE 5301. Other mixed culture fermentations did not yield sensorically acceptable products. Although a monoculture fermentation, using only B. subtilis strain BFE 5372, produced okpehe with very good sensory characteristics, the growth of B. cereus could be detected after 48 h fermentation, indicating that this starter did not sufficiently contribute to product safety. Mixed culture fermentation with the combination of bacteriocin-producing starter B. subtilis BFE 5301 and the non-bacteriocin-producing B. subtilis BFE 5372, produced a product with good sensory characteristics, in which growth of B. cereus was delayed. The bacteriocin produced by B. subtilis strain BFE 5301 was identified as subtilisin, using subtilisin-specific primers and PCR amplification of the subtilisin gene. The bacteriocin was heat-stable at 100 degrees C for 10 min and exhibited highest activity at pH values lower or equal to pH 6.0. The bacteriocin was sensitive to the proteolytic enzymes trypsin and alpha-chymotrypsin at concentrations of 10 mg/ml.

Draft Genome Sequence of Lactobacillus fermentum BFE 6620, a Potential Starter Culture for African Vegetable Foods, Isolated from Fermented Cassava.

We report the draft genome sequence of Lactobacillus fermentum BFE 6620 from fermented cassava used as a potential starter culture for African vegetable fermentation. Sequence analysis showed the assembled genome size to be 1,982,893 bp, encoding a predicted total of 2,003 protein-coding genes, 14 rRNAs, 54 tRNAs, and 3 noncoding RNAs (ncRNAs).

The Biotechnology of Ugba, a Nigerian Traditional Fermented Food Condiment.

Legumes and oil bean seeds used for the production of condiments in Africa are inedible in their natural state; they contain some anti-nutritional factors especially undigestible oligosaccharides and phytate. Fermentation impact desirable changes by reducing anti-nutritional factors and increasing digestibility. Ugba is an alkaline fermented African oil bean cotyledon (Pentaclethra macrophylla) produced by the Ibos and other ethnic groups in southern Nigeria. Seen as a family business in many homes, its preparation is in accordance with handed-down tradition from previous generations and serves as a cheap source of plant protein. Its consumption as a native salad is made possible by fermentation of the cotyledon for 2-5 days, but could also serve as a soup flavoring agent when fermentation last for 6-10 days. The fermentation process involved is usually natural with an attendant issue of product safety, quality and inconsistency. The production of this condiment is on a small scale and the equipment used are very rudimentary, devoid of good manufacturing procedures that call to question the issue of microbial safety. This paper therefore reviews the production process and the spectrum of microbial composition involved during fermentation. In addition, potential spoilage agents, nutritional and biochemical changes during production are examined. Furthermore, information that can support development of starter cultures for controlled fermentation process in order to guarantee microbiological safety, quality and improved shelf life are also discussed.

Produce from Africa's Gardens: Potential for Leafy Vegetable and Fruit Fermentations.

A rich variety of indigenous fruits and vegetables grow in Africa, which contribute to the nutrition and health of Africa's populations. Fruits and vegetables have high moisture and are thus inherently prone to accelerated spoilage. Food fermentation still plays a major role in combating food spoilage and foodborne diseases that are prevalent in many of Africa's resource disadvantaged regions. Lactic acid fermentation is probably the oldest and best-accepted food processing method among the African people, and is largely a home-based process. Fermentation of leafy vegetables and fruits is, however, underutilized in Africa, although such fermented products could contribute toward improving nutrition and food security in this continent, where many are still malnourished and suffer from hidden hunger. Fermentation of leafy vegetables and fruits may not only improve safety and prolong shelf life, but may also enhance the availability of some trace minerals, vitamins and anti-oxidants. Cassava, cow-peas, amaranth, African nightshade, and spider plant leaves have a potential for fermentation, as do various fruits for the production of vinegars or fruit beers and wines. What is needed to accelerate efforts for production of fermented leaves and vegetables is the development of fermentation protocols, training of personnel and scale-up of production methods. Furthermore, suitable starter cultures need to be developed and produced to guarantee the success of the fermentations.

Draft Genome Sequence of Lactobacillus plantarum BFE 5092 Isolated from Maasai Fermented Milk.

The draft genome of Lactobacillus plantarum BFE 5092 isolated from the Maasai traditional fermented milk product kule naoto was sequenced, and sequence analysis showed the assembled genome size to be 3,285,094 bp, containing a predicted total of 3,111 protein-encoding genes, 17 rRNAs, and 70 tRNAs.

Fermentation of African kale (Brassica carinata) using L. plantarum BFE 5092 and L. fermentum BFE 6620 starter strains.

Vegetables produced in Africa are sources of much needed micronutrients and fermentation is one way to enhance the shelf life of these perishable products. To prevent post-harvest losses and preserve African leafy vegetables, Lactobacillus plantarum BFE 5092 and Lactobacillus fermentum BFE 6620 starter strains were investigated for their application in fermentation of African kale (Brassica carinata) leaves. They were inoculated at 1×107cfu/ml and grew to a maximum level of 108cfu/ml during 24h submerged fermentation. The strains utilized simple sugars (i.e., glucose, fructose, and sucrose) in the kale to quickly reduce the pH from pH6.0 to pH3.6 within 24h. The strains continued to produce both d and l lactic acid up to 144h, reaching a maximum concentration of 4.0g/l. Fermentations with pathogens inoculated at 104cfu/ml showed that the quick growth of the starters inhibited the growth of Listeria monocytogenes and Salmonella Enteritidis, as well as other enterobacteria. Denaturing gradient gel electrophoresis and 16S rRNA gene (V3-V4-region) amplicon sequencing showed that in the spontaneous fermentations a microbial succession took place, though with marked differences in biodiversity from fermentation to fermentation. The fermentations inoculated with starters however were clearly dominated by both the inoculated strains throughout the fermentations. RAPD-PCR fingerprinting showed that the strains established themselves at approx. equal proportions. Although vitamins C, B1 and B2 decreased during the fermentation, the final level of vitamin C in the product was an appreciable concentration of 35mg/100g. In conclusion, controlled fermentation of kale offers a promising avenue to prevent spoilage and improve the shelf life and safety.

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

Analysis of bacterial communities of traditional fermented West African cereal foods using culture independent methods.

In this study, the microbial composition of kunu-zaki and ogi, two popular foods in Nigeria produced after natural, uncontrolled fermentation of cereals, was assessed by culture-independent molecular profiling methods. In particular, PCR-denaturing gradient gel electrophoresis and construction of 16S rRNA gene clone libraries revealed the presence of diverse bacterial communities. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE fingerprints identified species related to Weissella confusa, Lactobacillus fermentum, Lactobacillus amylolyticus, Lactobacillus delbrueckii subsp. bulgaricus, Bacillus spp. and Lactococcus lactis spp lactis from food samples obtained from northern and southern geographical locations. A more comprehensive analysis of 272 full-length 16S rRNA gene inserts revealed that 70% of them were assigned to the Lactobacillaceae family and 19% to the Streptococcaceae family. Interestingly, sequences associated with a particular food type were also identified. For example, L. plantarum, L. pantheris and L. vaccinostercus were found in ogi but not in kunu-zaki while W. confusa, Streptococcus lutetiensis and Streptococcus gallolyticus subsp. macedonicus were found in kunu-zaki but not in ogi. Phylotypes corresponding to potentially pathogenic bacteria, such as Clostridium perfringens and Bacillus cereus were also detected highlighting the need for controlled fermentation processes.