The predominant lactic acid bacteria and yeasts involved in the spontaneous fermentation of millet during the production of the traditional porridge Hausa koko in Ghana.

Spontaneous fermentation of cereals like millet involves a diverse population of microbes from various sources, including raw materials, processing equipment, fermenting receptacles, and the environment. Here, we present data on the predominant microbial species and their succession at each stage of the Hausa koko production process from five regions of Ghana. The isolates were enumerated using selective media, purified, and phenotypically characterised. The LAB isolates were further characterised by 16S rRNA Sanger sequencing, typed using (GTG)5 repetitive-PCR, and whole genome sequencing, while 28S rRNA Sanger sequencing was performed for yeast identification. The pH of the millet grains ranged from mean values of 6.02-6.53 to 3.51-3.99 in the final product, depending on the processors. The mean LAB and yeast counts increased during fermentation then fell to final counts of log 2.77-3.95 CFU/g for LAB and log 2.10-2.98 CFU/g for yeast in Hausa koko samples. At the various processing stages, the counts of LAB and yeast revealed significant variations (p < 0.0001). The species of LAB identified in this study were Limosilactobacillus pontis, Pediococcus acidilactici, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Pediococcus pentosaceus, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Schleiferilactobacillus harbinensis, and Weissella confusa. The yeasts were Saccharomyces cf. cerevisiae/paradoxus, Saccharomyces cerevisiae, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis. The identification and sequencing of these novel isolates and how they change during the fermentation process will pave the way for future controlled fermentation, safer starter cultures, and identifying optimal stages for starter culture addition or nutritional interventions. These LAB and yeast species are linked to many indigenous African fermented foods, potentially acting as probiotics in some cases. This result serves as the basis for further studies into the technological and probiotic potential of these Hausa koko microorganisms.

Hollow-Structured Microporous Organic Networks Adsorbents Enabled Specific and Sensitive Identification and Determination of Aflatoxins.

Aflatoxin (AFT) contamination, commonly in foods and grains with extremely low content while high toxicity, has caused serious economic and health problems worldwide. Now researchers are making an effort to develop nanomaterials with remarkable adsorption capacity for the identification, determination and regulation of AFT. Herein, we constructed a novel hollow-structured microporous organic networks (HMONs) material. On the basis of Fe3O4@MOF@MON, hydrofluoric acid (HF) was introduced to remove the transferable metal organic framework (MOF) to give hollow MON structures. Compared to the original Fe3O4@MOF@MON, HMON showed improved surface area and typical hollow cavities, thus increasing the adsorption capacity. More importantly, AFT is a hydrophobic substance, and our constructed HMON had a higher water contact angle, greatly enhancing the adsorption affinity. From that, the solid phase extraction (SPE-HPLC) method developed based on HMONs was applied to analyze four kinds of actual samples, with satisfied recoveries of 85-98%. This work provided a specific and sensitive method for the identification and determination of AFT in the food matrix and demonstrated the great potential of HMONs in the field of the identification and control of mycotoxins.

The changes of microbial diversity and flavor compounds during the fermentation of millet Huangjiu, a traditional Chinese beverage.

Huangjiu is a national alcoholic beverage in China. Millet has congenital advantages in development and utilization of nutrient. Brewing Huangjiu with millet can increase the value of millet. Microbial community plays crucial roles in millet Huangjiu fermentation. Flavor compounds reflect the quality and health function of Huangjiu. The flavor compounds of Huangjiu are complex and their formation is closely associated with microorganisms, but the relationship between them during fermentation has been unknown. In this research, this relationship during millet Huangjiu fermentation were deeply investigated. Totally 86 volatile compounds were detected. Bacillus, Weissella, Paenibacillus, Klebsiella, Prevotella was investigated as the dominant microbes through high-throughput sequencing. 537 correlations between major flavor compounds and microbes were established to reflect the dynamic change during millet Huangjiu fermentation. The top five dominant genus of flavor producing microbes were Chryseobacterium, Sporolactobacillus, Psychrobacter, Sphingobium and Anoxybacillus. The content of malic acid and citric acid was gradually improved all through the millet Huangjiu fermentation. Malic acid and citric acid generated from millet Huangjiu fermentation shows healthy properties as liver protection and eliminating fatigue. Our research provides essential information on microbial community succession and the flavor formation during millet Huangjiu fermentation, and beneficial for development of Huangjiu products.

Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus.

Supernumerary mini-chromosomes-a unique type of genomic structural variation-have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.

Parallel validation of a green-solvent extraction method and quantitative estimation of multi-mycotoxins in staple cereals using LC-MS/MS.

In this study, 15 different mycotoxins were estimated in three staple cereals from selected agro-ecological regions in Nigeria using a 'novel' green extraction method, pressurized hot water extraction (PHWE) in comparison to a conventional solvent extraction method. Discrimination of the results of PHWE and solvent extraction using principal component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA) did not yield any differential clustering patterns. All maize samples (n = 16), 32% (n = 38) of sorghum and 35% (n = 37) of millet samples were positive for at least one of the 15 tested mycotoxins. Contamination levels for the cereals were higher in the warm humid rain forest region and gradually decreased towards the hot and arid region in the north of the country. The results demonstrate the applicability of PHWE as a possible alternative extraction method to conventional methods of extraction, which are solvent based.

Effects of Gamma Irradiation on Ochratoxin A Stability and Cytotoxicity in Methanolic Solutions and Potential Application in Tunisian Millet Samples.

Gamma irradiation is a useful technology for degrading mycotoxins. The purpose of this study was to investigate the effect of irradiation on ochratoxin A (OTA) stability under different conditions. OTA was irradiated in methanolic solution and on millet flour at doses of 2 and 4 kGy. Residual OTA concentrations and possible degradation products in irradiated samples were analyzed by high-performance liquid chromatography with fluorescence detection and liquid chromatography coupled to mass spectrometry. The extent of in vitro cytotoxicity of OTA to HepG2 cells, with and without irradiation treatment, was assessed with an MTT assay. OTA was more sensitive to gamma radiation on Tunisian millet flour than in methanolic solutions. After irradiation of naturally contaminated millet flour, the OTA concentration was significantly reduced by 48 and 62% at a dose of 2 and 4 kGy, respectively. However, in the methanolic solution, OTA at concentrations of 1 and 5 µg mL-1 was relatively stable even at a dose of 4 kGy, with no degradation products detected in the chemical analysis. Analytical results were confirmed by cell culture assays. The remaining cytotoxicity (MTT assay) of OTA following irradiation was not significantly affected compared with the controls. These findings indicate that gamma irradiation could offer a solution for OTA decontamination in the postharvest processing chain of millet flour. However, the associated toxicological hazard of decontaminated food matrices needs more investigation.

Simultaneous Direct Analysis of Aflatoxins and Ochratoxin A in Cereals and Their Processed Products by Ultra-High Performance Liquid Chromatography with Fluorescence Detection.

Background: Mycotoxins such as aflatoxins (AFs) and ochratoxin A (OTA) can pose severe health hazards because of their toxicity. Given a wide range of food matrices susceptible to fungal infections and possible cooccurrence of mycotoxins at different concentrations, validated multimycotoxin and multimatrix methods are strongly warranted. Objective: The aim of this research was to develop a simple and fast ultra-high performance LC (UHPLC) fluorescence detection (FLD)-based method to simultaneously determine AFs (B1, G1, B2, and G2) and OTA and, furthermore, to carry out single-laboratory validation in a range of cereals and processed product matrices. Methods: The sample preparation involved homogenization and extraction with methanol-water (80 + 20). For cleanup, an aliquot (3 mL) was diluted with phosphate-buffered saline, loaded on an immunoaffinity column (AFLAOCHRA PREP®), and eluted with methanol (1 mL). The cleaned extract was diluted with 0.2% acetic acid (at a 1:1 ratio) before injection into an ultra-high performance liquid chromatograph. To perform simultaneous analysis of AFs and OTA, the FLD program was developed by switching the excitation wavelength in a single chromatographic run. Results: The method provided LOQs of 0.25 and 1 ng/g for AFs and OTA, respectively, without involving any derivatization. In rice, the recoveries of AFs ranged from 84 to 106%, whereas OTA had a recovery above 72%, with the repeatability relative SDs <12% for both analytes. The method was successfully applied to a range of naturally contaminated market samples. Conclusions: The method is suitable for regulatory testing because of its significant time and cost effectiveness and sensitivity in compliance with the regulatory maximum levels. Highlights: The study achieves high-throughput analysis of AFs and OTA in raw and processed cereals using simultaneous extraction, cleanup, and UHPLC-FLD. Method sensitivity complies with the regulatory maximum levels. Single-laboratory validation results meet analytical QC requirements.

Blast Fungal Genomes Show Frequent Chromosomal Changes, Gene Gains and Losses, and Effector Gene Turnover.

Pyricularia is a fungal genus comprising several pathogenic species causing the blast disease in monocots. Pyricularia oryzae, the best-known species, infects rice, wheat, finger millet, and other crops. As past comparative and population genomics studies mainly focused on isolates of P. oryzae, the genomes of the other Pyricularia species have not been well explored. In this study, we obtained a chromosomal-level genome assembly of the finger millet isolate P. oryzae MZ5-1-6 and also highly contiguous assemblies of Pyricularia sp. LS, P. grisea, and P. pennisetigena. The differences in the genomic content of repetitive DNA sequences could largely explain the variation in genome size among these new genomes. Moreover, we found extensive gene gains and losses and structural changes among Pyricularia genomes, including a large interchromosomal translocation. We searched for homologs of known blast effectors across fungal taxa and found that most avirulence effectors are specific to Pyricularia, whereas many other effectors share homologs with distant fungal taxa. In particular, we discovered a novel effector family with metalloprotease activity, distinct from the well-known AVR-Pita family. We predicted 751 gene families containing putative effectors in 7 Pyricularia genomes and found that 60 of them showed differential expression in the P. oryzae MZ5-1-6 transcriptomes obtained under experimental conditions mimicking the pathogen infection process. In summary, this study increased our understanding of the structural, functional, and evolutionary genomics of the blast pathogen and identified new potential effector genes, providing useful data for developing crops with durable resistance.

Lactococci dominate the bacterial communities of fermented maize, sorghum and millet slurries in Zimbabwe.

Maize, sorghum and millet fermented porridges are important as complementary foods for young children in Africa. The objective of the present study was to gain some insights into the bacterial communities of fermented slurries prepared from maize, sorghum and millets originating from different locations in Zimbabwe, and prepared either at household or laboratory level. A deep sequencing approach targeting the hypervariable V4 region of the 16S rRNA gene was used and yielded about 100,000 sequences per sample. Lactococcus dominated all the fermented slurries, flanked by other lactic acid bacteria such as Weissella, Leuconostoc and Enterococcus. Enterobacteriaceae detected in the water samples persisted throughout all the fermented cereals. Other sub-dominant bacteria identified in the fermented slurries included Aeromonas, Pseudomonas and Acinetobacter. In addition, some Proteobacteria, Actinobacteria and Bacteroidetes associated with the raw materials and environment were also detected. Fermented slurries could not be differentiated based on their origin nor on the type of fermentation, but clear differences were observed between red sorghum fermented slurries and fermented slurries prepared from other cereal flours. A thorough understanding of the functional capacities of the microbiota in African fermented slurries is highly needed in order to steer the fermentation for the production of standard, safe and nutritious fermented products.

Iron and zinc bioaccessibility of fermented maize, sorghum and millets from five locations in Zimbabwe.

The present study is an evaluation of iron and zinc bioaccessibility of fermented maize, sorghum, pearl millet and finger millet from five different locations in Zimbabwe. Iron and zinc contents ranged between 3.22 and 49.7 and 1.25-4.39mg/100gdm, respectively. Fermentation caused a reduction of between 20 and 88% of phytic acid (PA) while a general increase in soluble phenolic compounds (PC) and a decrease of the bound (PC) was observed. Bioaccessibility of iron and zinc ranged between 2.77 and 26.1% and 0.45-12.8%, respectively. The contribution of the fermented cereals towards iron and zinc absolute requirements ranged between 25 and 411% and 0.5-23% with higher contribution of iron coming from cereals that were contaminated with extrinsic iron. Populations subsisting on cereals could be more at risk of zinc rather than iron deficiency.

Quantitation of Mycotoxins Using Direct Analysis in Real Time Mass Spectrometry (DART-MS).

Ambient ionization represents a new generation of MS ion sources and is used for the rapid ionization of small molecules under ambient conditions. The combination of ambient ionization and MS allows the analysis of multiple food samples with simple or no sample treatment or in conjunction with prevailing sample preparation methods. Two ambient ionization methods, desorptive electrospray ionization (DESI) and direct analysis in real time (DART) have been adapted for food safety application. Both ionization techniques provide unique advantages and capabilities. DART has been used for a variety of qualitative and quantitative applications. In particular, mycotoxin contamination of food and feed materials has been addressed by DART-MS. Applications to mycotoxin analysis by ambient ionization MS and particularly DART-MS are summarized.

Indigenous endophytic seed bacteria promote seedling development and defend against fungal disease in browntop millet (Urochloa ramosa L.).

AIMS: This study was conducted to investigate indigenous seed endophyte effects on browntop millet seedling development. We report that seed-inhabiting bacterial endophytes are responsible for promoting seedling development, including stimulation of root hair formation, increasing root and shoot length growth and increasing photosynthetic pigment content of seedlings. Bacterial endophytes also improved resistance of seedlings to disease. METHODS AND RESULTS: A total of four endophytic bacteria were isolated from surface-sterilized seeds and identified by 16S rDNA sequencing as Curtobacterium sp. (M1), Microbacterium sp. (M2), Methylobacterium sp. (M3) and Bacillus amyloliquefaciens (M4). Removal of bacteria with streptomycin treatment from the seeds compromised seedling growth and development. When endophytes were reinoculated onto seeds, seedlings recovered normal development. Strains M3 and M4 were found to be most potent in promoting growth of seedlings. Bacteria were found to produce auxin, solubilize phosphate and inhibit fungal pathogens. Significant protection of seedlings from Fusarium infection was found using strain M4 in microcosm assays. The antifungal lipopeptide genes for surfactin and iturin were detected in M4; culture extracts of M4 showed a positive drop collapse result for surfactins. CONCLUSIONS: This study demonstrates that browntop millet seeds vector indigenous endophytes that are responsible for modulation of seedling development and protection of seedlings from fungal disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is significant and original in that it is the first report of seed-inhabiting endophytes of browntop millet that influence seedling development and function in defence against soilborne pathogens. This study suggests that conservation and management of seed-vectored endophytes may be important in development of more sustainable agricultural practices.

Manure and mineral fertilization change enzyme activity and bacterial community in millet rhizosphere soils.

Fertilization is a key agricultural practice for increasing millet yields and influencing soil properties, enzyme activities and rhizosphere bacterial communities. High throughput Illumina sequencing of the 16S rDNA was applied to compare the bacterial community structures and diversities among six different soil samples. The experiments involved the following: no fertilizer (CK), phosphate (P) and potassium (K) plus organic manure (M) (PKM), nitrogen (N) and K plus M (NKM), NPM, NPK and NPKM fertilization. The results showed that the NPKM fertilization of the millet field had a maximal yield of 3617 kg ha-1 among the six different treatments. The abundances of the Actinobacteria and Bacteroidetes phyla, especially the Devosia, Mycobacterium, Opitutus and Chitinophaga genera, were higher in NPKM than those in the other samples. Redundancy analysis showed that the soil organic matter (SOM), available phosphorus (AP), and urease (UR) activity were significantly correlated with bacterial communities, while SOM and AP were strongly correlated with soil enzyme activities. Pearson's correlation showed that the available nitrogen was strongly correlated with Devosia and Mycobacterium, and SOM was strongly correlated with Opitutus and Chitinophaga. Besides, catalase was significantly related to Iamia, the UR was significantly related to Devosia, phosphatase was significantly related to Luteimonas and Chitinophaga. Based on the soil quality and millet yield, NPKM treatment was a better choice for the millet field fertilization practices. These findings provide a better understanding of the importance of fertilization in influencing millet yield, soil fertility and microbial diversity, and they lead to a choice of scientific fertilization practices for sustainable development of the agroecosystem.

Taxonomic structure and functional association of foxtail millet root microbiome.

The root microbes play pivotal roles in plant productivity, nutrient uptakes, and disease resistance. The root microbial community structure has been extensively investigated by 16S/18S/ITS amplicons and metagenomic sequencing in crops and model plants. However, the functional associations between root microbes and host plant growth are poorly understood. This work investigates the root bacterial community of foxtail millet (Setaria italica) and its potential effects on host plant productivity. We determined the bacterial composition of 2882 samples from foxtail millet rhizoplane, rhizosphere and corresponding bulk soils from 2 well-separated geographic locations by 16S rRNA gene amplicon sequencing. We identified 16 109 operational taxonomic units (OTUs), and defined 187 OTUs as shared rhizoplane core OTUs. The ß-diversity analysis revealed that microhabitat was the major factor shaping foxtail millet root bacterial community, followed by geographic locations. Large-scale association analysis identified the potential beneficial bacteria correlated with plant high productivity. Besides, the functional prediction revealed specific pathways enriched in foxtail millet rhizoplane bacterial community. We systematically described the root bacterial community structure of foxtail millet and found its core rhizoplane bacterial members. Our results demonstrated that host plants enrich specific bacteria and functions in the rhizoplane. The potentially beneficial bacteria may serve as a valuable knowledge foundation for bio-fertilizer development in agriculture.

A Zymogram technique for preliminary screening and characterization of feruloyl esterases.

We report here, for the first time, a zymogram technique designed for rapid screening of feruloyl esterase using ethyl ferulate as enzyme substrate and casein precipitation as enzyme activity indicator.

Technological properties and probiotic potential of Lactobacillus fermentum strains isolated from West African fermented millet dough.

BACKGROUND: Throughout Africa, food fermentations are still driven by indigenous microorganisms which influence the nutritional, organoleptic and safety of the final products. However, for improved safety, consistent quality and beneficial health effects, a trend has emerged which involves the isolation of indigenous strains from traditional fermented products to be used as functional starter cultures. These functional starter cultures possess inherent functional characteristics and can contribute to food quality and safety by offering one or more organoleptic, nutritional, technological or health advantage (probiotics). With the aim of selecting potential probiotic starter cultures, Lactobacillus fermentum strains isolated from fermented millet dough were investigated for technological properties and probiotic traits in-vitro. RESULTS: A total of 176 L. fermentum strains were assessed for technological properties including rate of acidification, exopolysaccharide production and amylase activity. Following this, 48 strains showing desirable technological properties were first screened for acid resistance. Sixteen acid resistant strains were assessed for additional probiotic properties including resistance to bile salts, bile salt hydrolysis, antimicrobial property, haemolysis and antibiotics resistance. L. fermentum strains clustered into 3 groups represented by 36 %, 47 % and 17 % as fast, medium and slow acidifiers respectively. About 8 %, 78 % and 14 % of the strains showed strong, weak and no exopolysaccharides production respectively. Amylase activity was generally weak or not detected. After exposure of 48 L. fermentum strains to pH 2.5 for 4 h, 16 strains were considered to be acid resistant. All 16 strains were resistant to bile salt. Four strains demonstrated bile salt hydrolysis. Antimicrobial activity was observed towards Listeria monocytogenes and Staphylococcus aureus but not E. coli and Salmonella enteritidis. Lactobacillus fermentum strains were generally susceptible to antibiotics except 6 strains which showed resistance towards streptomycin, gentamicin and kanamycin. CONCLUSION: In vitro determination of technological and probiotic properties have shown strain specific difference among L. fermentum strains isolated from fermented millet dough. Sixteen (16) L. fermentum strains have been shown to possess desirable technological and probiotic characteristics in vitro. These strains are therefore good candidates for further studies to elucidate their full potential and possible application as novel probiotic starter cultures.