Charles Muyanja

Isolation, characterisation and identification of lactic acid bacteria from bushera: a Ugandan traditional fermented beverage

One hundred and thirteen strains of lactic acid bacteria (LAB) were selected from 351 isolates from 15 samples of traditionally fermented household bushera from Uganda and also from laboratory-prepared bushera. Isolates were phenotypically characterised by their ability to ferment 49 carbohydrates using API 50 CHL kits and additional biochemical tests. Coliforms, yeasts and LAB were enumerated in bushera. The pH, volatile organic compounds and organic acids were also determined. The LAB counts in household bushera varied between 7.1 and 9.4 log cfu ml(-1). The coliform counts varied between < 1 and 5.2 log cfu ml(-1). The pH of bushera ranged from 3.7 to 4.5. Ethanol (max, 0.27%) was the major volatile organic compound while lactic acid (max, 0.52%) was identified as the dominant organic acid in household bushera. The initial numbers of LAB and coliforms in laboratory-fermented bushera were similar; however, the LAB numbers increased faster during the first 24 h. LAB counts increased from 5.5 to 9.0 log cfu ml(-1) during the laboratory fermentation. Coliform counts increased from 5.9 to 7.8 log cfu ml(-1) at 24 h, but after 48 h, counts were less 4 log cfu ml(-1). Yeasts increased from 4.3 to 7.7 log cfu ml(-1) at 48 h, but thereafter decreased slightly. The pH declined from 7.0 to around 4.0. Lactic acid and ethanol increased from zero to 0.75% and 0.20%, respectively. Lactic acid bacteria isolated from household bushera belonged to Lactobacillus, Streptococcus and Enterococcus genera. Tentatively, Lactobacillus isolates were identified as Lactobacillus plantarum, L. paracasei subsp. paracasei, L. fermentum, L. brevis and L. delbrueckii subsp. delbrueckii. Streptococcus thermophilus strains were also identified in household bushera. LAB isolated from bushera produced in the laboratory belonged to five genera (Lactococcus, Leuconostoc, Lactobacillus, Weissella and Enterococcus. Eight isolates were able to produce acid from starch and were identified as Lactococcus lactis subsp. lactis (four strains), Leuconostoc mesenteroides subsp. mesenteroides (one strain), Leuconostoc mesenteroides subsp. dextranicum (one strain), Weissella confusa (one strain) and L. plantarum (one strain).

Survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure and manure‐amended soil under tropical climatic conditions in Sub‐Saharan Africa

Aims:  To establish the fate of Escherichia coli O157:H7 and Salmonella Typhimurium in manure and manure‐amended agricultural soils under tropical conditions in Sub‐Saharan Africa.

Fate of Escherichia coli O157:H7 and Salmonella enterica in the manure-amended soil-plant ecosystem of fresh vegetable crops: A review

Abstract Enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica have been implicated in several disease outbreaks linked to consumption of fresh vegetables. Both ruminant and non-ruminant animals carry EHEC and S. enterica in their gastrointestinal tracts and can shed the pathogens in the faecal matter both in symptomatic and asymptomatic states. Application of animal waste in soil fertility management and irrigation of crops with contaminated waste water has been recognised as an important route through which EHEC and S. enterica can contaminate fresh vegetables during primary production. The behavior of E. coli O157:H7 and S. enterica in the agricultural environment has been extensively studied in the last decades. Several microbiological detection methods have been applied. This review therefore puts together current knowledge on the behavior of E. coli O157:H7 and S. enterica in the manure-amended soil-plant ecosystem of fresh vegetable crops during cultivation under various environmental conditions. The review focuses on methodological issues involved in undertaking survival studies and makes comparative analysis of experimental results obtained from studies conducted under controlled environmental conditions integrating results obtained from field experiments. Finally, a theoretical discussion on the potential likely impact of climate change on pre-harvest safety of field-cultivated vegetables is highlighted.

The dominant microbial community associated with fermentation of Obushera (sorghum and millet beverages) determined by culture-dependent and culture-independent methods

Obushera includes four fermented cereal beverages from Uganda namely: Obutoko, Enturire, Ekitiribita and Obuteire, whose microbial diversity has not hitherto been fully investigated. Knowledge of the microbial diversity and dynamics in these products is crucial for understanding their safety and development of appropriate starter cultures for controlled industrial processing. Culture-dependent and culture-independent techniques including denaturating gradient gel electrophoresis (DGGE) and mixed DNA sequencing of polymerase chain reaction (PCR) amplified ribosomal RNA genes were used to study the bacteria and yeast diversity of Obushera. The pH dropped from 6.0-4.6 to 3.5-4.0 within 1-2 days for Obutoko, Enturire and Obuteire whereas that of Ekitiribita decreased to 4.4 after 4 days. Counts of lactic acid bacteria (LAB) increased from 5.0 to 11.0 log cfug(-1) and yeasts increased from 3.4 to 7.1 log cfug(-1) while coliform counts decreased from 2.0 to <1 log cfug(-1) during four days of fermentation. LAB and yeast isolates were identified by rRNA gene sequence analysis. LAB isolates included: Enterococcus spp., Lactobacillus (Lb.) plantarum, Lb. fermentum, Lb. delbrueckii, Lactococcus lactis, Leuconostoc lactis, Streptococcus (S.) infantarius subsp. infantarius, Pediococcus pentosaceus and Weisella (W.) confusa. DGGE indicated predominance of S. gallolyticus, S. infantarius subsp. infantarius, Lb. fermentum, Lb. delbrueckii, W. confusa, Lb. reuteri, Fructobacillus spp., L. lactis and L. lactis. Yeast isolates included Clavispora lusitaniae, Cyberlindnera fabianii, Issatchenkia orientalis and Saccharomyces cerevisiae. DGGE indicated predominance of S. cerevisiae in Obutoko, Enturire and Obuteire and also detected Pichia spp. and I. orientalis in Obutoko. Obushera produced in the laboratory was initially dominated by Enterobacteriaceae and later by Lactococcus spp. Enterobacteriaceae and Bacillus spp. were also detected in Ekitiribita. Development of starters for Obushera may require combinations of LAB and S. cerevisiae for Obutoko, Enturire and Obuteire and LAB for Ekitiribita.

Transfer and internalisation of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in cabbage cultivated on contaminated manure-amended soil under tropical field conditions in Sub-Saharan Africa

Surface contamination and internalisation of Escherichia coli O157:H7 and Salmonella Typhimurium in cabbage leaf tissues at harvest (120 days post-transplantation) following amendment of contaminated bovine manure to soil at different times during crop cultivation were investigated under tropical field conditions in the Central Agro-Ecological Zone of Uganda. Fresh bovine manure inoculated with rifampicin-resistant derivatives of non-virulent strains of E. coli O157:H7 and S. Typhimurium was incorporated into the soil to achieve inoculum concentrations of 4 and 7 log CFU/g at the point of transplantation, 56 or 105 days post-transplantation of cabbage seedlings. Frequent sampling of the soil enabled the accurate identification of the survival kinetics in soil, which could be described by the Double Weibull model in all but one of the cases. The persistence of 4 log CFU/g E. coli O157:H7 and S. Typhimurium in the soil was limited, i.e. only inocula applied 105 days post-transplantation were still present at harvest. Moreover, no internalisation in cabbage leaf tissues was observed. In contrast, at the 7 log CFU/g inoculum level, E. coli O157:H7 and S. Typhimurium survived in the soil throughout the cultivation period. All plants (18/18) examined for leaf contamination were positive for E. coli O157:H7 at harvest irrespective of the time of manure application. A similar incidence of leaf contamination was found for S. Typhimurium. On the other hand, only plants (18/18) cultivated on soil amended with contaminated manure at the point of transplantation showed internalised E. coli O157:H7 and S. Typhimurium at harvest. These results demonstrate that under tropical field conditions, the risk of surface contamination and internalisation of E. coli O157:H7 and S. Typhimurium in cabbage leaf tissues at harvest depend on the inoculum concentration and the time of manure application. Moreover, the internalisation of E. coli O157:H7 and S. Typhimurium in cabbage leaf tissues at harvest seems to be limited to the worst case situation, i.e., when highly contaminated manure is introduced into the soil at the time of transplantation of cabbage seedlings.

Kitchen practices used in handling broiler chickens and survival of Campylobacter spp. on cutting surfaces in Kampala, Uganda.

Cross-contamination during food preparation has been identified as an important factor associated with foodborne illnesses. Handling practices used during preparation of broiler chickens in 31 fast-food restaurants and 86 semisettled street stands (street vendors) were assessed by use of a standard checklist. These establishments used wood, plastic, or metal cutting surfaces during the preparation of broiler chickens. The survival of Campylobacter spp. on kitchen cutting surfaces was determined by inoculating approximately 10(6) CFU of Campylobacter jejuni onto sterile plastic, wooden, and metal cutting boards. The concentrations of the organisms were then assessed in triplicate on each type of cutting board over a 3-h period using standard microbiological methods for thermophilic Campylobacter spp. In 87% of food establishments, the same work area was used for preparation of raw and cooked chicken, and in 68% of these establishments the same cutting boards were used for raw and cooked chicken. None of the establishments applied disinfectants or sanitizers when washing contact surfaces. Campylobacter spp. survived on wooden and plastic but not on metal cutting boards after 3 h of exposure. The handling practices in food preparation areas, therefore, provide an opportunity for cross-contamination of Campylobacter spp. to ready-to-eat foods.

Organic Acids and Volatile Organic Compounds Produced During Traditional and Starter Culture Fermentation of Bushera, a Ugandan Fermented Cereal Beverage

Starter cultures of lactic acid bacteria (Lactobacillus fermentum MINF99, Weissella confusa MINF8, Lactobacillus plantarum MINF277, Lactobacillus brevis MINF226, and Lactobacillus paracasei subsp paracasei MINF98) were used to ferment Bushera during fermentation (96 h). Organic acids and volatile compounds produced during starter and natural fermentation were investigated. Microbial counts, pH, and sugars were also determined. LAB counts increased from 5.87 ± 0.00 to 8.32 ± 0.02 log cfu mL−1 while yeasts increased from 4.39 ± 0.02 to 7.10 ± 0.04 log cfu mL−1 during natural fermentation. The pH decreased from 6.5 to 3.55–4.0. W. confusa MINF8 attained similar final pH (3.55) as naturally fermented Bushera. Lactate was the dominant acid and varied between 0.34% and 0.66%. W. confusa MINF8 produced the highest amounts of lactate (0.66%). Lactate content in naturally fermented Bushera was 0.89%, 96 h. Glucose and maltose decreased from 8.64–9.27 g kg−1 to 0.13–2.11 gkg−1 and 7.95–8.42 g kg−1 to 0.06–2.66 g kg−1, respectively, in all starter fermented Bushera within 24 h. Glucose and maltose increased during the first 24 h of natural fermentation and then decreased. No citrate was detectable after 24 h. Succinate, pyruvate, and pyro-glutamate were detected and varied between 1.44 and 1495.93 mg kg−1. Methyl alcohols increased with fermentation time and ranged between 0.02 and 0.53 mg kg−1. Methyl alcohols concentration of naturally fermented Bushera were significantly higher (P < 0.05) than those of starters after 24 h. Acetaldehyde levels detected in starter fermented bushera was below 5 mg kg−1 compared to 17.6 mg kg−1 of naturally fermented Bushera. All starters except L. paracasei subsp. paracasei MINF98 reduced the methyl aldehydes within 12 h to levels ranging between 0.01 and 0.06 mg kg−1. Ethyl alcohol was the predominant volatile compound and ranged from 2.16 to 1406 mg kg−1. W. confusa MINF8 produced the highest amounts of ethyl alcohol (1406 g kg−1). Maximum ethyl alcohol amount detected in naturally fermented Bushera was 10.4 g kg−1. Other volatiles detected were butanone, 2, 3-pentanone, diacetyl and ethyl acetate. The study has shown that all starters have ability to singly ferment Bushera. The varying amounts of the volatile compounds observed may influence the sensory properties of Bushera and may be the basis for selecting suitable starters for commercial production.

Rhizosphere effect on survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure-amended soil during cabbage (Brassica oleracea) cultivation under tropical field conditions in Sub-Saharan Africa.

The effect of cabbage (Brassica oleracea) rhizosphere on survival of Escherichia coli O157:H7 and Salmonella Typhimurium in manure-amended soils under tropical field conditions was investigated in the Central Agro-Ecological Zone of Uganda. Three-week old cabbage seedlings were transplanted and cultivated for 120 days on manure-amended soil inoculated with 4 or 7 log CFU/g non-virulent E. coli O157:H7 and S. Typhimurium. Cabbage rhizosphere did not affect survival of the 4log CFU/g inocula in manure-amended soil and the two enteric bacteria were not detected on/in cabbage leaves at harvest. The 7 log CFU/g E. coli O157:H7 and S. Typhimurium survived in bulk soil for a maximum of 80 and 96 days, respectively, but the organisms remained culturable in cabbage rhizosphere up to the time of harvest. At 7 log CFU/g inoculum, E. coli O157:H7 and S. Typhimurium contamination on cabbage leaves occurred throughout the cultivation period. Leaf surface sterilisation with 1% AgNO(3) indicated that the organisms were present superficially and in protected locations on the leaves. These results demonstrate that under tropical field conditions, cabbage rhizosphere enhances the persistence of E. coli O157:H7 and S. Typhimurium in manure-amended soil at high inoculum density and is associated with long-term contamination of the leaves.

Influence of Cofermentation by Amylolytic Lactobacillus plantarum and Lactococcus lactis Strains on the Fermentation Process and Rheology of Sorghum Porridge

ABSTRACT Amylolytic lactic acid bacteria (ALAB) can potentially replace malt in reducing the viscosity of starchy porridges. However, the drawback of using ALAB is their low and delayed amylolytic activity. This necessitates searching for efficient ALAB and strategies to improve their amylolytic activity. Two ALAB, Lactobacillus plantarum MNC 21 and Lactococcus lactis MNC 24, isolated from Obushera, were used to ferment starches in MRS broth: sorghum, millet, sweet potato, and commercial soluble starch. The amylolytic activity of MNC 21 was comparable to that of the ALAB collection strain Lb. plantarum A6, while that of MNC 24 was extremely low. MNC 21, MNC 24, and their coculture were compared to A6 and sorghum malt for ability to ferment and reduce the viscosity of sorghum porridge (11.6% dry matter). ALAB and the coculture lowered the pH from 6.2 to <4.5 within 12 h, while malt as a carrier of wild starter took about 20 h. Coculturing increased lactic acid yield by 46% and 76.8% compared to the yields of MNC 21 and MNC 24 monocultures, respectively. The coculture accumulated significantly larger (P < 0.05) amounts of maltose and diacetyl than the monocultures. Sorghum malt control and the coculture hydrolyzed more starch in sorghum porridge than the monocultures. The coculture initiated changes in the rheological parameters storage modulus (G′), loss modulus (G″), phase angle (δ), and complex viscosity (η*) earlier than its constituent monocultures. The shear viscosity of sorghum porridge was reduced significantly (P < 0.05) from 1950 cP to 110 cP (malt), 281 cP (coculture), 382 cP (MNC 21), 713 cP (MNC 24), and 722 cP (A6). Coculturing strong ALAB with weak ALAB or non-ALAB can be exploited for preparation of nutrient-dense weaning foods and increasing lactic acid yield from starchy materials.

Kinetic model-based prediction of the persistence of Salmonella enterica serovar Typhimurium under tropical agricultural field conditions.

Aim:  Present a kinetic model‐based approach for using isothermal data to predict the survival of manure‐borne enteric bacteria under dynamic conditions in an agricultural environment.