Effect of milk fermentation by kefir grains and selected single strains of lactic acid bacteria on the survival of Mycobacterium bovis BCG.

Mycobacterium bovis that causes Bovine tuberculosis (BTB) can be transmitted to humans thought consumption of raw and raw fermented milk products from diseased animals. Lactic acid bacteria (LAB) used in popular traditional milk products in Africa produce anti-microbial compounds that inhibit some pathogenic and spoilage bacteria. M. bovis BCG is an attenuated non-pathogenic vaccine strain of M. bovis and the aim of the study was to determine the effect of the fermentation process on the survival of M. bovis BCG in milk. M. bovis BCG at concentrations of 6 log CFU/ml was added to products of kefir fermentation. The survival of M. bovis BCG was monitored at 12-h intervals for 72 h by enumerating viable cells on Middlebrook 7H10 agar plates enriched with 2% BD BACTEC PANTA™. M. bovis BCG was increasingly reduced in sterile kefir that was fermented for a period of 24h and longer. In the milk fermented with kefir grains, Lactobacillus paracasei subsp. paracasei or Lactobacillus casei, the viability of M. bovis BCG was reduced by 0.4 logs after 24h and by 2 logs after 48 h of fermentation. No viable M. bovis BCG was detected after 60 h of fermentation. Results from this study show that long term fermentation under certain conditions may have the potential to inactivate M. bovis BCG present in the milk. However, to ensure safety of fermented milk in Africa, fermentation should be combined with other hurdle technologies such as boiling and milk pasteurisation.

Prevention of the accumulation of Alicyclobacillus in apple concentrate by restricting the continuous process running time.

AIMS: To study the accumulation of vegetative cells and endospores of Alicyclobacillus, as well as viable aerobic counts during the continuous production of apple juice concentrate. METHODS AND RESULTS: Apples were processed for a continuous process running time of 108 h (processing rate 1·8-2·0 t h(-1) ) without clean-in-place (CIP) procedures in-between different batches. Samples from single-strength apple juice, concentrate after evaporation (± 30°Brix), the final product (concentrate pasteurized at 102-104°C for 90 s) and condensate water (by-product of the juice concentration process) were collected every 12 h. From 12 to 84 h of processing, vegetative Alicyclobacillus counts in single-strength apple juice increased significantly (P < 0·05) from 1 to 3·15 log(10) CFU ml(-1). Accumulation patterns of vegetative cells in apple concentrate and the final product were similar from 24 to 84 h of processing, with the respective counts increasing from 0·13 to 1·63 and 0·01 to 1·69 log(10) CFU ml(-1). The highest Alicyclobacillus endospore counts in single-strength juice, concentrate and the final product was at 84 h of processing with 1·32, 1·59 and 1·64 log(10) CFU ml(-1), respectively. CONCLUSIONS: Alicyclobacillus vegetative cells and endospores accumulate in fruit concentrates during a continuous process running time of 108 h. SIGNIFICANCE AND IMPACT OF THE STUDY: In conjunction with good manufacturing practices, fruit concentrate manufactures can minimize Alicyclobacillus accumulation in fruit concentrates by limiting the continuous process running time between clean-ups to under 84 h.

Physico-chemical, microbiological, textural and sensory attributes of matured game salami produced from springbok (Antidorcas marsupialis), gemsbok (Oryx gazella), kudu (Tragelaphus strepsiceros) and zebra (Equus burchelli) harvested in Namibia.

Differences and consumer acceptability of matured salami produced from game species were evaluated. The pH of the salami differed (p < 0.05) with springbok salami having the highest mean pH value. No differences (p > 0.05) were observed among the species for a(w), shear force, gumminess or cohesiveness. Microbiological counts of the game salami differed for coliform (p < 0.05) but not for E. coli (p > 0.05) counts. The most distinctive characteristics observed by the quantitative descriptive analyses were smoky, salty, pepper and salami flavour, combined with a smoky, salami aroma. Game flavour was not perceived as a strong attribute during the sensory analyses. Gemsbok salami was strongly associated with the attribute colour as described by the male and female consumer panels. The springbok salami scored the lowest for both colour and taste. Salami produced from gemsbok, kudu and zebra were superior to springbok salami.

Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide.

AIMS: The detection of viable Enterobacter sakazakii cells is important due to the association of this pathogen with outbreaks of life-threatening neonatal infections. The aim of this study was to optimize a PCR-based method for selective detection of only viable Ent. sakazakii cells in the presence of dead cells, utilizing propidium monoazide (PMA) or ethidium bromide monoazide (EMA). METHODS AND RESULTS: PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 microg ml(-1)) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 microg ml(-1), PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the amplification from viable cells. PMA was also effective in allowing selective PCR detection of only viable cells in mixtures of varying ratios of viable and dead cells. EMA was equally effective in preventing amplification from dead cells, however, it also inhibited DNA amplification from viable cells. CONCLUSIONS: This study demonstrated the efficiency of PMA for viable and dead differentiation of Ent. sakazakii, as well as the lack of selectivity of EMA for this purpose. SIGNIFICANCE AND IMPACT OF THE STUDY: PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment.

Evaluation of different growth media for the recovery of the species of Alicyclobacillus.

AIMS: Five different isolation media, namely potato dextrose agar (PDA), orange serum agar (OSA), K agar, yeast-starch-glucose agar and Bacillus acidocaldarius medium were evaluated for the recovery of Alicyclobacillus spp. from inoculated diluted and undiluted fruit-juice concentrates. METHODS AND RESULTS: Plates of PDA (pH 3.7), spread with vegetative cells (3.9 x 10(6) CFU ml(-1)) of Alicyclobacillus acidoterrestris from single-strength pear juice, recovered 2.9 x 10(6 )CFU ml(-1) after 5 days at 50 degrees C (74% recovery). The recovery of endospores from single-strength pear juice, after a heat treatment at 80 degrees C for 10 min, was higher on spread plates of OSA (pH 5.5) at 50 degrees C for 5 days (97% recovery). CONCLUSIONS: PDA (pH 3.7) and OSA (pH 5.5) at 50 degrees C for 3-5 days recovered the highest numbers of vegative cells and endospores of Alicyclobacillus spp. from sterilized fruit juices and concentrates. SIGNIFICANCE AND IMPACT OF THE STUDY: The most appropriate synthetic media for the recovery of Alicyclobacillus species from inoculated fruit juices and concentrates are shown.

PCR-based DGGE fingerprinting and identification of methanogens detected in three different types of UASB granules.

Methane is produced by various methanogenic bacteria present in upflow anaerobic sludge blanket (UASB) bioreactors. Methane can be used to predict and improve UASB bioreactor efficiency. The methanogen population in the granules can be influenced by the composition of the substrate. The aim of this study was to fingerprint and identify the methanogens present in three different types of UASB granules that had been used to treat winery, brewery and peach-lye canning effluents. This was done using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. The DGGE fingerprints obtained from the methanogen reference cultures of Methanosaeta concilii, Methanosaeta thermophila, Methanosarcina barkeri, Methanosarcina mazeii and Methanobacterium formicicum were compared to the DGGE profiles of the Archaea in the different granules. The positions of the DGGE bands that did not correspond well to the bands of the known species were sequenced and compared to sequences available on GenBank using the Blastn search option. The aligned DNA sequences were used to construct a phylogenetic tree. Based on the data obtained, a DGGE marker was constructed which was used to provide a quick method to identify the Archaeal members of the microbial consortium in UASB granules.

Microbial content of commercial South African high-moisture dried fruits.

AIM: The aim was to evaluate commercially available South African high-moisture dried fruits (HMDF) for the microbial, moisture and SO2 contents, as well as aw and pH. METHODS AND RESULTS: The microbial content of commercially available HMDF was evaluated using nine different growth media. The moisture content, aw) SO2 and pH of each product were determined using standard analytical methods. It was found that the highest total aerobic counts were generated from high-moisture dried (HMD) prunes and raisins. The most frequent spoilers were members of the genus Bacillus. Fungal counts were also very high in the apricot products, exceeding the limit of 1000 CFU g(-1) as set by HMDF producers. Members of the genus Staphylococcus were found in the HMD raisins and Salmonella and thermoduric organisms were isolated from the HMD prunes. CONCLUSIONS: The microbial levels of South African HMDF were within the limits set, with the exception of apricots. SIGNIFICANCE AND IMPACT OF STUDY: The study shows the presence of Salmonella, Staphylococcus and Clostridium in South African HMDF. The presence of thermoduric organisms indicated that the current pasteurization process is not adequate and that the addition of preservatives would be an additional method to ensure safety and quality.

Identification of probiotic microorganisms in South African products using PCR-based DGGE analysis.

Probiotic microorganisms in commercial yoghurts and other food products are currently identified by traditional methods such as growth on selective media, morphological and biochemical characteristics. In this study, PCR-based DGGE analysis was used for the rapid and accurate identification of probiotic microorganisms from South African yoghurts and lyophilized preparations in capsule and tablet form. To identify the microorganisms present in these products, the DGGE profiles obtained were compared to two reference markers (A and B) composed of five lactobacilli and seven Bifidobacterium species, respectively. The results obtained were confirmed by species-specific PCR, as well as sequence analyses of unknown bands not present in the reference markers. It was found that only 54.5% of the probiotic yoghurts contained the microorganisms stated on the label compared to only a third (33.3%) of the lyophilized probiotic products. Some Bifidobacterium species were incorrectly identified and various microorganisms were detected that were not listed on the label. Sequence analyses confirmed the presence of Streptococcus spp. other than the yoghurt starter, Streptococcus thermophilus, in some of these products and in some instances label information was vague and non-scientific. PCR-based DGGE analyses proved to be a valuable culture-independent approach for the rapid and specific identification of the microbial species present in South African probiotic products.

Treatment of winery effluent with upflow anaerobic sludge blanket (UASB)--granular sludges enriched with Enterobacter sakazakii.

Three upflow anaerobic sludge blankets (UASBs) were evaluated for the treatment of winery wastewater: the first was seeded with granular sludge enriched with Enterobacter sakazakii and reached a 90% COD removal within 17 d at hydraulic retention time of 24 h; the second was seeded with brewery granules and achieved 85% COD removal within 50 d, the third was seeded with just sludge and showed the typical problems encountered with conventional sludge seeding and had continuously to be re-seeded. A PCR-based technique was developed for the rapid detection of E. sakazakii in the granular sludge.

Deletion of the MAT-2 mating-type gene during uni-directional mating-type switching in Ceratocystis.

Ceratocystis eucalypti is strictly heterothallic, with single ascospore strains representing one of two opposite mating types. Most other Ceratocystis species, including C. virescens, C. pinicola, and C. fimbriata, are homothallic. In the homothallic species, the MAT-2 strains are self-fertile, while MAT-1 strains are self-sterile and grow more slowly than MAT-2 strains. The current hypothesis is that self-fertility of MAT-2 strains is due to the deletion of the MAT-2 mating-type gene, resulting in the expression of the MAT-1 mating type. These mutant MAT-1 strains are able to cross with MAT-2 strains. Part of the MAT-2 mating-type gene in C. eucalypti, C. pinicola, and C. fimbriata was amplified using degenerate primers designed from the conserved MAT-2 HMG DNA-binding motif. The expected approximately 300-bp PCR products were cloned and sequenced. Specific primers were designed that amplified 210-bp fragments only in MAT-2 isolates of C. eucalypti, C. virescens, C. pinicola, and C. finbriata. These fragments were present in self-fertile field isolates and self-fertile progeny but were absent in the self-sterile (MAT-1) progeny from selfings of C. virescens, C. pinicola, and C. fimbriata, thus supporting the hypothesis that the MAT-2 mating-type gene is deleted during uni-directional mating-type switching. A Southern-blot analysis was performed to confirm the deletion of MAT-2 gene in self-sterile progeny. The DNA sequence data for the C. eucalypti MAT-2 mating-type gene was increased to 1371-bp using TAIL-PCR and uneven PCR, representing a portion of the complete MAT-2 gene DNA sequence.