Hamid B. Ghoddusi

Enumeration of starter cultures in fermented milks

Some media available for the isolation and enumeration of starter cultures employed for the manufacture of cheese, yogurt and bio-yogurt were examined. Reddys medium or a modification of Ellikers medium was found to be most satisfactory for Lactococcus spp., while trypticase phytone yeast (TPY) agar with a mixture of antibiotics proved suitable for the discrete enumeration of Bifidobacterium spp. The inclusion of Prussian blue (PB) in reinforced clostridial medium or tryptone proteose peptone yeast extract (TPPY) agar gave excellent differential counts for the starter bacteria in yogurt even when the culture was imbalanced, while TPPY (PB) agar allowed the visible separation of all four of the organisms that might be found in a typical bio-yogurt, namely Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, a, Bifidobacterium sp. and Lb. acidophilus. It was noted that variation among different strains of any given species could change the expected reactions, so for quality control purposes the suggested media may need to be modified to cope with the specific cultures in use.

Preliminary study on the isolation of Clostridium butyricum strains from natural sources in the UK and screening the isolates for presence of the type E botulinal toxin gene.

Clostridia such as Clostridium tyrobutyricum, C. pasteurianum and C. butyricum may cause spoilage problems in certain types of food, but they are not normally regarded as dangerous. However some strains of C. butyricum have acquired the type E botulinum neurotoxin gene and have caused both infant and classical botulism in Italy (1986), China (1994) and India (1996). This study was carried out to examine a range of samples from fresh vegetables to food and environmental samples in the UK and test their ability to produce type E botulinal neurotoxin (BoNT) by probing for the presence of the toxin gene. Samples were enriched in modified Bhat and Barker (MBB) broth which is a minimal medium with lactate and acetate as a source of carbon and energy. In addition selective antibiotics are present in the medium to favour the growth of C. butyricum. This was followed by plating out onto iron sulphite agar (ISA) for isolation of C. butyricum from food and environmental samples. A total of 978 samples were tested and 302 (31%) yielded presumptive C. butyricum isolates. The highest percentage of positives came from soil, potato skins, Swede skin, yoghurt and cream. No positive isolates were obtained from pate, garlic or spring greens. A sub-sample of isolates was examined for the presence of gene encoding the type E botulinum neurotoxin using PCR. Only one of the many existing PCR methods was successful and therefore used for screening C. butyricum isolates for the presence of the type E toxin gene. None of the 93 tested isolates were found to be toxigenic (type E botulinal neurotoxin).

Assessing the effects of exposure to environmental stress on some functional properties of Bifidobacterium animalis ssp. lactis

This study assessed the effects of exposing a strain of Bifidobacterium animalis ssp. lactis to acid, bile and osmotic stresses on antagonistic properties, biofilm formation and antibiotic susceptibility/resistance profile. Exposure to each stress factor appeared to have no significant effect on the antagonism against Escherichia coli NCTC 12900 and Salmonella enterica serovar Enteritidis PT4. No suppression in biofilm formation due to exposure to stress was observed. Bile and osmotic stresses resulted in significantly higher biofilm formation. Expression of an exopolysaccharide synthesis gene, gtf 01207, was significantly higher when the B. animalis ssp. lactis strain was exposed to osmotic stress. Susceptibility of the B. animalis ssp. lactis strain to chloramphenicol, erythromycin, ampicillin and vancomycin, and resistance to tetracycline remained unchanged when exposed to each stress. The expression of a tetracycline resistance gene, tet(W), was significantly higher when exposed to each stress. These results may suggest that the potential for the B. animalis ssp. lactis strain to provide probiotic benefit, after exposure to the stressful conditions of the gastrointestinal tract, remains intact.

Growth Limiting pH, Water Activity, and Temperature for Neurotoxigenic Strains of Clostridium butyricum.

Some rare strains of Clostridium butyricum carry the gene encoding the botulinal type E neurotoxin and must be considered as possible hazards in certain types of food. The limiting growth conditions for C. butyricum were determined in peptone yeast glucose starch (PYGS) broth incubated anaerobically at 30°C for up to 42 days. The minimum pH values permitting growth depended on the acidulant and strain. Organic acids were more effective at inhibiting growth than HCl as expected. The lowest pH values at which growth of toxigenic and nontoxigenic strains of C. butyricum was observed in broth acidified with HCl were 4.1 and 4.2, respectively. In organic acids, however, the minimum pH varied between 4.4 and 5.1 depending on acid type and concentration. The minimum water activity for growth of toxigenic strains of C. butyricum was 0.96. The minimum growth temperatures of the toxigenic strains of C. butyricum (ca 10-11°C) were somewhat higher than for non-toxigenic ones (8°C). It was concluded that control of toxigenic C. butyricum in the food industry needs to allow for the greater pH tolerance of this species compared with proteolytic C. botulinum.