Effect of bacteriocin-producing lactobacilli on the survival of Escherichia coli and Listeria in a dynamic model of the stomach and the small intestine

The survival of Lactobacillus curvatus LTH 1174 (bac+) and (bac-) in combination with Escherichia coli LTH 1600 or Listeria innocua DSM20649 during transit through a dynamic model of the human stomach and small intestine (GIT model) was studied. Furthermore, we determined the digestion of curvacin A during gastro-intestinal transit and the effect of this bacteriocin on microbial survival. Lb. curvatus is rapidly killed in the gastric compartment at pH strongly agitated > agitated. Lactic acid and curvacin A enhanced the lethal effect of low pH on E. coli. Accordingly, cells from strongly agitated cultures were killed faster in the gastric compartment of the GIT model than those from agitated cultures, and inactivation was accelerated in the presence of curvacin A. E. coli tolerated the bile concentrations prevailing in the small intestinal compartments of the model. The survival of Listeria innocua in the GIT model was comparable to that of Lb. curvatus. The curvacin A produced by Lb. curvatus LTH1174 (bac+) killed > 90% of the L. innocua within 10 min after mixing of the cultures. Curvacin A was not degraded in the the gastric compartment, and could be detected in the ileal compartment during the first 180 min upon addition of the meal

The Effect of Lactulose on the Composition of the Intestinal Microbiota and Short-chain Fatty Acid Production in Human Volunteers and a Computer-controlled Model of the Proximal Large Intestine

The objective of this study was to compare the in vivo effect of lactulose on faecal parameters with the effect in a dynamic, computer-controlled in vitro model of the proximal large intestine (TIM-2). Faecal samples from 10 human volunteers collected before (non-adapted) and after 1 week of treatment (10 g/day) with lactulose (lactulose-adapted) were investigated. Parameters were compared immediately in the faecal samples, and after incubation in the in vitro model of the large intestine. After an adaptation period of the faecal microbiota in the in vitro model of the proximal colon, lactulose (10 g/day) was fed to the microbiota over a 48-h period. Samples taken from the model were investigated for microbiota composition and metabolite production (short-chain fatty acids (SCFAs) and lactate). No changes in the faecal parameters pH, dry weight or SCFA ratio were observed in the in vivo samples. However, the results show a major change in the ratio of SCFAs produced in the in vitro model, with a drastic reduction of butyrate production on lactulose. This was clear in the non-adapted microbiota by the observed arrest in butyrate production 24 h after the start of lactulose feeding. However, in the adapted microbiota butyrate production was already low from the start of the experiment. In fact, only the microbiota of one of the 10 individuals still produced significant amounts of butyrate after lactulose adaptation, the concentration in the other samples was extremely low. Similarly, in the in vitro model lactate production of the non-adapted microbiota started after approximately 24 h, whereas the adapted microbiota produced lactate from the start. In faecal (in vivo) samples no changes in microbiota composition were obvious, except for a significant increase in Bifidobacterium counts after lactulose feeding. With classic plating techniques, the in vitro samples showed an increase in Lactobacillus and Enterococcus species. With denaturing gradient gel electrophoresis, a clear change in banding pattern was observed, indicating a shift in microbiota composition. When the major bands that appeared after lactulose feeding in the in vitro model were excised and sequenced, the sequences showed homology to Lactobacillus and Enterococcus species. This is in agreement with the classic plating technique as well as with the observed increase in lactate production. Sampling in vivo at 'the site where it all happens' (the proximal colon) is difficult and inconvenient. We conclude that the in vitro model for the proximal colon reflects much better the fermentation of lactulose, in both metabolite production and changes in microbiota composition, than do faecal samples from an in vivo experiment. Therefore, the in vitro model is an excellent tool with which to study bioconversion of functional food components and/or drugs

In ovo inoculation of chicken embryos with probiotic bacteria and its effect on posthatch Salmonella susceptibility

The feasibility of establishing probiotic bacteria in the intestine of broiler chickens by in ovo inoculation was investigated, followed by verifying possible subsequent protection against Salmonella Enteriditis infection. In a first study, 7 commercially available probiotics were screened for compatibility with in ovo inoculation. Two of these probiotics, one being a Enterococcus faecium and the other a Bacillus subtilis, were selected for colonizing the chick gut without compromising hatchability. In a second study, these 2 products were administered in ovo and in the feed to chicks reared until 18 d in comparison with noninoculated chicks and with chicks fed an antibiotic. All chicks were orally challenged with Salmonella Enteritidis at 4 d of age. Results showed reduced performance of Salmonella Enteritidis challenged chicks fed no additives compared with challenged chicks fed antibiotic, but no significant differences in mortality was observed. Probiotics offered in ovo or through the diet could only partially recover performance compared with antibiotic-fed chicks. A significant reduction in the number of Salmonella Enteritidis positive chicks was observed when chicks were in ovo inoculated with E. faecium and continued receiving it in the diet. This work establishes standards for future in ovo colonization research and emphasizes its value as a promising method to deliver individual precise dose of probiotics to poultry in mass scale at the earliest possible age based on the competitive exclusion concept. In ovo colonization with probiotic can therefore become an important ally in combination with other approaches to combat Salmonella and other intestinal bacterial infections in poultry © 2014 Poultry Science Association Inc

Short communication : Growth of dairy isolates of Geobacillus thermoglucosidans in skim milk depends on lactose degradation products supplied by Anoxybacillus flavithermus as secondary species

Thermophilic bacilli such as Anoxybacillus and Geobacillus are important contaminants in dairy powder products. Remarkably, one of the common contaminants, Geobacillus thermoglucosidans, showed poor growth in skim milk, whereas significant growth of G. thermoglucosidans was observed in the presence of an Anoxybacillus flavithermus dairy isolate. In the present study, we investigated the underlying reason for this growth dependence of G. thermoglucosidans. Whole-genome sequences of 4 A. flavithermus strains and 4 G. thermoglucosidans strains were acquired, with special attention given to carbohydrate utilization clusters and proteolytic enzymes. Focusing on traits relevant for dairy environments, comparative genomic analysis revealed that all G. thermoglucosidans strains lacked the genes necessary for lactose transport and metabolism, showed poor growth in skim milk, and produced white colonies on X-gal plates, indicating the lack of β-galactosidase activity. The A. flavithermus isolates scored positive in these tests, consistent with the presence of a putative lactose utilization gene cluster. All tested isolates from both species showed proteolytic activity on milk plate count agar plates. Adding glucose or galactose to liquid skim milk supported growth of G. thermoglucosidans isolates, in line with the presence of the respective monosaccharide utilization gene clusters in the genomes. Analysis by HPLC of A. flavithermus TNO-09.006 culture filtrate indicated that the previously described growth dependence of G. thermoglucosidans in skim milk was based on the supply of glucose and galactose by A. flavithermus TNO-09.006

Short communication : Growth of dairy isolates of Geobacillus thermoglucosidans in skim milk depends on lactose degradation products supplied by Anoxybacillus flavithermus as secondary species

Thermophilic bacilli such as Anoxybacillus and Geobacillus are important contaminants in dairy powder products. Remarkably, one of the common contaminants, Geobacillus thermoglucosidans, showed poor growth in skim milk, whereas significant growth of G. thermoglucosidans was observed in the presence of an Anoxybacillus flavithermus dairy isolate. In the present study, we investigated the underlying reason for this growth dependence of G. thermoglucosidans. Whole-genome sequences of 4 A. flavithermus strains and 4 G. thermoglucosidans strains were acquired, with special attention given to carbohydrate utilization clusters and proteolytic enzymes. Focusing on traits relevant for dairy environments, comparative genomic analysis revealed that all G. thermoglucosidans strains lacked the genes necessary for lactose transport and metabolism, showed poor growth in skim milk, and produced white colonies on X-gal plates, indicating the lack of β-galactosidase activity. The A. flavithermus isolates scored positive in these tests, consistent with the presence of a putative lactose utilization gene cluster. All tested isolates from both species showed proteolytic activity on milk plate count agar plates. Adding glucose or galactose to liquid skim milk supported growth of G. thermoglucosidans isolates, in line with the presence of the respective monosaccharide utilization gene clusters in the genomes. Analysis by HPLC of A. flavithermus TNO-09.006 culture filtrate indicated that the previously described growth dependence of G. thermoglucosidans in skim milk was based on the supply of glucose and galactose by A. flavithermus TNO-09.006

Organic acids produced by lactic acid bacteria (Leuconostoc sp.) contribute to sensorial quality loss in modified-atmosphere-packed fresh-cut iceberg lettuce

The shelf-life of fresh-cut lettuce packed in a modified atmosphere (MA) is determined by its "overall visual quality" (OVQ), being a measure of its general appearance based on colour and shape criteria. In addition to the OVQ, the development of off-flavour and acid off-smell reduces consumer acceptance of such products. Concomitant with these changes in organoleptic properties, there is a rapidly developing microbial population inside the MA package (MAP), dominated by lactic acid bacteria. We studied the bacterial population dynamics of active MAP freshcut lettuce as well as the effect of metabolites produced by the bacteria (lactic acid and acetic acid) on lettuce quality aspects. Within 3 days of packaging, the oxygen concentration in the package was reduced to near zero, and this resulted in the selective advantage of lactic acid bacteria, in particular Leuconostoc and Lactococcus species. Leuconostoc, when cultivated on lettuce-enriched artificial medium, was found to produce both acetic and lactic acids. Low concentrations of acetic and lactic acids were found in MAP lettuce after 5 days of storage at 7°C. Freshly prepared fresh-cut product treated with comparably small amounts of acetic and lactic acids showed severe quality loss. This was reflected by rapid browning, yellowing and loss of texture. The experiments demonstrate that, under anaerobic conditions, organic acids are produced by lactic acid bacteria, affecting both off-flavour production and sensorial quality in fresh-cut lettuce.</p