Influence of Lactobacillus plantarum WCFS1 on post-acidification, metabolite formation and survival of starter bacteria in set-yoghurt.

The objectives of this study were to evaluate the growth and survival of the model probiotic strain Lactobacillus plantarum WCFS1 in co-culture with traditional yoghurt starters and to investigate the impact of preculturing on their survival and metabolite formation in set-yoghurt. L. plantarum WCFS1 was precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor before inoculation in milk. Adaptive responses of L. plantarum WCFS1 were evaluated by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated that sublethal preculturing did not significantly affect survival of L. plantarum WCFS1. On the other hand, incorporation of sublethally precultured L. plantarum WCFS1 significantly impaired the survival of Lactobacillus delbrueckii subsp. bulgaricus which consequently reduced the post-acidification of yoghurt during refrigerated storage. A complementary metabolomics approach using headspace SPME-GC/MS and (1)H NMR combined with multivariate statistical analysis revealed substantial impact of sublethally precultured L. plantarum WCFS1 on the metabolite profiles of set-yoghurt. This study provides insight in the technological implications of non-dairy model probiotic strain L. plantarum WCFS1, such as its good stability in fermented milk and the inhibitory effect on post-acidification.

Minimal inhibitory concentrations of undissociated lactic, acetic, citric and propionic acid for Listeria monocytogenes under conditions relevant to cheese.

Minimal inhibitory concentrations (MICs) of undissociated lactic acid were determined for six different Listeria monocytogenes strains at 30 °C and in a pH range of 4.2-5.8. Small increments in pH and acid concentrations were used to accurately establish the growth/no growth limits of L. monocytogenes for these acids. The MICs of undissociated lactic acid in the pH range of 5.2-5.8 were generally higher than at pH 4.6 for the different L. monocytogenes strains. The average MIC of undissociated lactic acid was 5.0 (SD 1.5) mM in the pH range 5.2-5.6, which is relevant to Gouda cheese. Significant differences in MICs of undissociated lactic acid were found between strains of L. monocytogenes at a given pH, with a maximum observed level of 9.0 mM. Variations in MICs were mostly due to strain variation. In the pH range 5.2-5.6, the MICs of undissociated lactic acid were not significantly different at 12 °C and 30 °C. The average MICs of undissociated acetic acid, citric acid, and propionic acid were 19.0 (SD 6.5) mM, 3.8 (SD 0.9) mM, and 11.0 (SD 6.3) mM, respectively, for the six L. monocytogenes strains tested in the pH range 5.2-5.6. Variations in MICs of these organic acids for L. monocytogenes were also mostly due to strain variation. The generated data contribute to improved predictions of growth/no growth of L. monocytogenes in cheese and other foods containing these organic acids.

Effect of sublethal preculturing on the survival of probiotics and metabolite formation in set-yoghurt.

The objective of this study was to investigate the effect of preculturing of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 under sublethal stress conditions on their survival and metabolite formation in set-yoghurt. Prior to co-cultivation with yoghurt starters in milk, the two probiotic strains were precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor. The activity of sublethally precultured probiotics was evaluated during fermentation and refrigerated storage by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated adaptive stress responses of the two probiotic strains resulting in their viability improvement without adverse influence on milk acidification. A complementary metabolomic approach using SPME-GC/MS and (1)H-NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Principal component analysis revealed substantial impact of the activity of sublethally precultured probiotics on metabolite formation demonstrated by distinctive volatile and non-volatile metabolite profiles of set-yoghurt. Changes in relative abundance of various aroma compounds suggest that incorporation of stress-adapted probiotics considerably influences the organoleptic quality of product. This study provides new information on the application of stress-adapted probiotics in an actual food-carrier environment.

Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt.

Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product.

The histone deacetylase SIRT2 stabilizes Myc oncoproteins.

Myc oncoproteins are commonly upregulated in human cancers of different organ origins, stabilized by Aurora A, degraded through ubiquitin-proteasome pathway-mediated proteolysis, and exert oncogenic effects by modulating gene and protein expression. Histone deacetylases are emerging as targets for cancer therapy. Here we demonstrated that the class III histone deacetylase SIRT2 was upregulated by N-Myc in neuroblastoma cells and by c-Myc in pancreatic cancer cells, and that SIRT2 enhanced N-Myc and c-Myc protein stability and promoted cancer cell proliferation. Affymetrix gene array studies revealed that the gene most significantly repressed by SIRT2 was the ubiquitin-protein ligase NEDD4. Consistent with this finding, SIRT2 repressed NEDD4 gene expression by directly binding to the NEDD4 gene core promoter and deacetylating histone H4 lysine 16. Importantly, NEDD4 directly bound to Myc oncoproteins and targeted Myc oncoproteins for ubiquitination and degradation, and small-molecule SIRT2 inhibitors reactivated NEDD4 gene expression, reduced N-Myc and c-Myc protein expression, and suppressed neuroblastoma and pancreatic cancer cell proliferation. Additionally, SIRT2 upregulated and small-molecule SIRT2 inhibitors decreased Aurora A expression. Our data reveal a novel pathway critical for Myc oncoprotein stability, and provide important evidences for potential application of SIRT2 inhibitors for the prevention and therapy of Myc-induced malignancies.

Utilization of a MAB for BRAF(V600E) detection in papillary thyroid carcinoma.

Identification of BRAF(V600E) in thyroid neoplasia may be useful because it is specific for malignancy, connotes a worse prognosis, and is the target of novel therapies currently under investigation. Sanger sequencing is the 'gold standard' for mutation detection but is subject to sampling error and requires resources beyond many diagnostic pathology laboratories. In this study, we compared immunohistochemistry (IHC) using a BRAF(V600E) mutation-specific MAB to Sanger sequencing on DNA from formalin-fixed paraffin-embedded tissue, in a well-characterized cohort of 101 papillary thyroid carcinoma (PTC) patients. For all cases, an IHC result was available; however, five cases failed Sanger sequencing. Of the 96 cases with molecular data, 68 (71%) were BRAF(V600E) positive by IHC and 59 (61%) were BRAF(V600E) positive by sequencing. Eleven cases were discordant. One case was negative by IHC and initially positive by sequencing. Repeat sequencing of that sample and sequencing of a macrodissected sample were negative for BRAF(V600E). Of ten cases positive by IHC but negative by sequencing on whole sections, repeat sequencing on macrodissected tissue confirmed the IHC result in seven cases (suggesting that these were false negatives of sequencing on whole sections). In three cases, repeat sequencing on recut tissue remained negative (including using massive parallel sequencing), but these cases demonstrated relatively low neoplastic cellularity. We conclude that IHC for BRAF(V600E) is more sensitive and specific than Sanger sequencing in the routine diagnostic setting and may represent the new gold standard for detection of BRAF(V600E) mutation in PTC.

Characterization of milk fatty acids based on genetic and herd parameters.

The objective of this study was to characterize the fatty acids (FA) in milk based on genetic and herd parameters to investigate the origin of the different FA in milk. Milk samples of 1912 Dutch Holstein-Friesian cows were analysed for 39 different FA including odd and branched-chain fatty acids. The proportion of variation caused by genetic and herd effects was calculated. In addition, genetic and herd correlations among the fatty acids were estimated and a clustering technique was used to visualise these correlations. The results indicated that in Dutch milk C12:0 is not completely synthesised de novo but also partly blood derived. It was suggested that C20:0 in milk is formed from the action of elongase enzymes on C18:0 and that the odd-chain FA C5:0-C13:0 and a part of C15:0 and C17:0 are synthesised de novo while the other part of C15:0 and C17:0 is blood derived. Furthermore, this work gives an overview of the opportunities to change the concentration of individual FA both by breeding and feeding. It is clearly shown that the extent to which the individual FA can be changed varies greatly and is dependent on the origin of the different FA in milk.

Attitudes of handicapped and nonhandicapped high school students toward physical education.

The attitudes of 381 handicapped and nonhandicapped high school students toward physical education in mainstreamed classes were compared using measures from the Kneer Attitude Inventory and Diagnostic Statements. A two-way analysis of variance yielded a significant difference between the groups' attitudes. Nonhandicapped high school students had significantly more positive attitudes toward physical education than their handicapped peers. No sex differences were significant.