Efficient isolation of Campylobacter bacteriophages from chicken skin, analysis of several isolation protocols.

The application of Campylobacter specific bacteriophages appears as a promising food safety tool for the biocontrol of this pathogen in the poultry meat production chain. However, their isolation is a complicated challenge since their occurrence appears to be low. This work assessed the efficiency of seven protocols for recovering Campylobacter phages from chicken skin samples inoculated at phage loads from 5.0 × 101 to 5.0 × 106 PFU/g. The enrichment of chicken skin in selective Bolton broth containing target isolates was the most efficient procedure, showing a low detection limit of 5.0 × 101 PFU/g and high recovery rates of up to 560%. This method's effectiveness increased as phage concentration decreased, showing its suitability for phage isolation. When this method was applied to isolate new Campylobacter phages from retail chicken skin, a total of 280 phages were recovered achieving an isolation success rate of 257%. From the 109 samples 68 resulted phage positive (62%). Chicken skin could be, therefore, considered a rich source in Campylobacter phages. This method is a simple, reproducible and efficient approach for the successful isolation of both group II and III Campylobacter specific bacteriophages, which could be helpful for the enhancement of food safety by reducing this pathogen contamination in broiler meat.

Wine aroma profile modification by Oenococcus oeni strains from Rioja Alavesa region: selection of potential malolactic starters.

Previously six selected Oenococcus oeni strains (P2A, P3A, P3G, P5A, P5C and P7B) have been submitted to further characterization in order to clarify their potential as malolactic starters. Laboratory scale vinifications gave an insight of the most vigorous strains: both P2A and P3A strains were able to conclude malolactic fermentation (MLF) in less than 15 days. The remaining strains showed good viability and were able to successfully finish MLF in the established analysis time, except for the strain P5A, which viability was totally lost after inoculation. Also spontaneous fermentation was not initiated. None of the strains was biogenic amine producer; however, P5C strain significantly increased the concentration of volatile phenol-precursor hydroxycinnamic acids after MLF. Regarding the evolution of wine aromatic compounds, main changes were detected for both ethyl and acetate esters after MLF; however, key aromatic compounds including alcohols, terpenes or acids were also found to significantly increase. Principal component analysis classified the strains in two distinct groups, each one correlated with different key volatile compounds. P2A, P3A, P3G and P5C strains were mainly linked to esters, while P7B and the commercial strain Viniflora OENOS showed higher score for diverse compounds as hexanoic acid, ß-damascenone, linalool or 2-phenylethanol. These results confirmed the specific impact of each strain on wine aroma profile, which could lead to the production of wines with individual characteristics, in which the reliability and safety of MLF is also ensured.

Evaluation of an extracting method for the detection of Hepatitis A virus in shellfish by SYBR-Green real-time RT-PCR.

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In the present study, we evaluated a rapid and simple extraction method to concentrate and purify enteric viruses from shellfish tissues for their detection by real-time RT-PCR. This procedure consists of an alkaline elution with a glycine buffer, solids removal by slow speed centrifugation, purification by chloroform extraction and virus concentration by ultracentrifugation. The efficiency of this method to recover Hepatitis A virus (HAV) from oysters seeded with this virus, was assessed by real-time RT-PCR and conventional RT-nested PCR after extracting viral RNA by a commercial isolation kit. Real-time RT-PCR yielded higher detection sensitivity than the obtained by conventional RT-nested PCR. Besides the improvements in detection sensitivity, the real-time RT-PCR, by quantifying HAV RNA, allowed to check the overall extraction procedure and the recovery efficiency after each processing step. After the last phase, i.e. virus concentration by ultracentrifugation, the RNA purity was high but the estimated HAV recovery efficiency was however low, probably due to virus losses and the presence of RT-PCR inhibitors in sample concentrates. In contrast, the HAV recovery percentage was higher after the virus elution step while the RNA purity was lower. Real-time RT-PCR detection could allow to eliminate some purification and concentration steps that are required for conventional RT-nested PCR detection. The overall procedure for detecting HAV could be then simplify avoiding virus losses during manipulation.

Methylthioacetaldehyde, a possible intermediate metabolite for the production of volatile sulphur compounds from L-methionine by Lactococcus lactis.

Volatile sulphur compounds (VSCs) production from L-methionine was studied in Lactococcus lactis. In vitro studies with radiolabelled L-methionine and resting cells of L. lactis revealed that L-methionine was initially converted to alpha-keto-gamma-methylthiobutyrate (KMBA) by a transamination reaction. A part of KMBA was subsequently chemically converted to methylthioacetaldehyde, methanethiol and dimethylsulphides. Chemical conversion of KMBA to methylthioacetaldehyde was dependent on pH, Mn(II) and oxygen. Since methanethiol and dimethylsulphide production was highly related to that of methylthioacetaldehyde, the latter compound was proposed as being an intermediate in VSCs production by L. lactis.

Catabolism of volatile sulfur compounds precursors by Brevibacterium linens and Geotrichum candidum, two microorganisms of the cheese ecosystem.

Two Brevibacterium linens strains and the cheese-ripening yeast Geotrichum candidum were compared with regard to their ability to produce volatile sulfur compounds (VSCs) from three different precursors namely L-methionine, 4-methylthio-2-oxobutyric acid (KMBA) and 4-methylthio-2-hydroxybutyric acid (HMBA). All microorganisms were able to convert these precursors to VSCs. However, although all were able to produce VSCs from L-methionine, only G. candidum accumulated KMBA when cultivated on this amino acid, contrary to B. linens suggesting that the transamination pathway is not active in this microorganism. Conversely, a L-methionine gamma-lyase activity--which catalyses the one step L-methionine to methanethiol (MTL) degradation route--was only found in B. linens strains. Several other enzymatic activities involved in the catabolism of the precursors tested were investigated. KMBA transiently accumulated in G. candidum cultures, and was then reduced to HMBA by a KMBA dehydrogenase (KDH) activity. This activity was not detected in B. linens. Despite no HMBA dehydrogenase (HDH) was found in G. candidum, a strong HMBA oxidase (HOX) activity was measured in this microorganism. This latter activity was weakly active in B. linens. KMBA and HMBA demethiolating activities were found in all the microorganisms. Our results illustrate the metabolic diversity between cheese-ripening microorganisms of the cheese ecosystem.

Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens.

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.