VAAM-Jahrestagung 2012 18.–21. März in Tübingen

169Salmonella spp by amplifying specific genes 16s rDNA gene. The PCRproducts were analyzed on 1% agarose gel. Thirty six samples were foundas a positive among of 1124 collected samples. The dada shown thatmolecular based approaches are more rapid and should thus be used forany initial detection of Salmonella SPP.OTP145Systematical approach to decipher the rationales behind thedetergent and solvent stability of a model lipaseA. Fulton* 1 , J. Frauenkron-Machedjou 2 , S. Ulrich 2 , W. Susanne 1 , J. Karl-Erich 11 Heinrich-Heine-University Düsseldorf, Insitute for molecular EnzymeTechnology, Research Center Jülich, Germany2 RWTH Aachen University, Lehrstuhl für Biotechnologie, GermanyThe stability and activity of the biocatalysts is often compromised by theuse of certain additives, e.g. detergents and organic solvents, to increasethe solubility of certain reactants.This effect is not surprising and due to the evolutionary design of theenzyme. Enzymes have been evolved by nature to work efficiently inaqueous environments and thus require a water shell surrounding theprotein surface to retain enzymatic activity. Solvents and detergentsinterfere with the surrounding water shell and protein electrostatics. Thisinterference can lead to the unfolding and aggregation and a loss ofactivity. Despite these effects the influence of solvents on the enzymestructure and function has neither been studied systematically nor beenunderstood theoretically so far.We aim to discover the potential of stabilizing a model enzyme in nonconventionalmedia through a systematic mutagenesis study. We areinterested in the development of a predictive stability model for thecustomized design of biocatalysts in respect to the intended application.The model enzyme for our purpose is BSLA (LipA from Bacillus subtilis),a minimal /-hydrolase which can be easily expressed in Escherichia coli.BSLA has already been well characterized and is of known structure, thebiotechnological potential has been demonstrated with the production ofenantiopure cyclohexane-trans-1,2-diol[1].In preparation of this screening we have performed a saturationmutagenesis along the whole sequence of BSLA. Degenerated codonswere used to substitute the wild type amino acid by every other naturallyoccurring amino acid, resulting in a total of 3439 BSLA variants (181amino acids x 19 possible substitutions). We are now developing a highthroughput screening system to monitor the stability of every variant indifferent detergents and organic solvents. The selection of the solvents isjustified through different interferences towards the intra proteininteractions that will be weakened. The results will give us an insight intothe contribution of every single amino acid towards the stability of thewhole enzyme. The library construction and mutant screening is performedin cooperation with a project partner(b) which will focus on the stability inother non-conventional media. We will present the results from thescreening of several exemplary mutants.[1] Jean Detry, Thorsten Rosenbaum, Stephan Lütz, Doris Hahn, Karl-Erich Jaeger, Michael Müller& Thorsten Eggert (2006) Biocatalytic production of enantiopure cyclohexane-trans-1,2-diol usingextracellular lipases from Bacillus subtilis. Appl Microbiol Biotechnol. 72:1107-16.PMID:16586103OTP146Evaluating Food Safety Management Performance in a MilkPasteurising Facility using a Microbiological Assessment SchemeT. KennedyVeterinary Public Health Inspection Service, Department of Agriculture,Food and the Marine, Dublin, United KingdomMilk and milk products are a heterogeneous group of food products.Depending on the heat treatment applied during production, differentpathogens pose risks. The pathogens of concern are Listeriamonocytogenes, Bacillus cereus, Salmonella spp, Staphylococcus aureusand Escherichia coli since these may survive pasteurisation treatments.The performance of a food safety management system (FSMS) in adrinking milk pasteurisation establishment was measured using amicrobiological assessment scheme (MAS). The MAS consisted ofmultiple sampling locations along the processing line consisting of highriskraw materials, the processing environment, process water and endproducts. A total of 1268 samples were analysed over an 18-month-period.Nine microbial parameters (Salmonella spp., Listeria spp., B cereus, Staph.aureus, Total Bacterial Counts (TBC), Enterobacteriaceae, E. coli, Faecalenterococci and coliforms) were assessed. Results were benchmarkedagainst legal, industry and best practice norms. 100% (n 0 = 233) of rawmilk samples met the EU TBC standard of < 10 5 cfuml -1 , however, Listeriainnocua was isolated in 3% (n 1=134) of raw milk samples. Listeria spp.(n 2=128), Salmonella spp. (n 3=118), Staph. aureus (n 4=118),Enterobacteriaceae (n 5=114), B. cereus (n 6=38) and E. coli (n 7=23) werenot detected in any end products. Listeria welshimeri (a poor hygieneindicator) was identified in 2% (n 8=153) of environmental samplesSalmonella was not isolated in 63 environmental sample. 6% and 1% ofoperator hand swabs (n 9=100) had TBC and Enterobacteriaceae countsrespectively in excess of best practice norms of 10 2 cm -1 and 10 1 cm -1respectively. One (2.2%) water sample (n 11=46) had a coliform count of201cfuml -1 whereas five samples (11%) had TBC counts above acceptablenorms. The results indicate that the FSMS is producing a safe product. TheMAS is an effective risk assessment tool that is useful to assess the overallperformance of the FSMS and allows a more targeted use of resources toimplement improvement. Satisfactory end product microbiological resultsindicate that cold chain control, post pasteurisation contamination from dryingredients (e.g. buttermilk cultures), packaging or unsanitary pipe workare not issues for this plant. However, the prerequisites of environmentalsanitation, raw material supply and control, water treatment and storageand staff hygiene are the areas within the FSMS that pose the greatestrisks.OTP147Salmonella contamination of a Category 3 fat rendering plant - acase studyT. KennedyVeterinary Public Health Inspection Service, Department of Agriculture,Food and the Marine, Dublin, United KingdomSafe petfood production is a key objective of manufacturers. Petfoods andtreats are often found in the home food preparation areas. Petfoods areoften handled by children and the elderly. Food safety issues involvingdirect human contact with processed petfoods is becoming a majorregulatory focus. This case study describes an intractable caseofSalmonellacontamination in a Category 3 animal by-products renderingfacility that produces tallow for the oleo-chemical industry and greaves forpetfood manufacture. The facility is located adjacent to a beefslaughterhouse operates a Hazard Analysis and Critical Control Point(HACCP) based manufacturing system. The HACCP plan identifies threeCritical Control Points (CCPs) - pre-rendering particle size, metaldetection and rendering temperature and duration. The facility is approvedunder Regulation (EC) 1774/2002 and is subject to official controls by theCompetent Authority. Over a period of 5 years 33 of 305 official greavessamples intermittently revealed the presence of Salmonella anatum, S.kentucky and S. newington. No deficiencies were detected in CCPimplementation. Due to the high rendering temperatures the source ofcontamination was believed to be post renderingcontamination.Salmonellawas not isolated from any of the environmentalsamples (n = 62) nor from the products taken within process (n= 88).Analysis of pre-requisites identified deficiencies in pest control, sanitation,zoning, operator hygienic practices and structure fabrication. Deepcleaning and corrections to operational pre-requisite resulted in temporaryimprovements. The establishment was decommissioned for 10 months.Prior to re-opening fabrication was improved by laying a smooth floor,removing roughened welded seams in equipment, smooth plastering thewalls and properly ducting cables and hoses. Post structural improvement,none of the 120 official greaves samples revealed the presenceofSalmonella. The likely contamination source is from intermittentshedding from nidi located in the deep recesses of blemishes within thefabric.Salmonellais capable of surviving for extended periods in a varietyof environments. Complete elimination of pathogens is dependent on thestrict adherence to HACCP and GMPs. However, some practices are easyto apply, however in this case restoration of control required significantinvestment and plant redesign.OTP148Salmonella as a process hygiene microbiological criterion inIrish Wild PheasantT. KennedyVeterinary Public Health Inspection Service, Department of Agriculture,Food and the Marine, Dublin, United KingdomMicrobiological criteria provide guidance on the acceptability offoodstuffs and their HACCP-based manufacturing processes. Regulation(EC) 2073/2005 establishes process hygiene criteria (PHC) for carcasses ofdomestic fowl. No such criteria exist for pheasant. It is thus appropriate toestablish PHC. The processor selected for the study procures pheasantshunted from protected reserves, which are stocked with 18-week-oldpullets from a rearing unit 3-4 months prior to the shooting season. Inseason 1 on each of 10 processing days 4g of the neck skin (NS) wereaseptically harvested from 35 pheasants selected at random post-chilling.The NS from 7 carcasses were pooled to create 5 x 25g final samples.Samples were analysed for the presence forSalmonellausing ISO method6579. One sample revealed the presence of Salmonella. This procedurewas repeated in seasons 2 and 3 with identical results. PHC for pheasantwere determined thus:n= number of units compromising the sample = 50derived from 10 consecutive sessions;c= number of samples whereSalmonella is detected = 1;m = M= absence in 25g of a pooled NS sample.Ongoing performance exceeding these criteria prompts the establishmentto implement timely corrective action to its processing procedures and toreview disease control and bio-security measures on the rearing farm. InBIOspektrum | Tagungsband 2012