Final Report sub-project (1)

Transcription

1 FACULTY OF BIOSCIENCE ENGINEERING Dhr. Dirk de Meester Dhr. Steven Hagens EBI FOOD SAFETY Johan v. Oldenbarneveltlaan NE Den Haag NEDERLAND Your reference Offer 13/11/2006 Our reference LV 07rep-03 date contactpersoon dr. ir. Lieve Vermeiren lieve.vermeiren@ugent.be tel. en fax T F Final Report sub-project (1) 1. Experimental set-up 1.1. Test product The test product was a model cooked ham product, made in a pilot plant at the Laboratory for Food Chemistry and Meat Technology of KAHO St. Lieven (Gent, Belgium). A batch of 20 kg of product was prepared. The recipe of the model cooked ham (per kg) was: 1 kg of lean pork meat g of brine composition of the brine = 100 ml of water, 12.4 g of nitrite salt, 3.5 g/l glucose syrup, 0.7 g Naascorbate and 2 g/l phosphate. The different steps for the production of the cooked meat product were: (1) Preparation of the pork meat by removal of fat and connective tissue (2) Mincing pork meat though a plate with a diameter of 16 mm (3) Preparing the brine (4) Adding brine to the minced meat (5) Vacuum tumble for 40 min. (6) Curing for 18 hours at 4 C (7) Vacuum tumble for 20 min. (8) Filling in cans of 250 g (9) Cooking at a core temperature of 71 C and a surrounding temperature of 74 C (10) Cooling at 2 C The product was transferred to the laboratory of UGent under refrigerated conditions and immediately used for the experiment. The product was manually sliced in a laminar flow cabinet. Obtained slices were circular with a diameter of ± 7 cm, a thickness of 3 to 3.5 mm and a weight of ± 15 g (surface 1

2 area/slice= 38.5 cm 2 ). Packages of 150 g of product (10 slices) were made. The total treated surface area (per package of 150 g) = 385 cm Strains The product was inoculated with a cocktail of five Listeria monocytogenes strains: Table 1. Investigated Listeria monocytogenes strains LFMFP 1 -code Species Origin/Serotype LFMFP 235 L. monocytogenes serotype 4b Isolate LFMFP Cooked ham LFMFP 182 L. monocytogenes serotype 4b Scott A LFMFP 34 L. monocytogenes serotype 4b LMG LFMFP 416 L. monocytogenes serotype 1/2a IHE2000/099 (Food isolate) LFMFP 417 L. monocytogenes serotype 1/2b IHE2000/098/04 (Food isolate) 1, LFMFP = Laboratory of Food Microbiology and Food Preservation 1.3. Description of the challenge test The test product was characterized by determining the ph, water activity, salt level, dry matter and nitrite level. The product was treated in three different ways: Control: product inoculated with a mixture of the five Listeria monocytogenes strains (at a level of 5 to 10 cfu/cm 2, corresponding to 13 to 26 cfu/g) (in triplicate) : product inoculated with P100 ( pfu/cm 2 ) and a mixture of the five Listeria monocytogenes strains (at a level of 5 to 10 cfu/cm 2, corresponding to 13 to 26 cfu/g) (in triplicate) : product inoculated with P100 ( pfu/cm 2 ) and a mixture of the five Listeria monocytogenes strains (at a level of 5 to 10 cfu/cm 2, corresponding to 13 to 26 cfu/g) (in triplicate) In the case of the : from the appropriate dilution of the mixture of the five L. monocytogenes strains ( cfu/ml), 220 µl was divided over and spread on the surface of ± 150 g of product to reach the desired inoculation level of L. monocytogenes. In the case of or 2: more or less seconds after inoculation with the L. monocytogenes mixture, the product was inoculated with the phage solution. From an appropriate dilution of the phage solution, 175 µl was divided over and spread on the surface of ± 150 g of product to reach the desired inoculation levels (: ± 1x10 7 pfu/cm 2, : 5x10 7 pfu/cm 2 ). After inoculation, the 150 g portions of product were vacuum packaged and stored at 7 ± 1 C in a ventilated refrigerator. Analyses (Methods for enumeration are described in Annex 1): Total aerobic psychrotrophic count on day 0, day 28, day 42, day 60, day 90 and day 120 Lactic acid bacteria on day 0, day 28, day 42, day 60, day 90 and day 120 Listeria monocytogenes (enumeration and detection if count below detection limit): day 0, day 14, day 21, day 28, day 42, day 60, day 90 and day 120 for the, and. phage number determination: day 0, day 14, day 21, day 28, day 42, day 60, day 90 and day 120 (only for and.) 2

3 1.4. Remark: change in experimental set-up When analysing the products on day 14 of the challenge test, it was noticed that the vacuum of the products was not good anymore. Oxygen concentrations of 10 to 15% were found inside the headspace of the packages. A defect of the packaging machine caused this problem. All products were immediately re-packaged (vacuum) and further stored at 7 C. By mutual agreement between UGent and EBI Food Safety, the challenge test was continued, however analyses were limited to enumeration of Listeria monocytogenes on day 21, day 28 and day Results 2.1. Product characterisation Table 2. Chemical parameters characterising the product ph a w NaCl (%) 1 DM (%) 2 Nitrite (mg/kg) (as NaNO 2 ) Duplicate Duplicate , NaCl is determined according to the method of Mohr (titrimetric determination of chloride ions) 2, DM = dry matter 2.2. Phage titer determination The titer of the P100-stock was determined one week before the start of the experiment, the result was pfu/ml. This concentration was used to calculate the inoculum of P100. The results of the phage titer determinations of the cooked ham samples that were treated with the phage solution are summarised in Table 3. Table 3. Phage titer as a function of time Exp.1 ( pfu/cm 2 ) Time(d) T1(pfu/cm 2 ) T2(pfu/cm 2 ) T3(pfu/cm 2 ) ( pfu/cm 2 ) Time(d) T1(pfu/cm 2 ) T2(pfu/cm 2 ) T3(pfu/cm 2 )

4 2.3. Microbial analyses The results of the microbial analyses are summarised in Tables 4, 5, 6 and 7. Table 4. Total aerobic psychrotrophic count as a function of time < < < < Table 5. Enumeration of lactic acid bacteria as a function of time 0 < < < < < < < < < <

5 Table 6. Enumeration of Listeria monocytogenes as a function of time < < < < < < < < < < < Table 7. Detection of Listeria monocytogenes on day 0 Absence/presence in N1 N2 N3 0.1 g present absent present 1 g present present present 10 g g Absence/presence in N1 N2 N3 0.1 g absent absent absent 1 g absent absent absent 10 g absent present absent 25 g present present absent Absence/presence in N1 N2 N3 0.1 g absent absent absent 1 g absent absent absent 10 g absent absent absent 25 g absent absent present Prof. dr. ir. F. Devlieghere dr. ir. L. Vermeiren 5

6 Annex 1: Methods for enumeration of bacteria and phages Microbiological analyses A 15 g sample was taken aseptically and a decimal dilution series in PPS was prepared to plate the appropriate dilutions on the appropriate agar media. Total aerobic psychrotrophic count and number of LAB were determined according to the pour plate technique on Plate Count Agar (PCA) (aerobic incubation at 22 C for 3-5 days) and de Man Rogosa Sharpe (MRS) agar (aerobic incubation at 30 C for 3-5 days), respectively. Quantitative enumeration of L. monocytogenes was done by spreadplating onto ALOA-plates (aerobic incubation for 2 days at 37 C) supplemented with ALOA Enrichment selective supplement. The presence of L. monocytogenes in 25 g was determined in three steps: (1) a primary enrichment (24h, 30 C) of a 10-fold diluted homogenised 25 g sample in demi-fraser broth, (2) subsequently, 0.1 ml of incubated demi-fraser broth was transferred to 10 ml Fraser broth for secondary enrichment (24h, 37 C) and (3) confirmation by investigating ALOA plates for typical colonies of L. monocytogenes.. Phage titer determination For phage number enumeration of phage-treated products, a soft agar overlay technique was used. As a positive, the Listex TM P100 stock solution was included in the test. The indicator strain used for the phage titer determination was Listeria innocua B1488, obtained from EBI Food Safety (Den Haag, The Netherlands). Samples (10 g) from phage-treated products were first diluted tenfold in sterile 0.1 M phosphate buffer of ph 7.4 and then filter sterilised through a membrane filter ( 0.45 µm) to avoid bacterial contamination of the soft agar double layer plates in the subsequent plaque assay. The phage titers of the filtrates of the phage-treated products and of the Listex TM P100 stock solution were determined by counting plaques from their serial 10-fold dilutions. Volumes of 20 µl of each phage dilution were mixed with 200 µl (10 6 cfu) of cells of the indicator strain L. innocua. After incubation for 30 min at 30 C, the cell-phage mixture was mixed with 3 ml of preheated 4YT (yeast trypton) semi-soft agar (32 g/l trypton, 20 g/l yeast extract, 5 g/l NaCl (VWR) and 7.5 g/l agar) and this final mixture was quickly poured onto pre-heated 4YT-agar plates (32 g/l trypton, 20 g/l yeast extract, 5 g/l NaCl and 15 g/l agar). Following incubation for 24h at 30 C, plaques were counted. 6 Department of Food Safety and Food Quality Laboratory of Food Microbiology 7 and Food Preservation