Search

Rapporten gir en grunnleggende innføring i mikroorganismenes systematiske inndeling, reproduksjon og forhold til miljøfaktorer som temperatur, fuktighet og næringstilgang. De mest aktuelle patogene bakterier som kan forekomme i fiskemel er beskrevet med data om toleranser og krav. Den naturlige flora av mikroorganismer i fiskeråstoff er varmesensitiv og blir inaktivert i koker. Floraen i fiskemel har en annen sammensetning og stammer fra smittekilder i produksjonslinjen etter koker. Smittekildene er belegg med produktkontakt i maskiner og transportører. Smittemåte og aktuelle forebyggende tiltak er beskrevet. Rapporten oppsummerer mikrobiologiske krav til fiskemel, herunder regelbundne krav som er basis for godkjenning av ferdigvarepartier, retningslinjer og kundekrav. Rapporten beskriver prøvetaking og analyseprinsipper som benyttes ifm. mikrobiologiske undersøkelser. Virksomhetenes forskriftsmessige plikter ifm. mikrobiologi og produktsikkerhet er oppsummert og det er beskrevet metoder for dekontaminering av varer som ikke kan omsettes pga. hygienesvikt. The report provides a basic introduction to microbiology and production hygiene in fish meal factories, with emphasis on contamination routes and preventive measures. The intrinsic microbial flora of raw fish is heat sensitive and readily inactivated by thermal treatment in the cooker. The flora associated with finished fishmeal is markedly different and originates from contaminated product deposits or surfaces in the process machinery after the cooker. The report summarizes basic legal requirements related to hygiene and product safety in food and feed processing facilities. The report also describes methods used in microbiological examinations, and microbiological standards and guidelines applicable to fishmeal. Methods for large-scale decontamination of products and process machinery are briefly described.

Metodens riktighet og presisjon ble vurdert som tilfredsstillende, basert på ekstern kollaborativ validering og intern verifisering av riktighet og presisjon. Riktighet Analyser av referansemateriale med ny metode (RYM), ga for gjær resultater sentralt i oppgitt initialt kontroll-intervall, mens mugg plasserte seg ved nedre kontrollgrense. Årsaken til lave muggresultater kan være tap av viabilitet etter fryselagring av referansematerialene i opptil 4 år. Analyser av naturlig kontaminerte prøver med ny metode (RYM) ga tilsvarende resultater som Nofimas etablerte metode (YM) for både mugg og gjær. AOACs og AFNORs kollaborative avprøvinger av RYM ga resultater for både mugg og gjær som ikke skilte seg statistisk fra resultatene av referansemetodene (US FDA BAM og ISO 21527). Presisjon Måleusikkerhet (U) for RYM ble beregnet til 0,12 for gjær og 0,23 for mugg som er lik eller noe lavere enn tidligere estimater for YM. AOACs og AFNORs interlaboratorie-studier konkluderte med at repeterbarhet og reproduserbarhet for RYM var tilsvarende som for referansemetodene (US FDA BAM og ISO 21527). Validation of a rapid method for enumeration of yeast and mold – Petrifilm Rapid Yeast and Mold (RYM) Count Plate

The Norwegian Seafood Federation (FHL) has applied for approval of a new alternative method for processing of category 2 animal by-products (ABP) of fish origin. The processing method is characterized by fish raw material that is grinded before mixing with formic acid at pH ≤4 and stored for ≥24 hours before heat treatment of the silage with a particle size ≤10 mm at a temperature ≥85 °C for ≥25 minutes. The method was assessed by EFSA (BIOHAZ-panel) who concluded that the risk related to pathogens present in fish ABP’s from aquaculture would be adequately reduced by the proposed process if the requirements of the HACCP-plan could be achieved in a full scale plant. The production trials verified the feasibility of the HACCP-plan under real processing conditions in a full scale plant. Another purpose of the production trials was to demonstrate that relevant pathogens are inactivated by the proposed process. For each of 20 batches produced, 5 samples of silage and 5 of end product were analyzed for Salmonella and Enterobacteriaceae. All results were negative, confirming earlier lab scale experiments showing that the formic acid treatment alone inactivate those bacteria. One composite sample of silage and one of end product for each batch were analyzed for C.perfringens. All results were negative, confirming a low prevalence of C.perfringens in fish silages, as also demonstrated in earlier surveys. Anaerobic sulfite-reducing bacteria were analyzed in the same composite samples. The concentration in silage ranged from 1.100 to 130.000 per gram, while heat treatment provided on average more than 3 log reductions. This reduction rate is comparable to that found in earlier lab scale inactivation experiments with C.perfringens and C.sporogenes spores. All batches of end product met the microbiological requirements used to assess inactivation effect of new processing methods according to method 7 in Regulation (EC) 1774/2002. Based on this, it is concluded that the risk related to pathogens present in fish ABP from aquaculture would be adequately reduced by the proposed process. Fiskeri og Havbruksnæringens Landsforening (FHL) har søkt om godkjenning av en ny alternativ metode for prosessering av kategori 2 bi-produkter fra fisk. Metoden består av oppmaling og maursyrebehandling av fisken ved pH ≤4 i ≥24 timer før varmebehandling av fiskeensilasje med partikkelstørrelse ≤10 mm ved ≥85 ⁰C i ≥25 minutter. Metoden ble vurdert av EFSA (BIOHAZ-panel) som konkluderte at risiko knyttet til patogener i biprodukter fra akvakultur ville bli tilstrekkelig redusert hvis kravene i HACCP-planen lot seg gjennomføre i fullskala. Rapporten beskriver en serie produksjonsforsøk som viste at HACCP-planen var gjennomførbar under realistiske forhold i et fullskala fabrikkanlegg og at relevante patogener ble inaktivert gjennom prosessen.

The aim of the study was to determine the hygienization effect of a new processing method intended for category 2 material of fish origin. The method includes storage (≥ 24 hours) and heat treatment (≥ 85 ºC/≥ 25 minutes) of grinded fish material with added formic acid to pH ≤ 4,0. C.sporogenes (ATCC 19404) spores replaced C.perfringens spores in D-value determinations and in examinations of hygienization effect during all phases of the new processing method. In order to minimize interference from the indigenous flora on the analysis of added spores, the fish minces were pre-treated with gamma irradiation (10 kGy). Average D-value for heat treatment of C.sporogenes spores at 85 ºC in fine fish mince adjusted to pH 7 was 580 minutes. Acidification of fish mince with formic acid to pH 4,0 had no inactivation effect per se, but heat treatment at 85 ºC in the presence of the same acid dosage resulted in a markedly reduced D-value; 8,9 minutes. The inactivation effect encountered during the complete new processing method was at least 3 Log10 reductions in fine as well as in coarse fish mince. This is regarded as satisfactory, considering the low incidence of C.perfringens spores in fish silages. Inactivation of Clostridium sporogenes spores in fish by-products by a new processing method

The aim of the project was to find a simple, inexpensive and safe method for the treatment of category 2 fish by-products without heat treatment. IPNV was chosen as indicator organism because it was considered to be the hardiest among fish-pathogenic microorganisms. Salmonella and Clostridium perfringens were included in the study to provide a basis for assessing whether the method could also be applicable to category 3 by-products intended for feed production. IPNV was inactivated (> 4 Log10 reductions) at pH below 1,5 or above 11,5. Depending on the composition of the by-products, this corresponds to dosage of 1,0-1,8 % HCl or 0,9-2,0 % KOH. Salmonella was inactivated at pH between 3,0 and 4,0 or above 11,0. This corresponds to less than 1,0 % HCl, or, depending on the composition of the by-products, 0,7-1,7 % KOH. Salmonella is therefore securely inactivated at conditions required to control IPNV. HCl treatment for 5 hours at pH 1,0 gave 1 Log10 reduction of viable C.perfringens spores. No inactivation was observed at high pH values, up to 12,0. The pH conditions required to control IPNV does not affect the viability of C.perfringens spores. The present study shows that the required inactivation of IPNV in fish by-products can be achieved by treatment with both inorganic acid and base. The effect of these treatments on processability and as product properties in relation to intended utilizations should be further examined. Inactivation of IPN-virus in fish by-products by inorganic acid and base

The present report is a consolidated version of former Nofima Report K-371, incorporating new kinetic data on thermal inactivation of IPNV (Nygaard and Myrmel, 2010). Report K-371 has been withdrawn. The project was initiated by the Norwegian Seafood Federation (FHL) after a request from the Norwegian Food Safety Authority to define a standard Norwegian fish meal process and criteria to kill infectious agents present in wild fish and aquaculture fish. The report summarizes inactivation data for Enterobacteriaceae/Salmonella and bacterial/viral pathogens of fish. Inactivation effects resulting from various temperature-time (T/T) combinations were estimated from available D-and z-values. Wild fish should be processed according to the “fishmeal method” as outlined in Regulation (EC) 1774/2002. The minimum conditions proposed for heat treatment of wild fish are 70 °C/20 minutes which provides 100 LOG10 reductions of Enterobacteriaceae/Salmonella. The minimum conditions proposed for heat treatment of aquaculture fish are 76 °C/20 minutes or other T/T combinations resulting in 3 LOG10 reductions of IPNV. The report describes heat treatment at two alternative stages of the manufacturing process; in cooker and in indirect steam drier. For production of fishmeal, fulfilment of minimum conditions for heat treatment may be documented either in the cooker or in the steam driers. For fishoil production, inactivation has to rely on heat treatment in the cooker. The report proposes conditions to be fulfilled in order to allow processing of category 3 materials from wild fish and aquaculture fish in the same processing unit. Standard Norwegian fishmeal- and fishoil process.Heat treatment requirements

Foreliggende rapport er en konsolidert versjon av tidligere Nofima rapport K-367, hvor nye kinetikkdata for termisk inaktivering av IPNV har blitt inkludert. Rapporten foreligger også i engelsk utgave (Nofima Report no. 33/2010). Rapport K-376 er trukket tilbake. Prosjektet ble initiert av FHL etter anmodning fra Mattilsynet til norsk fiskemelindustri om å definere en standard norsk fiskemelprosess med kriterier for å drepe aktuelle smittestoffer i villfisk og oppdrettsfisk. Rapporten oppsummerer drepekinetikkdata om Enterobacteriaceae/Salmonella og fiskepatogene bakterier/ virus. Inaktiveringseffekt av ulike temperatur-tid (T/T) kombinasjoner ble beregnet fra kjente D- og z-verdier. Villfisk skal gjennomgå en prosess som oppfyller kravene til ”fiskemelmetoden” i Forordning (EF) 1774/ 2002. Foreslåtte minimumsbetingelser for varmebehandling er 70 °C/20 minutter som gir 100 Log10 reduksjoner av Enterobacteriaceae/Salmonella. Foreslåtte minimumsbetingelser for oppdrettsfisk er 76 ºC/20 minutter eller andre T/T kombinasjoner som gir 3 Log10 reduksjoner av IPNV. Rapporten beskriver varmebehandling på to alternative trinn i fiskemelprosessen; i koker og i indirekte damptørke. For fiskemel kan oppfyllelse av minimumskravene til varmebehandling dokumenteres enten i koker eller i damptørke. For fiskeolje må inaktiveringen baseres på varmebehandling i koker. Rapporten beskriver vilkår som må oppfylles for å kunne prosessere villfisk og oppdrettsfisk i samme anlegg. Rapport/Report 35/2010 English summary Standard norsk fiskemel- og fiskeoljeprosess.Krav til varmebehandling

The aim of the present study was to determine the inactivation effect on IPNV resulting from heat treatment of fish by-products at different temperatures. Furthermore, the inactivation effect of a new alternative processing method intended for category 2 fish material was examined. The method involves size reduction, organic acid treatment and heat treatment (85 ºC for 25 minutes). Decimal reduction times (D-values) and temperature coefficients (z-values) were determined after heat treatment of IPNV in artificial media resembling fish silage with regard to its content of water soluble protein and salt. The inactivation curves obtained in this study were bi-phasic. A rapid initial loss of infectivity (approximately 0,7 Log10 reduction) was followed by a much slower exponential decrease. D-values for thermal inactivation at 60, 70, 80 and 90 ºC ranged from 290 to 0,5 minutes. The temperature increase (z-value) needed to obtain a ten-fold reduction of D-values was approximately 9,8 ºC. Surprisingly, the experiments showed that IPNV infectivity was better maintained at pH 4 (formic acid) than at pH 7. However, the heat resistance of IPNV was little influenced by pH. Based on the D- and z-values obtained during heat exposure of IPNV in artificial media, IPNV inactivation during the heating phase of the new processing method (85 ºC, 25 minutes) was calculated to 27 Log10 reductions. Even if IPNV is virtually unaffected by exposure to formic acid (pH 4), the heating phase alone will be fully sufficient to ensure adequate inactivation of IPNV during the complete process. In inactivation studies using fish suspension as a matrix, no infective IPNV could be recovered from the suspension at any step. The results indicate that the fish suspension reduces the IPNV infectivity. Extended studies on other batches of fish suspensions should be performed before a general conclusion on the effect of fish suspensions can be made. Inactivation of pathogenic microorganisms in fish by-products. Sub-project IPN-virus

Polymerase Chain Reaction (PCR) er en av de viktigste teknikker innen moderne molekylærbiologi. Ved bruk av PCR i mikrobiologisk analyse, amplifiseres (mangfoldiggjøres) DNA-segmenter som er unike for den søkte organisme. PCR-produktet kan detekteres på ulike måter. I ”Real Time PCR” blir produktet detektert automatisk under amplifiseringen. I den senere tid er det utviklet flere ”Real Time PCR” metoder for påvisning av Salmonella. Metodene er hurtige og tilpasset et stort analyseomfang. Fiskeriforskning har gjennomført validering av to ”Real Time PCR” metoder for analyse av Salmonella i fiskemel og andre fôrvarer. Light-Cycler Instrument (Rôche Diagnostics) og iCycler iQ System (BioRad Laboratories) ble benyttet parallellt med referansemetoden Malthus Konduktans Metode/NMKL 71. Prøve-materialet var ordinære fôrvarer og kulturer av ulike Salmonella-serovar i eller uten matrix. Begge PCR-metoder detekterte alle de 41 undersøkte Salmonella serovar som kunne påvises med referanse-metoden. Ved analyse av Salmonella i ordinære fôrvarer ga begge PCR-metodene innledningsvis en del falske negative resultater. Etter modifikasjon av prosedyrene for prøvepreparering ble frekvensen av falske negative resultater redusert til et akseptabelt nivå. Begge PCR-metoder ga over 99 % samsvar med referansemetoden når det ble benyttet modifisert prøve-preparering og kulturbasert anrikning/isolering/konfirmering av alle positive og ugyldige PCR-resultater. A validation study of LightCycler Instrument (Roche Diagnostics) and i-Cycler IQ System (Bio Rad Laboratories) for analysis of Salmonella in fishmeal and other feedingstuffs was undertaken. A total of 1193 samples, including naturally contaminated material and material spiked with different serovars of Salmonella, were analysed with the two PCR methods and Malthus Automated Conductance Method (reference method). With slight modifications to the DNA extraction procedures and with confirmation of PCR-positive and invalid samples, more than 99 % relative trueness was obtained.