Genaro García, DVM, MPVM, PhD Regional Food Safety Advisor Veterinary Public Health Unit Disease Prevention & Contr

1. Pan American Health Organization Regional Office of the World Health Organization Risk of Growth and Toxin Production by Clostridium botulinum Nonproteolytic Types B, C and F in Salmon Filets Stored under Modified Atmospheres (MA) at Low and Abused Temperatures • 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Porlamar, Margarita Island, Venezuela, 28 July- 1 August 2003) Genaro García, DVM, MPVM, PhD Regional Food Safety Advisor Veterinary Public Health Unit Disease Prevention & Control

2. Purposes • Review the application of modified-atmosphere (MA) technology for fresh fish preservation. • Discuss the mathematical microbiological modeling application for shelf life and safety in fish stored under MA. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

3. Background on Botulism and Modified Atmospheres (1) • Neuro-paralytic disease • Ingestion of preformed toxin (thermo-labile) • Taxonomy • Gram + • Spore-former rods • Anaerobes • 7 Serotypes: A, B, C, D, E, F &G. • A,B,E,F & G (human botulism) • C & D; G ?? (animal botulism) 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

4. Background on Botulism and Modified Atmospheres (2) • Symptoms General • Nausea. • Vomiting. • Weakness. • Lassitude. • Dry mouth. • Pharyngeal pain. • Neuro-muscular • Ocular: blurred vision, dyplopia. • Pharyngeal: dysphonia. • Laryngeal: dysphagia. • Respiratory: respiratory failure. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

5. Background on Botulism and Modified Atmospheres (3) • Modified Atmospheres (MA) • “A storage condition, where the atmospheres gas concentrations are altered before storage.” • Three basic categories • Vacuum-packaging. • Addition of a variety of gas blend. • Hypobaric storage. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

6. The Problem (1) • C. botulinum • High prevalence in seafood. • 85-100 % in trout farms. • A maximum of 100 spores per 100g of fish. • Non-proteolytic types B, E & F can grow to 3.3 C • Nature of fish flora and fish type affect toxigenesis. • Rate of toxigenesis increases with increase spore level and storage temperature. • Inhibition of the normal aerobic spoilage flora may change the pattern of spoilage flora and delay it. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

7. The Problem (2) • Toxigenesis may occur before signs of spoilage alarm the processor or the consumer. • Modified atmosphere ----> Aerobic & facultative anaerobes decrease ----> spoilage delayed. • Anaerobic ----> Toxigenesis may occur before spoilage. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

8. The Problem (3) • 2. Vacuum packaging (fresh meat and fish) • Extends shelf life by inhibiting aerobic spoilage. • Eventual spoilage at low temperature due to facultative and anaerobic bacteria. • Oxido-reduction potential (Eh) of the fish muscle. • Oxygen-consuming bacteria and vacuum-packaging may reduce the Eh and shorten the lag phase of clostridial growth. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

9. The Problem (4) • 3. Gas atmospheres • Effect of CO2 • Delays spoilage. • Bacteriostatic effects. • Concentration and temperature. • Initial bacterial population. • Type of food. • Once bacteria has passed the lag phase, inhibitory effect decreases. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

10. The Problem (5) • 3. Gas atmospheres • Effect of CO2 • Gram negative spoilage bacteria -----> more sensitive than lactobacilli and cocci, which are more sensitive than Clostridia. • Negligible anti-botulinal effect of CO2 when used alone in MA packaging. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

11. The Problem (6) • 3. An Important Question: “Will organoleptic spoilage precede toxin production and thus warn the consumer?” Studies have shown: • At < 10°C, spoilage by normal flora precedes toxigenesis. • At >10°C and increased storage, rate of toxigenesis and spoilage are similar. • At 21°C (abusive temperature), toxigenesis precedes spoilage. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

12. The Problem (6) • 4. Use of additional anti-microbial “hurdles” is being considered. • Modified atmosphere. • Low temperatures. • Preservatives. To delay C. botulinum and toxigenesis beyond the point of spoilage:Use of sorbates, tripolyphosphates, nitrites. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

13. Needs of the Project and Potential Use of Results (1) • Lack of quantitative evaluation of the risk of C. botulinum toxigenesis in fresh fish as it relates to type of fish, type and load of C. botulinum. • Storage temperature and selected preservatives known to delay toxigenesis. • Limiting factor preventing wider use of MA: • Potential of C. botulinum toxigenesis. • Limited knowledge of factors influencing toxigenesis. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

14. Needs of the Project and Potential Use of Results (2) 3. The project provided information to • answer complex questions; and • allow for quantitative estimation of the lack of evaluation of the risk of C. botulinum toxigenesis. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

15. General Objective Determine the effect of MA and temperature on the C. botulinum growth and toxigenesis in fish-muscle homogenates and filets. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

16. Specific Objectives(1) • 1. Define and quantify the effect of: • Type of C. botulinum. • Level of spore contamination. • Storage temperature and time. • Type of modified atmosphere. • Type of fish, as regards risk of C. botulinum growth. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

17. Specific Objectives(2) 2 . Correlate the time of spoilage and toxigenesis during MA for salmon filets and red snapper. 3. Determine the rate of toxigenesis and spoilage under all test conditions, using regression analysis to develop predictive formulas of the risk of toxigeneis. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

18. Specific Objectives(3) • Estimate the probability of growth. DR= log of inoculum spores minus log of spores initiating growth. • The reciprocal of the antilog of the DR expresses the probability of growth of one spore in a particular environment. Mathematical Models (Regression Analysis): (P)= a +b1(x1) +b2 (x2)+b3(x3) + bn (xn) + e 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

19. Specific Objectives(4) • Using this kind of model, we can answer such questions as: “How long does it take for 150 spores of type-E strains to develop detectable toxicity in salmon fillets stored at 8° C under pure CO2 ?” Meaningful quantitative conclusions on the action and interaction of the variables under study can thus be drawn. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

20. Discussion of Findings, Issues and Recommendations Source for all of the following tables and figures (slides 21–28): García, G. & C. Genigeorgis (1987)Quantitative Evaluation of Clostridium botulinum Nonproteolytic Types B, E and F Growth Risk in Fresh Salmon Tissue Homogenates Stored under Modified Atmospheres. Journal of Food Protection 50 (5/May): 390–397. 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

21. Lowest Inoculum Level 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

22. Shortages Observed 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

23. Summary of Regression Coefficients 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

24. Storage Time in Days (1) 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

25. Storage Time in Days (2) 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

26. Storage Time in Days (3) 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

27. Storage Time in Days (4) 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

28. Storage Time in Days (5) 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)

29. Thank you Obrigado Gracias 4th Annual Meeting of Inspection and Quality-Control Services and Institutions for Fish Products (Venezuela, summer 2003)