How to Manage Microorganisms with Complex Life Cycles in the Food Industry
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1 How to Manage Microorganisms with Complex Life Cycles in the Food Industry Organizers: L. Coroller, N. Desriac, F. Postollec Convenors: Frank Devlieghere and Louis Coroller
2 How to Manage Microorganisms with Complex Life Cycles in the Food Industry 15:30 Frank Devlieghere, Ghent University Food Spoilage By Fungi or forming Bacteria: Common Features and Differences 16:00 Maria Gougouli, Perrotis College, American Farm School Germination and Growth of Spoilage Fungi 16:30 Emilie Gauvry, University of Brest- UMT -Risk Growth Limits and Their Uses to Predict the Cycle of Life of forming Bacteria
3 Growth limits and their uses to predict the life cycle of spore-forming bacteria Emilie Gauvry A.G Mathot, O. Couvert, I. Leguérinel, N. Desriac, F. Postollec, L. Coroller LUBEM, Quimper, France University Laboratory of Biodiversity and Ecology IAFP s European Symposium on food safety March 2017 Brussels, Belgium 3
4 forming bacteria: microbial hazards Growth potential Surface adhesion forming bacteria Biofilm formation formation Persistance Resistance Food spoilage Food poisoning 2
5 forming bacteria: life cycle Vegetative cell 3
6 forming bacteria: life cycle GROWTH Vegetative cell 3
7 forming bacteria: life cycle GROWTH Vegetative cell SPORULATION 3
8 forming bacteria: life cycle GROWTH Vegetative cell SPORULATION GERMINATION 3
9 forming bacteria: life cycle GROWTH OUTGROWTH Vegetative cell SPORULATION GERMINATION 3
10 Effects of environmental factors (ph, a w, T C) forming bacteria: life cycle GROWTH OUTGROWTH Vegetative cell SPORULATION GERMINATION RESISTANCE 3
11 Effects of environmental factors (ph, a w, T C) OUTGROWTH forming bacteria: life cycle GROWTH Vegetative cell 1 SPORULATION GERMINATION 3
12 Effects of environmental factors (ph, a w, T C) forming bacteria: life cycle GROWTH 1 Vegetative cell A cellular process well described Physiology Quantitative prediction 3
13 Effects of environmental factors (ph, a w, T C) forming bacteria: life cycle GROWTH 1 Vegetative cell A cellular process well described Physiology Quantitative prediction Growth limits 3
14 Effects of environmental factors (ph, a w, T C) forming bacteria: life cycle GROWTH 1 OBJECTIVE: To extend the use of the growth limits to predict the other steps of the life-cycle Vegetative cell A cellular process well described Physiology Quantitative prediction Growth limits 3
15 Determination of the growth limits 4
16 The cardinal values: an evaluation of the growth limits 4
17 The cardinal values: an evaluation of the growth limits Growth kinetics 5
18 The cardinal values: an evaluation of the growth limits Growth kinetics 5
19 The cardinal values: an evaluation of the growth limits Effects of environmental factors on the growth rate Growth kinetics (Mtimet et al., 2015) Growth limits of Geobacillus stearothermophilus 5
20 The cardinal values: an evaluation of the growth limits Effects of environmental factors on the growth rate CARDINAL MODEL (Rosso et al. (1993, 1995, 1998)) Growth kinetics (Mtimet et al., 2015) Growth limits of Geobacillus stearothermophilus 5 5
21 The cardinal values: an evaluation of the growth limits Effects of environmental factors on the growth rate CARDINAL MODEL (Rosso et al. (1993, 1995, 1998)) Growth kinetics (Mtimet et al., 2015) 5 Growth limits of Geobacillus stearothermophilus 21
22 Prediction of bacterial growth using the growth limits growth rate Bacillus subtilis BSB1 (unpublished data, LUBEM Quimper) latency 6
23 (h -1 ) Prediction of bacterial growth using the growth limits growth rate Bacillus subtilis BSB1 (unpublished data, LUBEM Quimper) latency Temperature ( C) 60 (h -1 ) ph 6
24 (h -1 ) Prediction of bacterial growth using the growth limits growth rate Bacillus subtilis BSB1 (unpublished data, LUBEM Quimper) latency Temperature ( C) (h -1 ) Latency = growth rate * K (h -1 ) (h -1 ) ph 6
25 (h -1 ) Prediction of bacterial growth using the growth limits growth rate Bacillus subtilis BSB1 (unpublished data, LUBEM Quimper) (h -1 ) latency (h -1 ) Latency = growth rate * K Temperature ( C) 60 (h -1 ) (h -1 ) (h -1 ) 1/λ (h -1 ) ph ph
26 (h -1 ) Prediction of bacterial growth using the growth limits growth rate Bacillus subtilis BSB1 (unpublished data, LUBEM Quimper) (h -1 ) latency (h -1 ) Latency = growth rate * K Temperature ( C) (h -1 ) ph (h -1 ) (for high concentrations of the inocula) (h -1 ) 1/λ (h -1 ) ph
27 Prediction of bacterial sporulation using the growth limits Effects of environmental factors (ph, a w, T C) OUTGROWTH GROWTH Vegetative cell 1 2 SPORULATION A) Dynamics? GERMINATION B) Quantitatively? 3 RESISTANCE 7
28 Prediction of bacterial sporulation using the growth limits Effects of environmental factors (ph, a w, T C) OUTGROWTH GROWTH Vegetative cell 1 2 SPORULATION A) Dynamics? GERMINATION B) Quantitatively? 3 RESISTANCE 7
29 Prediction of bacterial sporulation using the growth limits Sporulation dynamics Effect of T C and ph on the time needed to observe the first spores in a bacterial culture of Bacillus subtilis BSB1 (unpublished data, LUBEM Quimper) 8
30 Prediction of bacterial sporulation using the growth limits Sporulation dynamics Effect of T C and ph on the time needed to observe the first spores in a bacterial culture of Bacillus subtilis BSB1 (unpublished data, LUBEM Quimper) (h -1 ) (h -1 ) 8
31 Prediction of bacterial sporulation using the growth limits Sporulation dynamics Effect of T C and ph on the time needed to observe the first spores in a bacterial culture of Bacillus subtilis BSB1 (unpublished data, LUBEM Quimper) (h -1 ) (h -1 ) 8
32 Prediction of bacterial sporulation using the growth limits Sporulation dynamics Effect of T C and ph on the time needed to observe the first spores in a bacterial culture of Bacillus subtilis BSB1 (unpublished data, LUBEM Quimper) Time for initiation of sporulation by vegetative cells to reach sufficient concentrations for QS + nutrient starvation Time to complete the process speed of the machinery 8
33 Prediction of bacterial sporulation using the growth limits Effects of environmental factors (ph, a w, T C) OUTGROWTH GROWTH Vegetative cell 1 2 SPORULATION A) Dynamics? GERMINATION B) Quantitatively? 3 RESISTANCE 9
34 Prediction of bacterial sporulation using the growth limits Concentration of spores 10
35 Prediction of bacterial sporulation using the growth limits Effect of T C on Bacillus weihenstephanensis KBAB4 Concentration of spores Concentration (LOG 10 ) (Baril et al., 2012) MNA (Nutrient agar) SMB (Sporulation mineral buffer) Limit of detection 10
36 Prediction of bacterial sporulation using the growth limits Effect of T C on Bacillus weihenstephanensis KBAB4 Concentration of spores Concentration (LOG 10 ) (Baril et al., 2012) MNA (Nutrient agar) SMB (Sporulation mineral buffer) Growth limits for temperature : Limit of detection Sporulation limits = growth limits 10
37 Prediction of bacterial sporulation using the growth limits Growth limits T C Bacillus weihenstephanensis Psychrophilic
38 Prediction of bacterial sporulation using the growth limits Growth limits T C Bacillus weihenstephanensis Psychrophilic Bacillus licheniformis Mesophilic Geobacillus stearothermophilus Thermophilic
39 Prediction of bacterial sporulation using the growth limits Growth limits T C Bacillus weihenstephanensis Psychrophilic Bacillus licheniformis Mesophilic Geobacillus stearothermophilus Thermophilic ph ? ? (Baril et al., 2012; Mtimet et al., 2015) 11
40 Prediction of bacterial sporulation using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH Vegetative cell 1 2 SPORULATION A) Dynamics? B) Quantitatively? 12
41 Prediction of bacterial sporulation using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH 1 Growth limits = sporulation limits Vegetative cell 2 SPORULATION A) Dynamics? B) Quantitatively? 12
42 Prediction of bacterial sporulation using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH 1 Growth limits = sporulation limits Sporulation VS growth : The same machinery Growth is needed prior to sporulation Cell division QS Vegetative cell 2 SPORULATION A) Dynamics? B) Quantitatively? 12
43 Prediction of bacterial sporulation using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH 1 Growth limits = sporulation limits Sporulation VS growth : The same machinery Growth is needed prior to sporulation Cell division QS Vegetative cell Growth limits can be used to predict the sporulation behaviour 2 SPORULATION A) Dynamics? B) Quantitatively? 12
44 Prediction of the resistance of spores using the growth limits Effects of environmental factors (ph, a w, T C) OUTGROWTH GROWTH Vegetative cell 1 SPORULATION 2 GERMINATION 3 RESISTANCE
45 Prediction of the resistance of spores using the growth limits Effect of T C on Bacillus weihenstephanensis KBAB4 Thermal resistance of spores 10 C 85 C 90 C 95 C 20 C 90 C 30 C 13
46 Prediction of the resistance of spores using the growth limits Thermal resistance of spores Effect of T C on Bacillus weihenstephanensis KBAB4 Initial concentration 10 (LOG) 10 C 85 C 90 C 95 C 20 C 90 C (Baril et al., 2011) 30 C 13
47 Prediction of the resistance of spores using the growth limits Thermal resistance of spores Effect of T C on Bacillus weihenstephanensis KBAB4 10 C 85 C 90 C 95 C Initial concentration 10 (LOG) Time needed to kill 90% of spores 20 C 90 C (Baril et al., 2011) 30 C δ 13
48 Prediction of the resistance of spores using the growth limits Effect of T C on Bacillus weihenstephanensis KBAB4 Thermal resistance of spores 10 C 85 C 90 C 95 C 20 C 90 C 30 C Time to kill 90% of spores (Baril et al., 2011) 13
49 Prediction of the resistance of spores using the growth limits Bacillus weihenstephanensis KBAB4 Effect of sporulation temperature Bacillus licheniformis AD978 Geobacillus stearothermophilus ATCC Time to kill 90% of spores Time to kill 90% of spores 85 C 90 C 95 C 100 C 90 C 95 C Time to kill 90% of spores 115 C 14 14
50 Prediction of the resistance of spores using the growth limits Bacillus weihenstephanensis KBAB4 Bacillus licheniformis AD978 Geobacillus stearothermophilus ATCC Time to kill 90% of spores Effect of sporulation temperature Time to kill 90% of spores 85 C 90 C 95 C 100 C 90 C 95 C Time to kill 90% of spores 115 C 14 14
51 Prediction of the resistance of spores using the growth limits Bacillus weihenstephanensis KBAB4 Bacillus licheniformis AD978 Geobacillus stearothermophilus ATCC Time to kill 90% of spores Effect of sporulation temperature Time to kill 90% of spores (Baril et al., 2011; Mtimet et al., 2015) 85 C 90 C 95 C 100 C 90 C 95 C Time to kill 90% of spores 115 C 14
52 Prediction of the resistance of spores using the growth limits Effects of environmental factors (ph, a w, T C) Growth limits = resistance limits GROWTH Vegetative cell SPORULATION 3 RESISTANCE 15
53 Prediction of the resistance of spores using the growth limits Effects of environmental factors (ph, a w, T C) Growth limits = resistance limits GROWTH Resistance of spores VS sporulation conditions : Resistance structures produced during sporulation Composition of these structures affected by environmental conditions Vegetative cell SPORULATION 3 RESISTANCE 15
54 Prediction of the resistance of spores using the growth limits Effects of environmental factors (ph, a w, T C) Growth limits = resistance limits GROWTH Resistance of spores VS sporulation conditions : Resistance structures produced during sporulation Composition of these structures affected by environmental conditions Vegetative cell SPORULATION Growth limits can be used to predict the resistance properties of spores 3 RESISTANCE 15
55 Prediction of the germination of spores using the growth limits Effects of environmental factors (ph, a w, T C) OUTGROWTH GROWTH Vegetative cell 1 SPORULATION 4 2 GERMINATION Dynamics? Quantitatively? 3 RESISTANCE
56 Prediction of the germination of spores using the growth limits Concentration of spores (LOG 10 ) Log 10 N Incubation Time (minutes) 16
57 Prediction of the germination of spores using the growth limits Concentration of spores (LOG 10 ) 7 Initial concentration Proportion of spores able to germinate Log 10 N Incubation Time (minutes) 16
58 Prediction of the germination of spores using the growth limits Concentration of spores (LOG 10 ) 7 Initial concentration % of germinated spores Proportion of spores able to germinate Log 10 N Time for 90% of germinated spores Incubation Time (minutes) 16
59 Prediction of the germination of spores using the growth limits Quantitatively Dynamics Bacillus weihenstephanensis KBAB4 Quantitatively Dynamics + B. licheniformis (Trunet et al., 2016) 17
60 Prediction of the germination of spores using the growth limits Quantitatively Dynamics Bacillus weihenstephanensis KBAB4 Quantitatively Dynamics 17
61 Prediction of the germination of spores using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH OUTGROWTH Vegetative cell 4 GERMINATION Dynamics? Quatitatively? 18
62 Prediction of the germination of spores using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH OUTGROWTH Vegetative cell Growth limits germination limits 4 GERMINATION Dynamics? Quatitatively? 18
63 Prediction of the germination of spores using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH OUTGROWTH 4 GERMINATION Dynamics? Quatitatively? Vegetative cell Growth limits germination limits Germination abilities : Germinant receptors, lytic enzymes + Mecanical/Physical process (changes in the conformation of germinant receptors) 18
64 Prediction of the germination of spores using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH OUTGROWTH 4 GERMINATION Dynamics? Quatitatively? Vegetative cell Growth limits germination limits Germination abilities : Germinant receptors, lytic enzymes + Mecanical/Physical process (changes in the conformation of germinant receptors) Growth limits can be used the germination. 18
65 Prediction of the germination of spores using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH 1 Dynamics? Quantitatively? OUTGROWTH 5 Vegetative cell SPORULATION 4 2 GERMINATION 3 RESISTANCE
66 Prediction of the outgrowth of germinated spores using the growth limits Concentration (LOG 10 CFU/mL) Germinated spores 19
67 Prediction of the outgrowth of germinated spores using the growth limits Concentration (LOG 10 CFU/mL) Initial concentration of germinated spores Germinated spores 19
68 Prediction of the outgrowth of germinated spores using the growth limits Concentration (LOG 10 CFU/mL) Initial concentration of germinated spores Germinated spores Proportion of germinated spores able to outgrow 19
69 Prediction of the outgrowth of germinated spores using the growth limits Concentration (LOG 10 CFU/mL) Initial concentration of germinated spores Germinated spores 90% of germinated spores able to outgrow Proportion of germinated spores able to outgrow Time for 90% of germinated spores able to outgrow 19
70 Prediction of the outgrowth of germinated spores using the growth limits Bacillus weihenstephanensis KBAB4 Quantitatively Dynamics Quantitatively Dynamics 20
71 Prediction of the outgrowth of germinated spores using the growth limits Bacillus weihenstephanensis KBAB4 Quantitatively Dynamics Quantitatively Dynamics 20
72 Prediction of the outgrowth of germinated spores using the growth limits Example for Bacillus weihenstephanensis KBAB4 Quantitatively Dynamics Quantitatively Dynamics Trunet et al., 2016) 20
73 Prediction of the germination of spores using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH Dynamics Concentration OUTGROWTH 5 Vegetative cell Growth limits = outrgowth limits 21
74 Prediction of the germination of spores using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH Dynamics Concentration OUTGROWTH 5 Vegetative cell Growth limits = outrgowth limits Outgrowth: Hydratation of the spore s become metabollically active Enzymatic activities recover Precedes vegetative growth 21
75 Prediction of the germination of spores using the growth limits Effects of environmental factors (ph, a w, T C) GROWTH Dynamics Concentration OUTGROWTH 5 Vegetative cell Growth limits = outrgowth limits Outgrowth: Hydratation of the spore s become metabollically active Enzymatic activities recover Precedes vegetative growth Growth limits can be used to predict the outgrowth of spores
76 The growth limits: a general tool to predict the life-cycle of spore forming bacteria GROWTH OUTGROWTH Vegetative cell SPORULATION GERMINATION RESISTANCE 22
77 The growth limits: a general tool to predict the life-cycle of spore forming bacteria GROWTH Latency Growth rate OUTGROWTH Vegetative cell SPORULATION GERMINATION RESISTANCE 22
78 The growth limits: a general tool to predict the life-cycle of spore forming bacteria GROWTH Latency Growth rate OUTGROWTH Vegetative cell Production of toxins, degradative enzymes SPORULATION Food poisonning and spoilage GERMINATION RESISTANCE 22
79 The growth limits: a general tool to predict the life-cycle of spore forming bacteria GROWTH Latency Growth rate OUTGROWTH Vegetative cell SPORULATION Dynamics: time necessary for the appearance of the first spores Concentration: maximal concentration of spores GERMINATION Thermal resistance RESISTANCE 22
80 The growth limits: a general tool to predict the life-cycle of spore forming bacteria GROWTH Latency Growth rate OUTGROWTH Vegetative cell Increase of the spores load and resistance Are subsequent treatments adapted? SPORULATION Dynamics: time necessary for the appearance of the first spores Concentration: maximal concentration of spores GERMINATION Thermal resistance RESISTANCE 22
81 The growth limits: a general tool to predict the life-cycle of spore forming bacteria GROWTH Latency Growth rate OUTGROWTH Vegetative cell Dynamics: time for 90% of germinated and/or outgrowing spores Concentration: Proportion of spores able to germinate and/or outgrow SPORULATION Dynamics: time necessary for the appearance of the first spores Concentration: maximal concentration of spores GERMINATION Thermal resistance RESISTANCE 22
82 The growth limits: a general tool to predict the life-cycle of spore forming bacteria OUTGROWTH Dynamics: time for 90% of germinated and/or outgrowing spores Concentration: Proportion of spores able to germinate and/or outgrow GROWTH Vegetative cell Recovery ability To keep inactivated forms OR Sensitivity to treatments Latency Growth rate SPORULATION Dynamics: time necessary for the appearance of the first spores Concentration: maximal concentration of spores GERMINATION Thermal resistance RESISTANCE 22
83 The growth limits: a general tool to predict the life-cycle of spore forming bacteria GROWTH Latency Growth rate OUTGROWTH Dynamics: time for 90% of germinated and/or outgrowing spores Concentration: Proportion of spores able to germinate and/or outgrow Vegetative cell Growth boundaries SPORULATION Dynamics: time necessary for the appearance of the first spores Concentration: maximal concentration of spores GERMINATION Thermal resistance RESISTANCE 22
84 Growth limits and their uses to predict the life cycle of spore-forming bacteria Thank you for your attention
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