MICROBIAL ECOLOGY FROM THE PERSPECTIVE OF FOOD SPOILAGE

MICROBIAL ECOLOGY FROM THE PERSPECTIVE OF FOOD SPOILAGE Professor Johanna Björkroth Department of Food Hygiene and Environmental Health University of Helsinki 29.7.2016 1

Trends in food manufacture and consumer preference The modern food logistics demands reasonably long product shelf lives for perishable foods MA-packaging (CO 2, O 2 and N 2 ) & cold chain Case ready products with suitable barriers Minimal processing if possible Low salt, few additives During shelf life, psychrotrophic, CO 2 -tolerating mainly Gram-positive spoilage bacterial communities develop in these products LAB Brochothrix thermosphacta Enterobacteria www.helsinki.fi/yliopisto

Estimated number of OTUs Total microbial cells 1000 Microbial succession in a high oxygen containing MAP meat product 1.E+09 900 1.E+08 800 Product spoiled 1.E+07 700 1.E+06 600 500 400 300 1.E+05 1.E+04 1.E+03 lot1 lot2 lot3 lot4 lot6 log 200 1.E+02 100 1.E+01 0 1.E+00 0 2 4 6 8 10 12 Time stored in MAP (days)

Rationale for studying bacterial interactomes related to food spoilage Which species are the fastest growers and what kind of interaction they have at the community level? Are there biomarkers for different growth stages of a spoilage community? Is the life style of a community fermentation driven throughout the shelf life Need to identify active pathways associated with community growth and development of spoilage changes during shelf life www.helsinki.fi/yliopisto

Microbial community analyses Chemical analyses Metabolome approach 29.7.2016 5 Eläinlääketieteellinen tiedekunta / Henkilön nimi / Esityksen nimi

Total RNA RNA extraction mrna Ribo zero depletion RNA seq 413 Gb of data from Illumina NextSeq Quality trimming and removal of Bos taurus reads Mapping to selected spoilage bacterial genomes Annotation against Greengenes database ph, gas content, CFU/g Commercial tenderized beef fillets packaged under high oxygen MAP analyzed on 11 days during the shelf life Two parallel packages per date Relative abundance, of gene/taxa of interest vs. the total reads Active species during beef storage and spoilage Anotation with MG-Rast and MEGAN Active genes and pathways during storage and spoilage

Day1_Na1 Day1_Na2 Day2_Na3 Day2_Na4 Day3_Na5 Day3_Na6 Day4_Na7 Day4_Na8 Day5_Na9 ay5_na10 day6_na11 ay6_na12 ay7_na13 ay7_na14 ay8_na15 ay8_na16 ay9_ Na17 ay9_na18 ay10_na19 ay10_na20 ay11_na21 ay11_na22 Day1_Na1 Day1_Na2 Day2_Na3 Day2_Na4 Day3_Na5 Day3_Na6 Day4_Na7 Day4_Na8 Day5_Na9 ay5_na10 ay6_na11 ay6_na12 ay7_na13 ay7_na14 ay8_na15 ay8_na16 ay9_ Na17 ay9_na18 y10_na19 y10_na20 y11_na21 y11_na22 Day1_Na1 Day1_Na2 Day2_Na3 Day2_Na4 Day3_Na5 Day3_Na6 Day4_Na7 Day4_Na8 Day5_Na9 Day5_Na10 day6_na11 Day6_Na12 Day7_Na13 Day7_Na14 Day8_Na15 Day8_Na16 Day9_ Na17 Day9_Na18 Day10_Na19 Day10_Na20 Day11_Na21 Day11_Na22 Day1_Na1 Day1_Na2 Day2_Na3 Day2_Na4 Day3_Na5 Day3_Na6 Day4_Na7 Day4_Na8 Day5_Na9 Day5_Na10 day6_na11 Day6_Na12 Day7_Na13 Day7_Na14 Day8_Na15 Day8_Na16 Day9_ Na17 Day9_Na18 Day10_Na19 Day10_Na20 Day11_Na21 Day11_Na22 % 1.00E+09 1.00E+08 1.00E+07 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Number of colony forming units Initial community Exponential growth Three phases during the study Spoiled After end of shelf life PCA cfu MRS cfu 80 70 60 50 40 30 20 10 0 Packaging gas content O2 CO2 5 4 3 Sensory evaluation 6.4 6.2 6 5.8 2 1 odor appearance 5.6 5.4 5.2 ph 0 5 4.8

Non-metric multidimensional scaling 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Active 16S rrna genes (Greengenes taxonomy) Vibrionales_Vibrionaceae_Photobacterium_Ph otobacterium profundum Vibrionales_Vibrionaceae_Photobacterium_Ph otobacterium phosphoreum Streptococcaceae_Lactococcus_Lactococcus raffinolactis Streptococcaceae_Lactococcus_Lactococcus piscium Leuconostocaceae_Leuconostoc_Leuconosto c lactis Leuconostocaceae_Leuconostoc_Leuconosto c inhae Leuconostocaceae_Leuconostoc_Leuconosto c kimchii Leuconostocaceae_Leuconostoc_Leuconosto c gelidum Leuconostocaceae_Leuconostoc_Leuconosto c gasicomitatum Leuconostocaceae_Leuconostoc_Leuconosto c citreum murinus mindensis manihotivorans helveticus cacaonum brevis aviarius agilis acidophilus alimentarius uncultured bacterium other Active mrna (SEED) 1r 3r 5r 7r 9r 11r 13r 15r 17r 19r 22r Virulence, Disease and Defense Sulfur Metabolism Stress Response RNA Metabolism Respiration Regulation and Cell signaling Protein Metabolism Phosphorus Metabolism Phages, Prophages, Transposable elements, Plasmids Nucleosides and Nucleotides Nitrogen Metabolism Miscellaneous Membrane Transport Fatty Acids, Lipids, and Isoprenoids DNA Metabolism Cofactors, Vitamins, Prosthetic Groups, Pigments Clustering-based subsystems Cell Wall and Capsule Cell Division and Cell Cycle Carbohydrates 29.7.2016 8 Amino Acids and Derivatives

Mapping to selected genomes Reads mapping to genomes Day11_Na22 Day11_Na21 Day10_Na20 Day10_Na19 Day9_Na18 Day9_ Na1 Day8_Na16 Day8_Na15 Day7_Na14 Day7_Na13 Day6_Na12 day6_na11 Day5_Na10 Day5_Na9 Day4_Na8 Day4_Na7 Day3_Na6 Day3_Na5 Day2_Na4 Day2_Na3 Day1_Na2 Day1_Na1 L. algidus L. piscium L. gasicomitatum 0 10 20 30 40 50 60 % of all reads mapping to selected genomes www.helsinki.fi/yliopisto

Lactate fermentation and pentose phosphate pathway active throughout Fermentation lactate Pentose phosphate pathway www.helsinki.fi/yliopisto 29.7.2016 10

Stress responses changing during shelf life Cold shock, CspA family Oxidative stress www.helsinki.fi/yliopisto 29.7.2016 11

Protein biosynthesis Bacterial cell division Sugar alcohols Respiration 29.7.2016 12

Conclusions of the main activities during shelf life Cold shock Universal stress protein family Oxidative stress Redox-dependent regulation of nucleus processes Osmotic stress Respiration Universal stress protein family Utilization of sugars and fermentation Oxidative stress Redox-dependent regulation of nucleus processes Respiration Osmotic stress Utilization of sugars and fermentation Respiration Osmotic stress Oxidative stress Redox-dependent regulation of nucleus processes Universal stress protein family Utilization of sugars and fermentation Cold shock Cold shock 29.7.2016 13

Thanks to team members and collaborators! Jenni Hultman Per Johansson Elina Jääskeläinen Elina Säde Erja Merivirta Henna Niinivirta Petri Auvinen Margarita Andreevskaya Olli-Pekka Smolander Kaisa Koskinen Food and packaging industry Academy of Finland GROWTHREG www.helsinki.fi/yliopisto