Characterization of the Fungal Microflora in Quebec Terroir Milks Steve Labrie Departement of Food and Nutrition Sciences Université Laval STELA Symposium 2011
Overview Introduction Milk and cheeses from small-scale operations, produits du terroir Milk characteristics The milk project 1.Characterization of the fungal flora in raw milk; 2.Persistence of fungal flora; 3.Persistence of milk yeasts and molds in cheese; 4.Cooperation between fungal flora and probiotic lactic acid starters; 5.Production of anti-listeria compounds; 6.Influence of the "terroir" milk composition. 2
Milk and cheese from the terroir A terroir product is a product or the main components of a product that comes from a limited and homogeneous territory. It has distinctive characteristics based on regional specificity and that significantly differentiate it from similar products. Its characteristics come from regional particularities such as geology, climate, landscape, as well as the culture, history and traditional and emerging knowledge and know-how of the inhabitants of the region. * Conseil des appellations réservées et des termes valorisants (CARTV) 3
Milk Microbiology Milk contains numerous microbial organisms: Bacteria Viruses (phages) Fungi (yeasts and molds) Each of these groups may contain: Undesirable organisms Pathogenic organisms Beneficial organisms 4
What do we know about the fungal flora of milk? Numerous species have been identified as dairy product contaminants. Pichia burtonii Few studies have been conducted to characterize fungal flora of milk and its persistence in cheese. Phoma glomerata 5
How is the Contribution of the Fungal Flora Quantified? 1 Fernandez Del Pozo. et al. 1988. J. Dairy Res. 2 Poullet et al. 1991. J. Dairy Res. 6
Our Research Project 7
Our Questions Which fungal species are dominant in milk from Québec? For one specific dairy production, is the fungal flora stable or does it vary over time? Cladosporium herbarum Can the fungal flora of milk grow in cheese and influence its ripening? Does milk chemistry influence growth of fungal flora in cheese? Penicillium pupurogenum 8
The aims of our study 1. Obtain samples over a 10-month period from dairy farmers who make and sell cheese from the milk they produce. 2. Determine the chemical composition of the milk. 3. Determine the microbiological composition of the milk. 4. Determine the interactions between fungal and lactic microflora. 5. Identify strains with anti-listeria activity 9
Methodology 19 dairy small-scale producer-processors Milk samples received each month over a 10-month period (Jan. 2009 - Oct. 2009) On-farm cheesemakers in six administrative regions of the province of Québec. Some targeted cheesemakers also provided specialty cheeses (bloomy rind / washed rind) ripened for 1 week. 10
Méthodology (cont d) 111 milk samples Microbiological Analysis Physico-Chemical Analysis ph, titrable acidity, buffering; Composition (Infrared Analyzer FT-120); Soluble and Total Minerals (ICP); Casein Profile (HPLC in reverse phase); Serum Proteins (Kjeldahl); Micelle size and zeta potential; Coagulation kinetics; Ash Mesophilic Bacteria Psychrophilic Bacteria Coliforms Staphylococcus aureus Yeasts and Molds Characterization of fungal flora Frequency of species Time Monitoring Dairy Fungal Bank Identification of strains with anti-listeria activity Effect of fungal flora on probiotic bacteria 11
1. Characterization of the Fungal Flora of Raw Milk Mesophilic bacteria (PCA) Psychrophilic bacteria (PCA) Coliforms (VRBA) Staphylococcus aureus (Baird-Parker) Yeasts and molds (YEGC and DRBC) 12
Fungal Diversity of Raw Milk Isolation of 505 yeasts and molds. Isolation by morphological grouping 13
Yeast and Mold Identification Identification of species by sequencing ITS1-5.8S-ITS2 regions. ITS1 ITS4 18S SSU 5.8S 28S LSU 5S 14
Fungal Diversity of Raw Milk 67% yeasts 33% molds Yeasts 11 genera, 37 species Main species Candida sp. (44%) C. parapsilosis (8%) 12 farms Debaryomyces hansenii (21%) 14 farms Cryptococcus sp. (6%) 17 farms 15
Fungal Diversity of Raw Milk Mouls 24 genera, 33 species Principal species Eurotium sp. (8%) 16 farms Cladosporium sp. (6%) 14 farms 16
2. Persistence of Fungal Flora? 4 farms selected Monitoring during 5 months (June to October) Semi-quantitative analysis Systematic isolation of yeasts and molds (total of 272) Identification: sequencing ITS1-5.8S-ITS2 17
Differences Between Producers Producer A 18
Differences Between Producers Producer C 19
Observed Variations Between Producers The yeast proportion: molds vary from one producer to another Yeasts are dominant for 3/4 of producers Molds dominate slightly for 1/4 of producers The most prevalent species differ from one producer to another Milk from producers A shows dominance of Candida glaebosa yeast over 5 months. The yeast Debaryomyces hansenii in dominant in milk from producer C. 20
3. Transfer of Yeasts and Molds from Milk to Cheese Can the fungal flora grow in cheese and participate in ripening like bacteria do? Strain genotyping for species other than those added for ripening Issatchenkia orientalis (yeast) Genotyping of strains isolated from raw milk and raw milk cheese by MLST 21
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4. Cooperation between Natural Flora and Probiotic Lactic Culture Probiotics Lb. rhamnosus R001 B. lactis BB12 Ripening Cultures A : B : C : P. camemberti G. candidum LMA-664 D. hansenii LMA-668 G. candidum LMA-563 D. hansenii LMA-695 G. candidum GEO17 D. hansenii LAF3 Location 1 : core 2 : surface See Pierre-Luc Champigny s poster
5. Production of Anti-Listeria Compounds 95 strains of yeasts and molds were tested for their anti-listeria potential. Preliminary tests reduced the number of candidates to 22 strains, then to 4 strains. 24
Inhibition of Listeria ivanovii 25 See Rima Hatoum s Poster
6. Influence of the Composition of "Terroir" Milk 26
Main Conclusions The yeast and mold counts must be treated separately. Each producer has a typical fungal flora that may change slightly over time. The natural milk fungi can grow in cheese in the first stages of ripening (NSYM, nonstarter yeast & molds). Cladosporium cladosporioides Eurotium sp. 27
Main conclusions Camembert allows probiotic survival. Alternaria alternata Some natural strains in milk have anti-listeria activity. Milk composition affects the colour of the Camembert ripening flora. Aspergillus versicolor 28
We thank Pierre-Luc Champigny Kathya Dupont Rima Hatoum Karine Lavoie Marilyne Touchette Claude Champagne Ismail Fliss Daniel St-Gelais Gaétan Bélanger Annie Caron Caroline Lapointe Mathieu Lapointe Yves Raymond Sophie Turcot Catherine Viel 29
We also thank The 19 dairy producer-processors who participated in the study. 30
Thank you! 31