New developments in the knowledge of cheese microbial ecosystems. Prof. HE Spinnler,
www.agroparistech.fr New developments in the knowledge of cheese microbial ecosystems. Prof. HE Spinnler, AgroParisTech / INRA 78850 Thiverval Grignon France www.agroparistech.fr Goals and outlines • The cheese curd and the ripening environment determines the growth of a microbial ecosystem inside the cheese and at the cheese surface. • This growth : • Determines the expression of metabolisms • And consequently the sensory caracteristics of the ripened cheeses • Even if relatively complex, these microbial communities are composed of a limited number of cultivable moulds, yeasts and bacteria. • In soft cheeses where the ratio S/V is high the rôle of the surface microbial ecosystem is particularly important. 2 Methods used to understand the development and expression of the ecosystem www.agroparistech.fr Milk ecosystems Factories ecosystems Direct ecosystem approach - Effect of microbial combination - Effect of species omission on functions - Understanding interactions and modelling - Sequencing the genome of complete ecosystems ( High throuput sequencing) -Transcriptomics and expression of complete ecosystems ( High Throuput sequencing) - Metabolite analysis (flavour produced) -Sensory analysis Cheese ecosystems Individual species studies - Complete Genomes - Transcriptomics and regulation of expression - Proteomics - Metabolite analysis (flavour produced, metabolomics) - Physiology (régulations) PLAN www.agroparistech.fr 1. Questions about Cheese Microbial Ecosystems 2. Diversity observed and main functions 3. Important parameters for the selection of the important species 4. Relations with organoleptic properties From Where these micro-organisms are coming from? The diversity of flora occuring in/on cheese are collected all along the process from paddock to plate Farm plants cheese technology cheese Starters Milk Coagulation and Moulding Salting and Surface drying Ripening 5 www.agroparistech.fr Wolfe et al, 2014 www.agroparistech.fr Geographic distribution Wolfe et al, 2014 www.agroparistech.fr Geographic distribution Wolfe et al, 2014 Inoculated Diversity www.agroparistech.fr • Some species necessary to make a pasteurised milk camembert cheese Lactococcus (Lc) lactis ssp lactis Lc lactis ssp lactis biovar diacetylactis, Lc lactis ssp cremoris Leuconostoc mesenteroïdes Kluyveromyces lactis Debaryomyces hansenii Geotrichum candidum (Yarrowia lipolytica) Penicillium camemberti Brevibacterium linens (Arthrobacter spp.) Hafnia alvei Factory : Microbial diversity in Munster cheeses (Single Strand Conformation Polymorphism) January * * ** * * April Brevibacterium linens Arthrobacter arilaitensis Carnobacterium maltaromaticum Lactobacillus sakei Streptococcus thermophilus L.lactis subsp.lactis ?? : pic b : pic a : pic e : pic i : pic g : pic h : pic c ?? : pic d h Milk + starters Octob er Feurer C. et al. JDS, 2004 b Massage solution g Inoculum i Quite stable and résilient flora: 50 % resident flora (*) 50 % inoculated flora ( ) 10 www.agroparistech.fr What are they? On 137 cheese microbial communities Most of them are consistantly convergents Wolfe et al, 2014, Cell, 422-433 Why do they grow? www.agroparistech.fr • What favours selection of bacteria and yeast in cheese? • How they adapt to dairy curds and their environment e.g. how to: - promote ripening starters with growth rates more competitive than pathogens - use ecosystems more resistant to unexpected germs and be more resilient if a technological factor (Dry matter, salt level, distribution of fat) is different to the one expected 12 When are they growing in the cheese? www.agroparistech.fr 1. Lactic acid bacteria are normally the first to grow and they produce the lactic acid that will protect the curd for a few days 2. As the curd is acid the yeasts and moulds are the only one able to grow for a while (4-8 days) at the surface. 3. Inside the cheese only anaerobic bacteria such as Propionibacterium are able to grow when the temperature is over . 4. When the pH is not too low (over 5.3) Ripening bacteria start to grow but it is only over pH= 5.8 that they will grow significantly. www.agroparistech.fr Succession within a Natural Rind Community Is Highly Reproducible (A and B) Reproducible succession in an in situ rind community was observed as three batches of a natural rind cheese aged (1–63 days). (A) Relative abundance of community members... Wolfe et al, Cell, Volume 158, Issue 2, 2014, 422 - 433 PLAN www.agroparistech.fr 1. Questions about Cheese Microbial Ecosystems 2. Diversity observed and main functions 3. Important parameters for the selection of the important species 4. Relations with organoleptic properties www.agroparistech.fr What are their functions? 1. Protection against the other micro-organisms 2. Aspect and colour : White : Penicillium camemberti, Penicillium nalgiovensis Toad skin: G. candidum Orange : B. linens 3. Odour : Debaryomyces hansenii, Kluyveromyces lactis, Brevibacterium linens, B. aurantiacum, Pseudoalteromonas haloplanctis 4. Taste : Penicillium camemberti, Lc lactis, Pseudomonas sp. (bitterness) 5. Flavour : B. linens, G.candidum,P.c. , P.r. What is doing what? www.agroparistech.fr But the relative expression of their genomes in cheese is still poorly known and this knowledge could help : To prioritize the research and consider the organisms that are more active To establish the relationship between the expression and the sensory properties To get better control of cheese organoleptic properties 17 PLAN www.agroparistech.fr 1. Questions about Cheese Microbial Ecosystems 2. Diversity observed and main functions 3. Important parameters for the selection of the important species 4. Relations with organoleptic properties 3.1. Moisture •Consistancy of the moiture level is probably the more important parameter for the cheese ripening and properties. www.agroparistech.fr •To much moisture accelarate the ripening processes •The stucture of the curd and the regularity of the porosity around the fat globule is also important. Pockets of whey can be the source of spoilage (in pressed cheese mainly) •The important parameter is the relative Humidity of the cheese but more informative is the Moisture in the Defated Cheese (MDC). The consistancy of this value integrate the variability of milk composition and of the process, up to the end of moulding. www.agroparistech.fr Humidity from surface to inner cheeese Water concentration profiles at different times of salting. J. Santapaola , S. Maldonado , J.L. Medina NaCl diffusion kinetics in dry salting of goat cheese Journal of Food Engineering, Volume 118, Issue 2, 2013, 172 - 177 Correlations between genius accurence and moisture level www.agroparistech.fr Taxonomic groups show different responses to gradients in moisture across cheese rinds. A plot of Pearson’s r depicts significant (p < 0.05, with false discovery rate correction) negative and positive correlations between abundance of particular genera and percent moisture. Wolfe et al, 2014 www.agroparistech.fr Correlations between Bacteria and Fungi? Wolfe et al, 2014 3.2. Salt www.agroparistech.fr -Salt will be mainly selective when the dry salt or brine is applied :many species are killed some are just « chocked ». (e.g. : Geotrichum candidum) and need a few days before to grow again. - The salt diffuse inside the cheese and its concentration in the cheese is finally low (1.5 to 3%) -However if you consider salt/moisture, this quantity is sensitive especially for the bacterial development – bacteria are very sensitive to salt www.agroparistech.fr Salt in cheeses www.agroparistech.fr Correlation between salt level and moisture Bloomy Natural Wash rind Ratio Salt/moisture is a parameter that can be useful to control the ripening Wolfe et al, 2014 a) In cheddar cheese www.agroparistech.fr Starter Bacteria Non Starter Bacteria Starter bacteria count (a) and non-starter bacteria count (b) in cheeses with different NaCl addition levels throughout ripening: ♦, 3.0%; ■, 2.5%; ▴, 2.25%; □, 1.8%; ▵, 1.25%; ○, 0.5%. Values presented are the means of triplicate trials. A. Rulikowska et al. International Dairy Journal, Volume 28, Issue 2, 2013, 45 55 26 b) Adaptation of reconstituted microbial ecosystem of washed rind cheeses - Effect of salt 1.7 % salt 2.8 % salt Straub et al, submitted 27 3.3. pH www.agroparistech.fr Context : Important changes in curd, composition, structure, aspect, texture, colour and taste due to biological activity and transport phenomena. CO2 O2 Yeasts, Penicillium, Ripening bacteria Surface Water core Lactose + - NH4+ Lactate + + + Flavour compoun ds - + 28 3.3. pH Cheese pH Soft cheeses www.agroparistech.fr Solubilised cheeses PPNC : Non cooked pressed cheeses PPC : Pressed cooked cheeses Cheese pH Limit of pH for the growth of different groups Evolution of the pH in différent cheeese during ripening (B. Mietton) 29 pH www.agroparistech.fr 3.3. Wolfe et al, 2014 30 www.agroparistech.fr On these 3 parameters moisture is the more important selection factor for the cheese surface diversity- Salt cc is the least significant Wolfe et al, 2014 3.4. Adaptation to the chemical environment Genetic adaptation => increase in fitness www.agroparistech.fr a) Data from complete genomes - Arthrobacter arilaitensis (Monnet et al, 2010, Plos1) • Adaptation to low iron concentration (siderophores) • High capabilities to catabolize fatty acids • Adaptation to osmotic shocks (glycyl betaïne transporters) - Corynebacterium casei (Monnet et al, 2012, J. Bacteriol., 194(3):738) • Adaptation to low iron concentration (siderophores, low number of enzymes needing iron as cofactors) 32 Capture of Iron through siderophores www.agroparistech.fr Monnet et al, 2012, AEM, 78:3185 Arthrobacter arilaitensis (7/7), Corynebacterium casei ( 2/2), C. variabile (1/2) Brevibacterium aurantiacum (4/5), B. antiquum (1/1) B. linens (1/1) are stimulated by FeCl3, 6H20 (1mg/kg of cheese), Growth of all the strains of these species were stimulated with 50mmol/kg of cheese of the siderophore desferrioxamine B. The expression of Iron ABC Siderophore transporters AARI_02870, AARI_3970, AARI_3980 are also amplified by the desferroxiamine B. 33 % of DNA mapped unique reads (70% of total reads) (Sequenced by SOLiD™, INRA MetaQuant Platform, 35bp) Straub et al, submitted 34 Expression (RNA) of the different genomes Straub et al, submitted 35 PLAN www.agroparistech.fr 1. Questions about Cheese Microbial Ecosystems 2. Diversity observed and main functions 3. Important Parameters for the selection of the important species 4. Relations with organoleptic properties www.agroparistech.fr Main functions with impact • • • • • Lactic acid production or lactic acid uptake Proteolysis amino acid breakdown Lipolysis and free fatty acid accumulation Flavour production Production of colouring molecule Example of the expression of flavour properties in Camembert (Molimard et al, 1997) Cabage, Cow shed 5 4 DIM 2 (26.7%) 0 P4G2 P3G3 -2 P4G1 P2G1 P4 P3G1 P2 P1G2 P2G3 P4G3 -1 Intensity, Blue Cheese P3G2 3 Butter, Milk 2 Cream, 1 Mushroom P2G2 P1G3 Cardboard, Plastic P1G1 P3 P1 -3 -4 -6 -4 -2 0 2 DIM 1 (45.8%) 4 6 8 38 L-methionine degradation pathways in cheeseripening micro-organisms L-methionine (CH3-S-CH2-CH2-CH(NH2)-COOH) www.agroparistech.fr L-methionine g-Lyase (MGL) a-HB Methylthioacetaldehyde a-KB (CH 3-CH 2-CO-CO 2H) + Acyl-CoA + METHANETHIOL (CH 3SH) Glu KMBA (CH 3S-CO-R) reduction (CH 3S-CH 2-CH 2-CO-CO 2H) KMBAdemethiolase a-KB + Thioesters Aminotransferase (+ acceptor) GDH NH3 reduction a-KG HMBA KMBAdecarboxylase Methional (CH 3S-CH 2-CH 2-CHO) METHANETHIOL (CH3SH) Auto-oxidation AA, FFA, Sugars DMDS, DMTS Other VSC 39 www.agroparistech.fr Wolfe et al, 2014 Conclusions : There is a large diversity of microbes in cheese and at the cheese surface but the selection factors of their development is not clear www.agroparistech.fr The data from genomics help to understand adaptation of bacteria, yeasts and moulds to the chemical environment of dairy products This knowledge may help control contaminant outbreaks and permit a « microbial driven ecology » The new information obtained could help to increase a range of starters available, more adapted to cheese technologies. Which means : - a better growth and propagation in the cheese environment For example with starter with a better moisture/salt Or more adapted to low iron environment -a adaptation to a range of better functional expression : for flavour, colour, texture, … 41 Conclusions : www.agroparistech.fr The transcriptomic approaches are helping to understand the base of the development of specific qualities in pure culture and in ecosystems of limited complexity. Understanding ecosystems means also understanding the basis of microbial interactions. Genomics or transcriptomics are, at the moment, insufficient to elucidate the types of interactions between micro-organisms Other constraints related to differential kinetics of mass transfer are still not quantitative. These depend on the curd composition, structure and on environmental parameters. . 42
© Copyright 2024