Milk, Molecules and Microbiomes...Milk, Molecules and Microbiomes Metabolomic and proteomic insights...
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Milk, Molecules and Microbiomes Metabolomic and proteomic insights into milk biochemistry: genetic, seasonal and feed induced changes in milk Simone Rochfort Molecular Phenomics Biosciences Research, AgriBio Department of Economic Development, Jobs, Transport and Resources School of Applied Systems Biology, La Trobe University
Transcript of Milk, Molecules and Microbiomes...Milk, Molecules and Microbiomes Metabolomic and proteomic insights...
Milk, Molecules and Microbiomes
Metabolomic and proteomic insights into milk biochemistry: genetic, seasonal and feed induced
changes in milk
Simone Rochfort
Molecular Phenomics
Biosciences Research, AgriBio
Department of Economic Development, Jobs, Transport and Resources
School of Applied Systems Biology, La Trobe University
Biosciences Research DivisionOutline
• Project background• Techniques – microbiome, OS, proteins, non-polar and polar lipids, NMR• Time course data• Within herd variation – how different are individual animals at one time point?• Seasonal variation – do profiles change over a milking season• Between breed variation – Holstein vs. Jersey• Feed induced variation• Data correlations• Next Steps
Biosciences Research DivisionAgriBio, Centre for AgriBioscience
- grains, dairy, meat and horticulture
• Supporting the agriculture and food sector
• Bioscience innovation for high impact outcomes in plant and animal industries
- driving productivity growth
- supporting market access
- enhancing farm business profitability
• Training the knowledge workforce of thefuture
• Two nationally recognised dairy research centres
- AgriBio
- Ellinbank
• Research priorities are improved;
- forage and feeding systems
- animal performance
- soil, water and nutrient use
- processor innovation
Major investment in dairy R&D
Gippsland - 38ºS latitude, 1100 mm rainfall, 500 cows
Melbourne area – glasshouses and laboratories
• Characterisation of milk for processing improvements• How does on-farm variation and manufacturing processes contribute to milk
characteristics and how does this affect product quality
Milk Systems Biology Project
Products
Plant SystemRumen Microbes
Animal
Milk quality
ProcessorMilk composition
• Components important for quality and processing are known, but not measured systematically
– enzymes (e.g. lipases)– peptides– immunoglobulins– complex sugars– metabolites– microbes
• New tools provide a unique opportunity to understand and better control milk quality in the supply chain
– Metagenomic sequencing – 16s– Sensitive mass spectrometry (lipids, intact protein, bottom up proteomics, OS)– NMR (Nuclear Magnetic Resonance)
• How do milk constituents vary – between animals, in a season, by diet?
Knowledge gap in the dairy supply chain
O P O
O−
O
H2C
CH
H2C
OCR1
O O C
O
R2
X
glycerophospholipid
Structure of phospholipids
H2CHC
OH
CH
N+ CH
C
CH2
CH3
H
H3
OH
( )12
sphingosine
H2CHC
O
CH
NH CH
C
CH2
CH3
H
OH
( )12
C
R
O
PO O
OH2C
H2CN+
CH3
H3C
CH3
Sphingomyelin
phosphocholine
sphingosine
fatty acid
−
Structure of glycosphingolipids
H2CHC
OH
CH
NH CH
C
CH2
CH3
H
OH
( )12
C
R
O
ceramide
Structure of sphingolipids
LCMS and GCMS Lipidomics Approaches Structure of triacylglycerols
LCMS Proteomics ApproachesFTMS full scan 15000 resolution TICAcclaim PepMap100, 75μm x 2cm, C18 3μm 100Å, Dionex50 min nLC run, 3-40%ACN separation in 35min
3%
40%
90%
Bottom up proteomics: nano LCMSMS
Identify 100s of proteins including caseins, beta-lactoglobulin, alpha-lactalbumin, lactoferrin, and minor proteins including glycoproteins and enzymes.
bCN_15k_sm_45C_200uLmin_P1C-9_01_3550.d
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PR0566pH4_15k_sm_200uLmin_7_PC-1_01_3565.d
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25.0
27.5
Time [min]
1092.9335
1414.07351602.4837
1716.95251848.7864 2002.9597
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LCMS Proteomics ApproachesTop down/intact proteomics: Adapting LC methods for intact protein analysis for LCMS analysis
Isoforms of casein, glycosylation κ casein, phosphorylation variants
Spectral deconvolution
β-casein standard A1
A2
Raw milkA1
no A2 β-casein
NMR (Nuclear Magnetic Resonance)
1H NMR Spectrum of Skim Milk
Citrate
Lactose
Aromatic compounds Organic acids
Linking biochemistry to structure and function
Which proteins and what modifications affect casein micelle size? What about minor interacting constituents?
Which lipids and proteins affect milk fat globule size?
How are physical factors affected e.g. UHT shelf life by microbial content, protein and lipid interaction in cheese making and spray drying
Deeper knowledge to underpin on farm or in factory manipulation
Time course analysis shows increase in protein, decrease in some lipids, increase in choline, betaine, decrease in PC, GPC
0%
10%
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30%
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90%
100%
0 24 48 72 24 48 72 24 28 72 time (hours)
4 10 25 temp ( oC )
4oC Profiles are comparable: lactobacillales, bifidobacteriales10oC pseudomonadales25oC enterobacteriales, steptococcaceae
Time course
Within herd variation: microbial diversityoligosaccharides
lipids
0%
10%
20%
30%
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100%
Lactobacillales
Enterobacteriales
Clostridiales
Pseudomonadales
BacillalesActinomycetales
Within herd variation: microbial diversity
12 animals on the same farm at the same time• Largely similar complexity and composition• Variation in relative abundances
Order
Within herd variation: microbial diversity
Family
Oligosaccharide – low abundance compared to lactose but nutritionally important . E.g. sialyllactose for brain development
LC-MS profiles of some of the OS
Rela
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nt(io
n in
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ity 1
e6)
Content of 3-sialyllactose in milk
Cow number
Within herd variation: OS diversity
Methods: Liu et al. 2014. Journal of Agricultural and Food Chemistry 62: 11568-11574
RT: 0.00 - 39.97
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38Time (min)
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10024.35
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20.60
14.76
9.02
PS (C18:0/C18:1)
SM (C18:1/C16:0)
PE (C18:0/C18:2)
PC (C16:0/C18:1)
LPC (C18:1)
PI (C18:0/C18:2)
LacCer (C18:1/C23:0)
GluCer (C18:1/C16)
phosphatidylcholine
phosphatidylethanolamine
phosphatidylserine
phosphatidylinositol
sphingomyelin
lysophophatidylcholine
lactosylceramide
glucosylceramide
Polar lipids – low abundance compared to TAG but, functionally and nutritionally important . E.g. Phosphoinositol levels regulated in infant formula.
Rela
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amou
nt(io
n in
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ity 1
e4)
Content of one PI species in milk(C45H83PO13; C18:0/C18:2)
Content of one LacCer species in milk(C53H103NO13; C18:0/C24:0)
Rela
tive
amou
nt(io
n in
tens
ity 1
e4)
Cow number
Cow number
Within herd variation: Polar lipid diversity
Content of one TAG species (C39H74O6: C4/C16/C16) in milk
Significant variation in lipid species concentration.
Significant differences in concentration between milks from individual cows
- Impact on milk properties?- Heritable
Rela
tive
amou
nt(io
n in
tens
ity 1
e7)
Rela
tive
amou
nt
Content of free cholesterol in milk
Chol
este
rol u
g/m
L
Cow numberCow number
Within herd variation: Non-Polar lipid diversity
Liu et al. 2015. Journal of Chromatography B 912: 8-15.
Significant variation in lipid species concentration.
Significant differences in concentration between milks from individual cows
- Impact on milk properties?- Heritable
mg/
L
Cow number
Cow number
Within herd variation: FAMES lipid diversity
mg/
L
Seasonal variation: microbial diversityoligosaccharides
LipidsNMR profiles
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Oct Nov Dec Jan Feb Mar Apr
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danc
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Oct Nov Dec Jan Feb Mar Apr
Rela
tive
abun
danc
eSeasonal Variation – OS and FAMEs examples
3-SL
6-SLN
FAM
E co
ncen
trat
ion
mg/
LFA
ME
conc
entr
atio
n m
g/L
butyric acid methyl ester
oleic acid methyl ester
Seasonal variation: Lipids
-3 -2 -1 0 1 2 3 4 5
x 109
-1.5
-1
-0.5
0
0.5
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2x 10
9
Scores on PC 1 (65.41%)
Sco
res
on P
C 2
(17.
86%
)
Samples/Scores Plot of Non-Polar Lipids Seasonal Study
1. Oct2. Nov3. Dec4. Jan5. Feb6. Mar7. Apr8. May95% Confidence Level
-2 -1 0 1 2 3 4
x 108
-1.5
-1
-0.5
0
0.5
1
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2x 10
8
Scores on PC 1 (79.25%)
Sco
res
on P
C 2
(12.
89%
)
Samples/Scores Plot of Polar Lipids Seasonal Study
1. Oct2. Nov3. Dec4. Jan5. Feb6. Mar7. Apr8. May95% Confidence Level
Non-polar lipids Polar lipids
Seasonal variation: FAMES and metagenomics
-20 -10 0 10 20 30 40 50-40
-30
-20
-10
0
10
20
30
Scores on PC 1 (17.42%)
Sco
res
on P
C 2
(6.3
5%)
Samples/Scores Plot of taxa
AprilDecFebJanMarMayNovOct95% Confidence Level
-6 -4 -2 0 2 4 6-6
-4
-2
0
2
4
6
Scores on PC 1 (37.60%)
Sco
res
on P
C 2
(20.
00%
)
Samples/Scores Plot of FAMES
AprilDecFebJanMarMayNovOct95% Confidence Level
FAMES
Microbial taxa
1H NMR Spectrum Over a Season
-2 -1.5 -1 -0.5 0 0.5 1
x 108
-6
-4
-2
0
2
4
6x 10
7
Scores on PC 1 (68.38%)
Sco
res
on P
C 2
(18.
49%
)
Samples/Scores Plot of data
1. Oct2. Nov3. Dec4. Jan5. Feb6. Mar7. Apr8. May95% Confidence Level
1234567891011
-0.05
0
0.05
0.1
0.15
ppm
PC
1 (6
8.38
%)
Variables/Loadings Plot for data
The profile changes significantly over the year. What does this mean for function?
Seasonal variation: NMR
Breed variation: major proteins
microbial diversity
LCMS
Analysis of most abundant intact proteins using LC-MS/MS (Q-TOF).
Clear breed differences between Holstein and Jersey animals e.g. aS1 and bCNA1/A2
Between breed variation: proteins
A-E: Actinobacteria F&G: Bacteroidetes H-R: Firmicutes S-Z: Proteobacteria
A-D Actinobacteria E-G: Bacteroidetes H: Cyanobacteria I-O: Firmicutes P-Z: Proteobacteria
Bifidobacteriaceae
Holstein
Jersey
Between breed variation: metagenomics
Diet variation: lipid
Citrate and urea may play a role in heat stability – quantitative methods being optimised
Between diet variation: lipids
*α linolenic acid is decreased in the almond diet compared to control (p<0.05)
Diet supplemented with either citrus or almond
Citrate and urea may play a role in heat stability – quantitative methods being optimised
Between diet variation: lipids
Data Correlations
Which proteins and what modifications affect casein micelle size? What about minor interacting constituents?
Which lipids and proteins affect milk fat globule size?
How are physical factors affected e.g. UHT shelf life by microbial content, protein and lipid interaction in cheese making and spray drying
Deeper knowledge to underpin on farm or in factory manipulation
What is the genetic heritability?
Within dataset correlations –biochemical pathway linkages
Between dataset correlations –cause/effect/interaction between different components, prediction of properties
Data Correlations - metagenomicsCorrelation Map, Variables in Original Order
f __;f __Cenarchaeaceae;f __SAGMA-X;f __Nitrososphaeraceae;f __Halobacteriaceae;f __Methanobacteriaceae;f __WSA2;f __Methanocellaceae;f __;f __Methanocorpusculaceae;f __Methanoregulaceae;f __Methanospirillaceae;f __Methanosaetaceae;f __Methanosarcinaceae;f __Marinef __[Methanomassiliicoccaceae];f __;f __;f __;f __;f __;f __;f __;f __RB40;f __mb2424;f __Acidobacteriaceae;f __Koribacteraceae;f __;f __;f __;f __Holophagaceae;f __;f __;f __;f __;f __AKIW659;f __MVS-65;f __PAUC26f ;f __Solibacteraceae;f __[Bry obacteraceae];f __;f __;f __;f __;f __;f __;f __;f __Ellin6075;f __[Chloracidobacteraceae];f __;f __;f __;f __;f __;f __AKIW874;f __C111;f __EB1017;f __Iamiaceae;f __JdFBGBact;f __Microthrixaceae;f __koll13;f __ntu14;f __;f __;f __ACK-M1;f __Actinomy cetaceae;f __Actinopoly sporaceae;f __Actinospicaceae;f __Actinosy nnemataceae;f __Beutenbergiaceae;f __Bogoriellaceae;f __Brev ibacteriaceae;f __Cellulomonadaceae;f __Cory nebacteriaceae;f __Cry ptosporangiaceae;f __Dermabacteraceae;f __Dermacoccaceae;f __Dermatophilaceae;f __Dietziaceae;f __Frankiaceae;f __Geodermatophilaceae;f __Gly comy cetaceae;f __Gordoniaceae;f __Intrasporangiaceae;f __Jonesiaceae;f __Kineosporiaceae;f __Microbacteriaceae;f __Micrococcaceae;f __Micromonosporaceae;f __My cobacteriaceae;f __Nakamurellaceae;f __Nocardiaceae;f __Nocardioidaceae;f __Nocardiopsaceae;f __Promicromonosporaceae;f __Propionibacteriaceae;f __Pseudonocardiaceae;f __Rarobacteraceae;f __Ruaniaceae;f __Sanguibacteraceae;f __Sporichthy aceae;f __Streptomy cetaceae;f __Streptosporangiaceae;f __Thermomonosporaceae;f __Tsukamurellaceae;f __Williamsiaceae;f __Yaniellaceae;f __Bif idobacteriaceae;f __;f __At425_EubF1;f __Coriobacteriaceae;f __;f __Euzeby aceae;f __Nitriliruptoraceae;f __;f __Rubrobacteraceae;f __;f __;f __AK1AB1_02E;f __Gaiellaceae;f __;f __Conexibacteraceae;f __Patulibacteraceae;f __Solirubrobacteraceae;f __;f __Armatimonadaceae;f __;f __;f __Chthonomonadaceae;f __;f __;f __;f __;f __;f __;f __[Fimbriimonadaceae];f __;f __;f __;f __;f __;f __;f __BA008;f __BS11;f __Bacteroidaceae;f __GZKB119;f __ML635J-40;f __Marinilabiaceae;f __Porphy romonadaceae;f __Prev otellaceae;f __RF16;f __Rikenellaceae;f __S24-7;f __SB-1;f __VC21_Bac22;f __[Barnesiellaceae];f __[Odoribacteraceae];f __[Paraprev otellaceae];f __p-2534-18B5;f __;f __Cy clobacteriaceae;f __Cy tophagaceae;f __Flammeov irgaceae;f __[Amoebophilaceae];f __;f __Blattabacteriaceae;f __Cry omorphaceae;f __Flav obacteriaceae;f __[Weeksellaceae];f __;f __Sphingobacteriaceae;f __;f __Rhodothermaceae;f __[Balneolaceae];f __;f __Chitinophagaceae;f __Saprospiraceae;f __;f __;f __;f __Criblamy diaceae;f __Parachlamy diaceae;f __Rhabdochlamy diaceae;f __Waddliaceae;f __;f __;f __;f __;f __Ignav ibacteriaceae;f __;f __;f __;f __;f __S47;f __Anaerolinaceae;f __Ardenscatenaceae;f __;f __Caldilineaceae;f __;f __;f __;f __;f __A4b;f __SHA-31;f __SJA-101;f __oc28;f __;f __;f __;f __;f __;f __;f __Chlorof lexaceae;f __FFCH7168;f __Oscillochloridaceae;f __;f __;f __[Kouleothrixaceae];f __;f __Dehalococcoidaceae;f __;f __;f __;f __;f __;f __;f __Ktedonobacteraceae;f __;f __Thermogemmatisporaceae;f __;f __;f __5B-12;f __;f __Dolo_23;f __;f __FFCH4570;f __;f __;f __;f __;f __;f __;f __;f __Chry siogenaceae;f __;f __;f __;f __;f __;f __;f __;f __Chlamy domonadaceae;f __Trebouxiophy ceae;f __;f __;f __;f __;f __;f __;f __Nostocaceae;f __Riv ulariaceae;f __;f __Chroococcaceae;f __Cy anobacteriaceae;f __Gomphosphaeriaceae;f __Microcy staceae;f __Xenococcaceae;f __Phormidiaceae;f __;f __Pseudanabaenaceae;f __Acary ochloridaceae;f __Chamaesiphonaceae;f __Sy nechococcaceae;f __Def erribacteraceae;f __;f __Elusimicrobiaceae;f __;f __;f __;f __;f __;f __;f __;f __Fibrobacteraceae;f __TSCOR003-O20;f __;f __;f __Alicy clobacillaceae;f __Bacillaceae;f __Listeriaceae;f __Paenibacillaceae;f __Planococcaceae;f __Sporolactobacillaceae;f __Staphy lococcaceae;f __Thermoactinomy cetaceae;f __[Exiguobacteraceae];f __[Thermicanaceae];f __;f __Gemellaceae;f __Haloplasmataceae;f __;f __Aerococcaceae;f __Carnobacteriaceae;f __Enterococcaceae;f __Lactobacillaceae;f __Leuconostocaceae;f __Streptococcaceae;f __Turicibacteraceae;f __;f __;f __Caldicoprobacteraceae;f __Christensenellaceae;f __Clostridiaceae;f __Dehalobacteriaceae;f __EtOH8;f __Eubacteriaceae;f __Gracilibacteraceae;f __JTB215;f __Lachnospiraceae;f __Peptococcaceae;f __Peptostreptococcaceae;f __Ruminococcaceae;f __SBYG_4172;f __Sy mbiobacteriaceae;f __Sy ntrophomonadaceae;f __Veillonellaceae;f __[Acidaminobacteraceae];f __[Mogibacteriaceae];f __[Tissierellaceae];f __Halanaerobiaceae;f __;f __Anaerobrancaceae;f __YAB3B13;f __;f __;f __;f __Ery sipelotrichaceae;f __;f __;f __Fusobacteriaceae;f __Leptotrichiaceae;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __A1-B1;f __Ellin5301;f __Gemmatimonadaceae;f __;f __;f __;f __;f __Victiv allaceae;f __;f __R4-45B;f __;f __;f __;f __0319-6A21;f __;f __FW;f __Nitrospiraceae;f __[Thermodesulf ov ibrionaceae];f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __;f __Gemmataceae;f __Isosphaeraceae;f __Pirellulaceae;f __Planctomy cetaceae;f __;f __;f __;f __;f __;f __;f __;f __Caulobacteraceae;f __;f __;f __;f __;f __Aurantimonadaceae;f __Bartonellaceae;f __Beijerinckiaceae;f __Brady rhizobiaceae;f __Brucellaceae;f __Cohaesibacteraceae;f __Hy phomicrobiaceae;f __Methy lobacteriaceae;f __Methy locy staceae;f __Phy llobacteriaceae;f __Rhizobiaceae;f __Rhodobiaceae;f __Xanthobacteraceae;f __Hy phomonadaceae;f __Rhodobacteraceae;f __;f __Acetobacteraceae;f __Rhodospirillaceae;f __;f __Rickettsiaceae;f __mitochondria;f __;f __Ery throbacteraceae;f __Sphingomonadaceae;f __;f __EB1003;f __UD5;f __;f __;f __Alcaligenaceae;f __Burkholderiaceae;f __Comamonadaceae;f __Oxalobacteraceae;f __;f __Gallionellaceae;f __Hy drogenophilaceae;f __;f __;f __;f __;f __;f __Methy lophilaceae;f __Neisseriaceae;f __;f __Nitrosomonadaceae;f __Procabacteriaceae;f __Rhodocy claceae;f __;f __;f __;f __Thiobacteraceae;f __Tremblay aceae;f __;f __Bacteriov oracaceae;f __Bdellov ibrionaceae;f __Desulf arculaceae;f __Desulf obacteraceae;f __Desulf obulbaceae;f __Desulf omicrobiaceae;f __Desulf ov ibrionaceae;f __Desulf uromonadaceae;f __Geobacteraceae;f __Pelobacteraceae;f __;f __;f __;f __;f __;f __0319-6G20;f __;f __Cy stobacteraceae;f __Cy stobacterineae;f __Haliangiaceae;f __My xococcaceae;f __Nannocy staceae;f __OM27;f __Poly angiaceae;f __;f __JTB38;f __;f __;f __Sy ntrophaceae;f __Sy ntrophobacteraceae;f __Sy ntrophorhabdaceae;f __;f __Campy lobacteraceae;f __Helicobacteraceae;f __;f __;f __;f __Aeromonadaceae;f __Succiniv ibrionaceae;f __125ds10;f __211ds20;f __;f __Alteromonadaceae;f __HTCC2188;f __Idiomarinaceae;f __OM60;f __Psy chromonadaceae;f __Shewanellaceae;f __[Chromatiaceae];f __Cardiobacteriaceae;f __;f __Chromatiaceae;f __Ectothiorhodospiraceae;f __Enterobacteriaceae;f __;f __;f __HTCC2089;f __;f __Coxiellaceae;f __Legionellaceae;f __;f __Crenotrichaceae;f __Methy lococcaceae;f __Alcaniv oracaceae;f __Hahellaceae;f __Halomonadaceae;f __Oceanospirillaceae;f __Oleiphilaceae;f __SUP05;f __Saccharospirillaceae;f __;f __;f __Pasteurellaceae;f __Moraxellaceae;f __Pseudomonadaceae;f __Salinisphaeraceae;f __Piscirickettsiaceae;f __Thiotrichaceae;f __Pseudoalteromonadaceae;f __Vibrionaceae;f __Sinobacteraceae;f __Xanthomonadaceae;f __[Marinicellaceae];f __;f __;f __;f __;f __;f __Sphaerochaetaceae;f __Spirochaetaceae;f __Leptospiraceae;f __Sediment-4;f __Dethiosulf ov ibrionaceae;f __Sy nergistaceae;f __TTA_B6;f __;f __;f __;f __;f __;f __;f __;f __;f __F16;f __;f __;f __Rs-045;f __;f __;f __Acholeplasmataceae;f __Anaeroplasmataceae;f __Entomoplasmataceae;f __My coplasmataceae;f __;f __;f __;f __Thermotogaceae;f __;f __;f __;f __Opitutaceae;f __Puniceicoccaceae;f __[Cerasicoccaceae];f __[Pelagicoccaceae];f __;f __;f __;f __;f __;f __RFP12;f __WCHB1-25;f __Verrucomicrobiaceae;f __;f __LD19;f __;f __;f __Ellin515;f __Ellin517;f __OPB35;f __R4-41B;f __[Pedosphaeraceae];f __auto67_4W;f __01D2Z36;f __[Chthoniobacteraceae];f __zEL20;f __;f __;f __;f __KSB4;f __;f __PRR-10;f __;f __;f __;f __SHA-116;f __[Cloacamonaceae];f __;f __;f __Deinococcaceae;f __Trueperaceae;f __Thermaceae;
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_Ellin
6075
;f_
_[C
hlor
acid
obac
tera
ceae
];f_
_;f_
_;f_
_;f_
_;f_
_;f_
_AKI
W87
4;f_
_C11
1;f_
_EB1
017;
f__I
amia
ceae
;f_
_JdF
BGBa
ct;
f__M
icro
thrix
acea
e;f_
_kol
l13;
f__n
tu14
;f_
_;f_
_;f_
_AC
K-M
1;f_
_Act
inom
ycet
acea
e;f_
_Act
inop
olys
pora
ceae
;f_
_Act
inos
pica
ceae
;f_
_Act
inos
ynne
mat
acea
e;f_
_Beu
tenb
ergi
acea
e;f_
_Bog
orie
llace
ae;
f__B
revi
bact
eria
ceae
;f_
_Cel
lulo
mon
adac
eae;
f__C
oryn
ebac
teria
ceae
;f_
_Cry
ptos
pora
ngia
ceae
;f_
_Der
mab
acte
race
ae;
f__D
erm
acoc
cace
ae;
f__D
erm
atop
hila
ceae
;f_
_Die
tzia
ceae
;f_
_Fra
nkia
ceae
;f_
_Geo
derm
atop
hila
ceae
;f_
_Gly
com
ycet
acea
e;f_
_Gor
doni
acea
e;f_
_Int
rasp
oran
giac
eae;
f__J
ones
iace
ae;
f__K
ineo
spor
iace
ae;
f__M
icro
bact
eria
ceae
;f_
_Mic
roco
ccac
eae;
f__M
icro
mon
ospo
race
ae;
f__M
ycob
acte
riace
ae;
f__N
akam
urel
lace
ae;
f__N
ocar
diac
eae;
f__N
ocar
dioi
dace
ae;
f__N
ocar
diop
sace
ae;
f__P
rom
icro
mon
ospo
race
ae;
f__P
ropi
onib
acte
riace
ae;
f__P
seud
onoc
ardi
acea
e;f_
_Rar
obac
tera
ceae
;f_
_Rua
niac
eae;
f__S
angu
ibac
tera
ceae
;f_
_Spo
richt
hyac
eae;
f__S
trept
omyc
etac
eae;
f__S
trept
ospo
rang
iace
ae;
f__T
herm
omon
ospo
race
ae;
f__T
suka
mur
ella
ceae
;f_
_Willi
amsi
acea
e;f_
_Yan
iella
ceae
;f_
_Bifi
doba
cter
iace
ae;
f__;
f__A
t425
_Eub
F1;
f__C
orio
bact
eria
ceae
;f_
_;f_
_Euz
ebya
ceae
;f_
_Nitr
ilirup
tora
ceae
;f_
_;f_
_Rub
roba
cter
acea
e;f_
_;f_
_;f_
_AK1
AB1_
02E;
f__G
aiel
lace
ae;
f__;
f__C
onex
ibac
tera
ceae
;f_
_Pat
ulib
acte
race
ae;
f__S
oliru
brob
acte
race
ae;
f__;
f__A
rmat
imon
adac
eae;
f__;
f__;
f__C
htho
nom
onad
acea
e;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_[Fi
mbr
iimon
adac
eae]
;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_BA0
08;
f__B
S11;
f__B
acte
roid
acea
e;f_
_GZK
B119
;f_
_ML6
35J-
40;
f__M
arin
ilabi
acea
e;f_
_Por
phyr
omon
adac
eae;
f__P
revo
tella
ceae
;f_
_RF1
6;f_
_Rik
enel
lace
ae;
f__S
24-7
;f_
_SB-
1;f_
_VC
21_B
ac22
;f_
_[Ba
rnes
iella
ceae
];f_
_[O
dorib
acte
race
ae];
f__[
Para
prev
otel
lace
ae];
f__p
-253
4-18
B5;
f__;
f__C
yclo
bact
eria
ceae
;f_
_Cyt
opha
gace
ae;
f__F
lam
meo
virg
acea
e;f_
_[Am
oebo
phila
ceae
];f_
_;f_
_Bla
ttaba
cter
iace
ae;
f__C
ryom
orph
acea
e;f_
_Fla
voba
cter
iace
ae;
f__[
Wee
ksel
lace
ae];
f__;
f__S
phin
goba
cter
iace
ae;
f__;
f__R
hodo
ther
mac
eae;
f__[
Baln
eola
ceae
];f_
_;f_
_Chi
tinop
haga
ceae
;f_
_Sap
rosp
irace
ae;
f__;
f__;
f__;
f__C
ribla
myd
iace
ae;
f__P
arac
hlam
ydia
ceae
;f_
_Rha
bdoc
hlam
ydia
ceae
;f_
_Wad
dlia
ceae
;f_
_;f_
_;f_
_;f_
_;f_
_Ign
avib
acte
riace
ae;
f__;
f__;
f__;
f__;
f__S
47;
f__A
naer
olin
acea
e;f_
_Ard
ensc
aten
acea
e;f_
_;f_
_Cal
dilin
eace
ae;
f__;
f__;
f__;
f__;
f__A
4b;
f__S
HA-
31;
f__S
JA-1
01;
f__o
c28;
f__;
f__;
f__;
f__;
f__;
f__;
f__C
hlor
ofle
xace
ae;
f__F
FCH
7168
;f_
_Osc
illoch
lorid
acea
e;f_
_;f_
_;f_
_[Ko
uleo
thrix
acea
e];
f__;
f__D
ehal
ococ
coid
acea
e;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_Kte
dono
bact
erac
eae;
f__;
f__T
herm
ogem
mat
ispo
race
ae;
f__;
f__;
f__5
B-12
;f_
_;f_
_Dol
o_23
;f_
_;f_
_FFC
H45
70;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__C
hrys
ioge
nace
ae;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__C
hlam
ydom
onad
acea
e;f_
_Tre
boux
ioph
ycea
e;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_Nos
toca
ceae
;f_
_Riv
ular
iace
ae;
f__;
f__C
hroo
cocc
acea
e;f_
_Cya
noba
cter
iace
ae;
f__G
omph
osph
aeria
ceae
;f_
_Mic
rocy
stac
eae;
f__X
enoc
occa
ceae
;f_
_Pho
rmid
iace
ae;
f__;
f__P
seud
anab
aena
ceae
;f_
_Aca
ryoc
hlor
idac
eae;
f__C
ham
aesi
phon
acea
e;f_
_Syn
echo
cocc
acea
e;f_
_Def
errib
acte
race
ae;
f__;
f__E
lusi
mic
robi
acea
e;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_Fib
roba
cter
acea
e;f_
_TSC
OR
003-
O20
;f_
_;f_
_;f_
_Alic
yclo
baci
llace
ae;
f__B
acilla
ceae
;f_
_Lis
teria
ceae
;f_
_Pae
niba
cilla
ceae
;f_
_Pla
noco
ccac
eae;
f__S
poro
lact
obac
illace
ae;
f__S
taph
yloc
occa
ceae
;f_
_The
rmoa
ctin
omyc
etac
eae;
f__[
Exig
uoba
cter
acea
e];
f__[
Ther
mic
anac
eae]
;f_
_;f_
_Gem
ella
ceae
;f_
_Hal
opla
smat
acea
e;f_
_;f_
_Aer
ococ
cace
ae;
f__C
arno
bact
eria
ceae
;f_
_Ent
eroc
occa
ceae
;f_
_Lac
toba
cilla
ceae
;f_
_Leu
cono
stoc
acea
e;f_
_Stre
ptoc
occa
ceae
;f_
_Tur
icib
acte
race
ae;
f__;
f__;
f__C
aldi
copr
obac
tera
ceae
;f_
_Chr
iste
nsen
ella
ceae
;f_
_Clo
strid
iace
ae;
f__D
ehal
obac
teria
ceae
;f_
_EtO
H8;
f__E
ubac
teria
ceae
;f_
_Gra
cilib
acte
race
ae;
f__J
TB21
5;f_
_Lac
hnos
pira
ceae
;f_
_Pep
toco
ccac
eae;
f__P
epto
stre
ptoc
occa
ceae
;f_
_Rum
inoc
occa
ceae
;f_
_SBY
G_4
172;
f__S
ymbi
obac
teria
ceae
;f_
_Syn
troph
omon
adac
eae;
f__V
eillo
nella
ceae
;f_
_[Ac
idam
inob
acte
race
ae];
f__[
Mog
ibac
teria
ceae
];f_
_[Ti
ssie
rella
ceae
];f_
_Hal
anae
robi
acea
e;f_
_;f_
_Ana
erob
ranc
acea
e;f_
_YAB
3B13
;f_
_;f_
_;f_
_;f_
_Ery
sipe
lotri
chac
eae;
f__;
f__;
f__F
usob
acte
riace
ae;
f__L
epto
trich
iace
ae;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__A
1-B1
;f_
_Ellin
5301
;f_
_Gem
mat
imon
adac
eae;
f__;
f__;
f__;
f__;
f__V
ictiv
alla
ceae
;f_
_;f_
_R4-
45B;
f__;
f__;
f__;
f__0
319-
6A21
;f_
_;f_
_FW
;f_
_Nitr
ospi
race
ae;
f__[
Ther
mod
esul
fovi
brio
nace
ae];
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__G
emm
atac
eae;
f__I
sosp
haer
acea
e;f_
_Pire
llula
ceae
;f_
_Pla
ncto
myc
etac
eae;
f__;
f__;
f__;
f__;
f__;
f__;
f__;
f__C
aulo
bact
erac
eae;
f__;
f__;
f__;
f__;
f__A
uran
timon
adac
eae;
f__B
arto
nella
ceae
;f_
_Bei
jerin
ckia
ceae
;f_
_Bra
dyrh
izob
iace
ae;
f__B
ruce
llace
ae;
f__C
ohae
siba
cter
acea
e;f_
_Hyp
hom
icro
biac
eae;
f__M
ethy
loba
cter
iace
ae;
f__M
ethy
locy
stac
eae;
f__P
hyllo
bact
eria
ceae
;f_
_Rhi
zobi
acea
e;f_
_Rho
dobi
acea
e;f_
_Xan
thob
acte
race
ae;
f__H
ypho
mon
adac
eae;
f__R
hodo
bact
erac
eae;
f__;
f__A
ceto
bact
erac
eae;
f__R
hodo
spiri
llace
ae;
f__;
f__R
icke
ttsia
ceae
;f_
_mito
chon
dria
;f_
_;f_
_Ery
thro
bact
erac
eae;
f__S
phin
gom
onad
acea
e;f_
_;f_
_EB1
003;
f__U
D5;
f__;
f__;
f__A
lcal
igen
acea
e;f_
_Bur
khol
deria
ceae
;f_
_Com
amon
adac
eae;
f__O
xalo
bact
erac
eae;
f__;
f__G
allio
nella
ceae
;f_
_Hyd
roge
noph
ilace
ae;
f__;
f__;
f__;
f__;
f__;
f__M
ethy
loph
ilace
ae;
f__N
eiss
eria
ceae
;f_
_;f_
_Nitr
osom
onad
acea
e;f_
_Pro
caba
cter
iace
ae;
f__R
hodo
cycl
acea
e;f_
_;f_
_;f_
_;f_
_Thi
obac
tera
ceae
;f_
_Tre
mbl
ayac
eae;
f__;
f__B
acte
riovo
raca
ceae
;f_
_Bde
llovi
brio
nace
ae;
f__D
esul
farc
ulac
eae;
f__D
esul
foba
cter
acea
e;f_
_Des
ulfo
bulb
acea
e;f_
_Des
ulfo
mic
robi
acea
e;f_
_Des
ulfo
vibr
iona
ceae
;f_
_Des
ulfu
rom
onad
acea
e;f_
_Geo
bact
erac
eae;
f__P
elob
acte
race
ae;
f__;
f__;
f__;
f__;
f__;
f__0
319-
6G20
;f_
_;f_
_Cys
toba
cter
acea
e;f_
_Cys
toba
cter
inea
e;f_
_Hal
iang
iace
ae;
f__M
yxoc
occa
ceae
;f_
_Nan
nocy
stac
eae;
f__O
M27
;f_
_Pol
yang
iace
ae;
f__;
f__J
TB38
;f_
_;f_
_;f_
_Syn
troph
acea
e;f_
_Syn
troph
obac
tera
ceae
;f_
_Syn
troph
orha
bdac
eae;
f__;
f__C
ampy
loba
cter
acea
e;f_
_Hel
icob
acte
race
ae;
f__;
f__;
f__;
f__A
erom
onad
acea
e;f_
_Suc
cini
vibr
iona
ceae
;f_
_125
ds10
;f_
_211
ds20
;f_
_;f_
_Alte
rom
onad
acea
e;f_
_HTC
C21
88;
f__I
diom
arin
acea
e;f_
_OM
60;
f__P
sych
rom
onad
acea
e;f_
_She
wan
ella
ceae
;f_
_[C
hrom
atia
ceae
];f_
_Car
diob
acte
riace
ae;
f__;
f__C
hrom
atia
ceae
;f_
_Ect
othi
orho
dosp
irace
ae;
f__E
nter
obac
teria
ceae
;f_
_;f_
_;f_
_HTC
C20
89;
f__;
f__C
oxie
llace
ae;
f__L
egio
nella
ceae
;f_
_;f_
_Cre
notri
chac
eae;
f__M
ethy
loco
ccac
eae;
f__A
lcan
ivor
acac
eae;
f__H
ahel
lace
ae;
f__H
alom
onad
acea
e;f_
_Oce
anos
piril
lace
ae;
f__O
leip
hila
ceae
;f_
_SU
P05;
f__S
acch
aros
piril
lace
ae;
f__;
f__;
f__P
aste
urel
lace
ae;
f__M
orax
ella
ceae
;f_
_Pse
udom
onad
acea
e;f_
_Sal
inis
phae
race
ae;
f__P
isci
ricke
ttsia
ceae
;f_
_Thi
otric
hace
ae;
f__P
seud
oalte
rom
onad
acea
e;f_
_Vib
riona
ceae
;f_
_Sin
obac
tera
ceae
;f_
_Xan
thom
onad
acea
e;f_
_[M
arin
icel
lace
ae];
f__;
f__;
f__;
f__;
f__;
f__S
phae
roch
aeta
ceae
;f_
_Spi
roch
aeta
ceae
;f_
_Lep
tosp
irace
ae;
f__S
edim
ent-4
;f_
_Det
hios
ulfo
vibr
iona
ceae
;f_
_Syn
ergi
stac
eae;
f__T
TA_B
6;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_;f_
_F16
;f_
_;f_
_;f_
_Rs-
045;
f__;
f__;
f__A
chol
epla
smat
acea
e;f_
_Ana
erop
lasm
atac
eae;
f__E
ntom
opla
smat
acea
e;f_
_Myc
opla
smat
acea
e;f_
_;f_
_;f_
_;f_
_The
rmot
ogac
eae;
f__;
f__;
f__;
f__O
pitu
tace
ae;
f__P
unic
eico
ccac
eae;
f__[
Cer
asic
occa
ceae
];f_
_[Pe
lagi
cocc
acea
e];
f__;
f__;
f__;
f__;
f__;
f__R
FP12
;f_
_WC
HB1
-25;
f__V
erru
com
icro
biac
eae;
f__;
f__L
D19
;f_
_;f_
_;f_
_Ellin
515;
f__E
llin51
7;f_
_OPB
35;
f__R
4-41
B;f_
_[Pe
dosp
haer
acea
e];
f__a
uto6
7_4W
;f_
_01D
2Z36
;f_
_[C
htho
niob
acte
race
ae];
f__z
EL20
;f_
_;f_
_;f_
_;f_
_KSB
4;f_
_;f_
_PR
R-1
0;f_
_;f_
_;f_
_;f_
_SH
A-11
6;f_
_[C
loac
amon
acea
e];
f__;
f__;
f__D
eino
cocc
acea
e;f_
_Tru
eper
acea
e;f_
_The
rmac
eae;
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
Data Correlations - metagenomics
Data Correlations/Prediction
2 3 4 5 6 7 82
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
Y Measured 2 Fat (%)
Y C
V P
redi
cted
2 F
at (%
)
Samples/Scores Plot of FAMES
R 2̂ = 0.802
Correlation between fames profile and total fat r=0.90
2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
Y Measured 3 Protein (%)
Y C
V P
redi
cted
3 P
rote
in (%
)
Samples/Scores Plot of FAMES
Class Set: study
R 2̂ = 0.660
Correlation between fames profile and total protein r=0.77
Data Correlations/Prediction
2.5 3 3.5 4 4.5 5 5.52.5
3
3.5
4
4.5
5
5.5
Y Measured 1 MFGY
CV
Pre
dict
ed 1
MFG
Samples/Scores Plot of FAMES
R 2̂ = 0.650
Correlation between fames profile MFG size r=0.81
2 3 4 5 6 7 8 93.8
4
4.2
4.4
4.6
4.8
5
5.2
Y Measured 4 Lactose (%)
Y C
V P
redi
cted
4 L
acto
se (%
)
Samples/Scores Plot of FAMES
Class Set: study
R 2̂ = 0.011
Correlation between fames profile and lactose r=0.1
Data Correlations/Prediction
2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.42
2.5
3
3.5
4
4.5
Y Measured 3 Protein (%)
Y C
V P
redi
cted
3 P
rote
in (%
)
Samples/Scores Plot of taxa
R 2̂ = 0.300
Fit1:1AprilDecFebJanMarMayNovOct
Correlation between metagenomicsprofile and protein r=0.55
Next Steps
• Finalise on going studies (e.g. cow heat stress, UHT longevity)• Complete analysis • Reanalyse and integrate datasets• Identify relationship between physiochemical traits and biochemistry
(e.g. Lipid content and MFG size)• A subset (420 animals) have been genotyped – GWAS
Future Steps
• Breeding values for specific products (e.g. cheese, spray drying)• On farm or In plant options for product optimisation• In plant tools to identify problems (e.g. microbial contamination)
How else are can these measurements be used?
• Indicators of animal health and fertility
• GWAS studies of metabolite heritability
• Milk and cheese provenance determination (adulteration)
• Metabolic changes in product manufacture e.g. cheese ripining
Some recent literature:• Curtis, S.D.; Curini, R.; Delfini, M.; Brosio, E.; D’Ascenzo, F.; Bocca, B. Amino acid profile in the ripening of Grana Padano cheese: a NMR
study. Food Chem. 2000, 71, 495–502.• Brescia, M.A.; Monfreda, M.; Buccolieri, A.; Carrino, C. Characterisation of the geographical origin of buffalo milk and mozzarella cheese
by means of analytical and spectroscopic determinations. Food Chem. 2005, 89, 139–147.• Mulas, G.; Roggio, T.; Uzzau, S.; Anedda, R. A new magnetic resonance imaging approach for discriminating Sardinian sheep milk cheese
made from heat-treated or raw milk. J. Dairy Sci. 2013, 96, 7393–7403.
AcknowledgementsDEDJTR AgriBioZhiqian LiuVilnis EzernieksPaul ConilioneNga NyguenMyrna DeseoKeith SavinDelphine VincentCarolina LeytonAaron ElkinsJianghui WangDoris RamJody ZawadzkiBen CocksGerman Spangenberg
DEDJTR EllinbankBill WalesPeter MoateMartin Auldist
CSIROAmy LoganRoderick WilliamsLi Day
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