New bioanalytical methods to assure food safety and ... · to assure food safety and quality Neue...
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Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
New bioanalytical methodsto assure food safety and quality
Neue bioanalytische Methodenzur Sicherung der Lebensmittelqualität
Michael W. Pfaffl
Lehrstuhl für Physiologie, TU München Weihenstephan, 85354 Freising
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceWhat is Food Quality & Safety ?
Food Safety
Protecting the food supply from microbial, chemical, and physical hazards orcontamination that may occur during all stages of food production: growing,harvesting, processing, transporting, preparing, distributing, storing, andhandling.The goal of food safety monitoring is to keep food wholesome, safe to eat andfree of disease causing agents such as:
– infectious agents (bacteria, viruses, parasites, fungus, e.g. Salmonella, E.coli, EHEC, Hepatitis A, …)
– toxic chemicals (natural toxins, pesticide, heavy metals, trace elements,antibiotics, preservatives, drugs, hormones, …)
– foreign objects (e.g. bone, rocks, metal, …)
Food Quality
Food quality is the quality characteristics of food that is acceptable toconsumers. This includes food appearance (size, shape, colour, gloss, andconsistency), texture, flavour, factors such as federal grade standards, andingredients (chemical, physical, microbial, secondary plant components, pre- orprobiotics, synbiotics, …)
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceFactors influencing GIT health
waterwater
climateclimate
nutrientsnutrients
stressstress
pathogenspathogens
liver & pancreatic enzymesliver & pancreatic enzymes
medicationmedication
intestinal epitheliumintestinal epithelium
healthy nutrientshealthy nutrients
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceEffect of food quality on GIT eubiosis
EUBIOSISEUBIOSIS
DYSBIOSISDYSBIOSIS
unbalanced
• benefit for digestion
• prevention of pathogenic bacterial
colonization & adhesion
• natural synthesis of vitamins
• stimulation of the immune system
• dysfunction
• loss of efficiency
• loss of resistance
• predisposition for disease• disease due to infection & toxins
bad boys good boys
balanced
sec. plant ingredients
pre- and probiotics
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceImmune response in the GIT
food ingredients
secondary plant components => healthy intestine
prebiotics & probiotics
NFB
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceEGCG & Catechin in tissues
Tissues treated with polyphenols, EGCG and red-wine extracts:
treated with DMACA
staining of the cell nucleus
EGCG and Catechin binds to Histon complexes ???
effects on mRNA and microRNA gene expression ???
liver:
6 m cryo slice, EGCG
jejunum:
5-6 m cryo slice, EGCG
spleen:
5-6 m paraffin-embedded slice, red wine extract
Polster et al., AFS 2002, 24, 73-78
Catechin
EGCG
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceEffects in piglets GIT
1. Lamina muscularis mucosae2. Crypt of Lieberkuhn3. Villus
ileum control groupapple-pomace
groupred-wine-pomace
group
Y = 0.0015x + 0.204 Y = 0.000005x + 0.203 Y = 0.0003x + 0.222
21% (p<0.007) 0% (n.s.) 3.8% (n.s.)
Peyer Patch area
Apple and red wine pomace can reduce the activation of the immune
system via the Peyer Patches in the ileum.
Red wine pomace has inhibitory effects in the jejunum villi growth and
apple pomace has a stimulating effect on colon villi and crypt growth.
Flavanoid rich feeding regimen showed positive effects on gut health.
Flavanoids seems to be a good alternative to improve piglet health in
agriculture after the ban on antibiotics.
Sehm et al., (2007) Journal of Animal Physiology and Animal Nutrition 91, 289–296
Effect of 26 day apple-pomace or red-wine-pomace treatment
Growth rate
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
Influence of apple- or red-wine pomaceon GIT eubiosis
Th3
Tr
BTGF-ß
IL-10
dIgA
M
NfKB
IL-18
IFN-γ
IL-12
TNF-α
in small gut (jejunum & ileum)
transcription factor NFB
IL-10 in the jejunum
TNF in the ileum
surface of peyer plates
=> promote GIT eubiosis
in hind gut (colon)
transcription factor NFB
growth promoter IGF-1
IL-10 in the colon
in stomach
transcription factor NFB
pro-inflammatory vs. anti-inflammatorymarker genes marker genes
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
Estrogens => development of primary andsecondary sexual characteristics
Prolactin => breast development, milk production
Gynecomastia: development of abnormally largemammary glands
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
Classical analytical methods fordrug/hormone residue analysis
Use of specific anabolic agents is licensed in Canada, USA, Australia, South Africa...
Since 1988 the use of anabolic agents is prohibited in the EU !
Misuse of anabolics => sensitive screening and test system
– RIA
– ELISA
– LC-MS/MS
– GC-MS
– GC-MS/MS
=> analysis of specific known anabolics
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceAIM
Establishment of a new hormone screening method
Screening of physiological response- in various hormone sensitive tissues
- in easy accessible tissues, e.g. body liquids, blood, hair roots
Regulative impact of hormones on transcriptome
qRT-PCR as most sensitive detection method(=> 1 molecule detection limit)
Evaluation and analysis of regulated genesmRNA & microRNA candidates
Candidate verification- via statistical methods, e.g. HCA or PCA- via independent methods (NGS)
Specification of “Expressed Biomarkers”
Identification of gene networks
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceSteroid hormone action
Riedmaier I, Becker C, Pfaffl MW, Meyer HHD. - ReviewThe use of omic technologies for biomarker development to tracefunctions of anabolic agents. Journal of Chromatogr A. 2009
mRNA
microRNA
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
Riedmaier I, Becker C, Pfaffl MW, Meyer HHD. (2009) ReviewThe use of -omic technologies for biomarker development to tracefunctions of anabolic agents. Journal of Chromatography A
Work flow TBA+E2
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceFunctional target gene groups
• steroid receptors
• steroid hormone metabolism
• proliferation / apoptosis
• angiogenesis
• tissue remodelling
• immune relevant factors
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
mRNA expressionfrom heifers ovary samples
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
microRNA expression profilingsingle assays and microRNA qPCR array
miR-x-fold regulation
(2-ΔΔCq)p -value
significancelevel
412 3.18 0.006 **
103-2* 2.16 0.010 **
15a 1.23 0.010 **
192* 1.72 0.029 *
378* 22.04 0.030 *
532-5p 1.38 0.027 *
103 1.20 0.032 *
885-5p 1.50 0.030 *
let-7f2 1.23 0.032 *
152 1.62 0.041 *
493 1.97 0.036 *
744* 1.71 0.046 *
215 1.61 0.049 *
572 39.49 0.054 *
138 3.42 0.060
192 1.49 0.061
1247 1.79 0.066
194 1.50 0.087
505 1.52 0.094
146-5p 1.27 0.090
125a-3p 4.43 0.089 single assay PCR
0 1 2 3 4 5 6
PC
Ra
rra
y
0
1
2
3
4
5
miRNA regulation in bovine liver underthe influence of TBA+E2
correlated regulationsingle microRNA assay vs. qPCR array
slope = 0.773r = 0.662p<0.001
miR 378: promotes cell survival, tumor growth,angiogenesis
miR 412: regulates Uterine leiomyomas growth,benign uterine smooth muscle tumor
miR 15a: tumorgenesis, unarrested cell cycle
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceWBC expression data analysis
Significantly regulated genes in heiferblood (16 days after treatment)
PCA & HCA
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
Group Icontrol animals (n=2)
Group Icontrol animals (n=2)
Group IItreated animals (n=3)
Group IItreated animals (n=3)
sequencingsequencing
data analysisdata analysis
RNA isolationRNA isolation
TU MünchenTU München
EMBLEMBL
GenomatixGenomatix
Alteration of gene regulation in bovine liverby treatment with anabolic steroids
* mapping* expression analysis
Conformational study => NGS
Experimental setup
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceSequence statistics
Sequencestatistics
C5 C8 T12 T14 T17
Number of sequences 23,429,884 22,352,193 25,350,551 25,310,479 17,810,394
Sequence length 76 76 76 76 76
Average GC content 52.21% 53.35% 52.66% 52.28% 52.92%
Sequences with ambiguities 1,500,337 1,408,133 1,136,989 181,228 116,747
Nucleotidedistribution
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
gene name abr x-foldNGS
sign.NGS
x-fold PCRn=9
sign. PCRn=9
x-fold PCRn=3
sign. PCRn=3
interferon-stimulated gene 15 ISG15 0.326 1 0.54 0.058 0.58 0.114
myxovirus (influenza virus) resistance 2 MX2 0.280 1 0.47 0.019 0.35 0.094
cut-like homeobox 2 CUX2 26.76 1 6.326 0.00 12.34 0.004
serpin peptidase inhibitor (pancpin) 2 SERPINI2 26.18 1 82.357 0.01 153.84 0.032
cytochrome P450 (CYP2E1) CYP2E1 0.02 1 0.38 0.015 0.02 0.009
fatty acid desaturase 2 (FADS2) FADS2 0.09 1 0.37 0.004 0.06 0.011
Correlation qRT-PCR vs. NGS
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural Science
BiblioSphere
Cocitation based network of genesregulated at least 2 fold betweencontrol and treatment. (and min NE of0.5 in at least one condition)
4.7
-6.8
0.0
Genes from input list are coloredbased on ratio between control andtreatment. Red – induced , blue –repressed with treatment.
White boxes represent transcriptionfactors that are cocited with at least10 input genes.Green lines indicate potential bindingsites in the promoter region.
Gene network regulated by Anabolics
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceConclusion & Perspectives
• Transcriptome analysis discover promising expression pattern whichcan be used as biomarkers
• New markers via microRNA profiling NGS
• The more regulated mRNA & microRNA transcripts are discoveredthe better a results prediction via PCA or HCA is possible
• Tissues that are directly influenced by steroid hormones are better forthe development of gene expression biomarkers
• Further trials with more animals and a panel of anabolic agents
• Large number of untreated control animals
• Combination of transcriptomics with other „-omic“ technologies, likeproteomics or metabolomics
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceFood quality & Food safety
Th3
Tr
BTGF-ß
IL-10
dIgA
M
NfKB
IL-18
IFN-γ
IL-12
TNF-α
pro-inflammatory vs. anti-inflammatorymarker genes marker genes
“Expressed Biomarkers” areable to identify treated animals
=> ensures food safety
secondary plant compoundshave impact on epithelial
morphology and GIT health
=> ensures healthy food quality
Agrarwissenschaftliches Symposium - 20. Sept. 2010
Hans-Eisenmann-Zentrumfor Agricultural ScienceAcknowledgements